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WO2001062911A2 - Antisense and catalytically acting nucleic acid molecules targeted to grb2- related with insert domain (grid) proteins and their uses - Google Patents

Antisense and catalytically acting nucleic acid molecules targeted to grb2- related with insert domain (grid) proteins and their uses Download PDF

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Publication number
WO2001062911A2
WO2001062911A2 PCT/US2001/005957 US0105957W WO0162911A2 WO 2001062911 A2 WO2001062911 A2 WO 2001062911A2 US 0105957 W US0105957 W US 0105957W WO 0162911 A2 WO0162911 A2 WO 0162911A2
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nucleic acid
acid molecule
cugaugag gccguuaggc
gccguuaggc cgaa
ucaaggacaucguccggg
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PCT/US2001/005957
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WO2001062911A3 (en
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Thale Jarvis
Ira Von Carlowitz
James A. Mcswiggen
Paul Andrew Hamblin
Jonathon Henry Ellis
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Ribozyme Pharmaceuticals, Inc.
Glaxo Group Limited
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Priority to AU39862/01A priority Critical patent/AU3986201A/en
Priority to CA002397813A priority patent/CA2397813A1/en
Publication of WO2001062911A2 publication Critical patent/WO2001062911A2/en
Publication of WO2001062911A3 publication Critical patent/WO2001062911A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/121—Hammerhead
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/332—Abasic residue

Definitions

  • the present invention concerns compounds, compositions, and methods for the study, diagnosis, and treatment of conditions and diseases related to the expression of the T-cell co-stimulatory adapter protein GRID (Grb2-related with Insert Domain).
  • One of the emerging paradigms for signal transduction in lymphocytes is that receptors and other signaling molecules do not operate in isolation, but through the recruitment of a complex of other proteins (Pawson and Scott, 1997; Science, 278, 2075; Rudd, 1999, Cell, 96, 5). These other proteins serve to amplify and diversify the signal into a number of biochemical cascades.
  • the archetypal adapter protein is Grb2, which serves to regulate downstream pathways such as Ras activation and Ca2+ mobilization (Lowenstein et al, 1992, Cell, 70, 431), and is ultimately responsible for modulating gene expression required for proliferation and differentiation.
  • Grb2 is recruited to LAT and SLP-76 which are downstream targets in the signaling cascade initiated by ligation of the T-cell receptor by MHC-antigen. These functions are mediated by specialized domains which bind specific motifs and include the phosphotyrosine binding SH2 (Src homology) domain and SH3 domain which are associated with proline-rich PXXP motifs.
  • Grb2 whose sole function appears to be the formation of bridges between other proteins, is entirely comprised of such domains having an SH3-SH2-SH3 structure (Peterson et al, 1998, Curr. Opin. Immunol, 10, 337; Koretzky, 1997, Immunol Today, 18, 401).
  • GRID Grb2-related with Insert Domain
  • GRID is recruited to the T cell co- stimulatory receptor CD28 upon activation of this receptor by cross-linking antibodies.
  • GRID shares significant similarity at the protein level with Grb2, possessing an SH3-SH2-SH3 domain structure, GRID also contains a unique proline-glutamine rich domain situated between the SH2 and C-terminal SH3 domain. The association of GRID with activated CD28 is absolutely dependent upon the integrity of the SH2 domain and phosphorylation of residue Y173 in the cytoplasmic tail of CD28.
  • GRID has been shown to associate with other T cell signaling proteins including SLP-76 and LAT (Asada et al, supra; Liu et al, supra; Law et al, supra), it's role in T cell signaling pathways is not well defined.
  • the invention features novel nucleic acid-based techniques [e.g., enzymatic nucleic acid molecules (for example, ribozymes or DNAzymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups] and methods for their use to modulate the expression of GRID (Grb2- related with Insert Domain).
  • enzymatic nucleic acid molecules for example, ribozymes or DNAzymes
  • antisense nucleic acids for example, ribozymes or DNAzymes
  • 2-5A antisense chimeras triplex DNA
  • antisense nucleic acids containing RNA cleaving chemical groups antisense nucleic acids containing RNA cleaving chemical groups
  • the various aspects and embodiments are also directed to other genes which express GRID -like adapter proteins involved in T- cell co-activation. Those additional genes can be analyzed for target sites using the methods described for GRID. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.
  • the invention features the use of one or more of the nucleic acid-based techniques independently or in combination to inhibit the expression of the genes encoding GRID.
  • the nucleic acid-based techniques of the present invention can be used to inhibit the expression of GRID gene sequences found at GenBank Accession NOS. AJ011736, NM_004810, Y18051, AF121002, AF042380, AF129476, AF090456).
  • the invention features the use of an enzymatic nucleic acid molecule, preferably in the hammerhead, NCH (Inozyme), G-cleaver, amberzyme, zinzyme and/or DNAzyme motif, to inhibit the expression of GRID gene.
  • inhibit it is meant that the activity of GRID or level of GRID RNAs or equivalent RNAs encoding one or more protein subunits of GRID or GRID-like proteins is reduced below that observed in the absence of the nucleic acid molecules of the invention.
  • the inhibition with enzymatic nucleic acid molecule preferably is below that level observed in the presence of an enzymatically inactive or attenuated molecule that is able to bind to the same site on the target RNA, but is unable to cleave that RNA.
  • inhibition with antisense oligonucleotides is preferably below that level observed in the presence of, for example, an oligonucleotide with scrambled sequence or with mismatches.
  • inhibition of GRID or GRID-like genes with the nucleic acid molecule of the instant invention is greater than in the presence of the nucleic acid molecule than in its absence.
  • enzymatic nucleic acid molecule it is meant a nucleic acid molecule which has complementarity in a substrate-binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave target RNA. That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. These complementary regions allow sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA and thus pe mit cleavage.
  • nucleic acids can be modified at the base, sugar, and/or phosphate groups.
  • enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA ?
  • RNA enzymatic RNA
  • catalytic DNA aptazyme or aptamer-binding ribozyme
  • regulatable ribozyme catalytic oligonucleotides
  • nucleozyme DNAzyme
  • minizyme minizyme
  • leadzyme oligozyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity.
  • enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving and/or ligation activity to the molecule (Cech et al., U.S. Patent No. 4,987,071; Cech et al, 1988, 260 JAMA 3030).
  • nucleic acid molecule as used herein is meant a molecule having nucleotides.
  • the nucleic acid can be single, double, or multiple stranded and may comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.
  • enzymatic portion or “catalytic domain” is meant that portion or region of the enzymatic nucleic acid molecule essential for cleavage of a nucleic acid substrate (for example, see Figures 1-5).
  • substrate binding arm or “substrate binding domain” is meant that portion or region of a enzymatic nucleic acid which is able to interact, for example, via complementarity (i.e., able to base-pair with), with a portion of its substrate.
  • complementarity i.e., able to base-pair with
  • such complementarity is 100%, but can be less if desired.
  • as few as 10 bases out of 14 can be base-paired (see for example Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al, 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). Examples of such arms are shown generally in Figures 1-5.
  • these arms contain sequences within an enzymatic nucleic acid which are intended to bring enzymatic nucleic acid and target RNA together through complementary base-pairing interactions.
  • the enzymatic nucleic acid of the invention can have binding arms that are contiguous or non-contiguous and can be of varying lengths.
  • the length of the binding arm(s) are preferably greater than or equal to four nucleotides and of sufficient length to stably interact with the target RNA.
  • the binding arm(s) are 12-100 nucleotides in length.
  • the binding arms are 14-24 nucleotides in length (see, for example, Werner and Uhlenbeck, supra; Hamman et al, supra; Hampel et al, EP0360257; Berzal-Herrance et al, 1993, EMBO J., 12, 2567-73).
  • the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., five and five nucleotides, or six and six nucleotides, or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).
  • Inozyme or "NCH” motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as NCH Rz in Figure 2. Inozymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCH/, where N is a nucleotide, C is cytidine and H is adenosine, uridine or cytidine, and / represents the cleavage site. H is used interchangeably with X.
  • Inozymes can also possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCN/, where N is a nucleotide, C is cytidine, and / represents the cleavage site.
  • "I” in Figure 2 represents an Inosine nucleotide, preferably a ribo-Inosine or xylo-Inosine nucleoside.
  • G-cleaver motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as G-cleaver in Figure 2.
  • G-cleavers possess endonuclease activity to cleave RNA substrates having a cleavage triplet NYN/, where N is a nucleotide,
  • Y is uridine or cytidine and / represents the cleavage site.
  • G-cleavers may be chemically modified as is generally shown in Figure 2.
  • amberzyme motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in Figure 3.
  • Amberzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NG/N, where N is a nucleotide, G is guanosine, and / represents the cleavage site.
  • Amberzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in Figure 3.
  • differing nucleoside and/or non-nucleoside linkers can be used to substitute the 5'-gaa-3' loops shown in the figure.
  • Amberzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2' -OH) group within its own nucleic acid sequence for activity.
  • “zinzyme” motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in Figure 4.
  • Zinzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet including but not limited to YG Y, where Y is uridine or cytidine, and G is guanosine and / represents the cleavage site.
  • Zinzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in Figure 4, including substituting 2'-0-methyl guanosine nucleotides for guanosine nucleotides.
  • differing nucleotide and/or non-nucleotide linkers can be used to substitute the 5'-gaaa-2' loop shown in the figure.
  • Zinzymes represent a non- limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2' -OH) group within its own nucleic acid sequence for activity.
  • DNAzyme' is meant, an enzymatic nucleic acid molecule that does not require the presence of a 2'-OH group for its activity.
  • the enzymatic nucleic acid molecule can have an attached linker(s) or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2' -OH groups.
  • DNAzymes can be synthesized chemically or expressed endogenously in vivo, by means of a single stranded DNA vector or equivalent thereof. An example of a DNAzyme is shown in Figure 5 and is generally reviewed in Usman et al, International PCT Publication No.
  • sufficient length is meant an oligonucleotide of greater than or equal to 3 nucleotides that is of a length great enough to provide the intended function under the expected condition.
  • sufficient length means that the binding arm sequence is long enough to provide stable binding to a target site under the expected binding conditions. Preferably, the binding arms are not so long as to prevent useful turnover.
  • stably interact is meant interaction of the oligonucleotides with target nucleic acid (e.g., by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions) that is sufficient to the intended purpose (e.g., cleavage of target RNA by an enzyme).
  • RNA to GRID is meant to include those naturally occurring RNA molecules having homology (partial or complete) to GRID proteins or encoding for proteins with similar function as GRID in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites.
  • the equivalent RNA sequence also includes in addition to the coding region, regions such as 5 '-untranslated region, 3 '-untranslated region, introns, intron-exon junction and the like.
  • nucleotide sequence of two or more nucleic acid molecules is partially or completely identical.
  • antisense nucleic acid a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al, 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al, US patent No. 5,849,902).
  • antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule.
  • an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop.
  • the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both.
  • antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex.
  • the antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA.
  • Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof.
  • RNase H activating region is meant a region (generally greater than or equal to 4-25 nucleotides in length, preferably from 5-11 nucleotides in length) of a nucleic acid molecule capable of binding to a target RNA to form a non-covalent complex that is recognized by cellular RNase H enzyme (see for example Arrow et al, US 5,849,902; Arrow et al, US 5,989,912).
  • the RNase H enzyme binds to the nucleic acid molecule- target RNA complex and cleaves the target RNA sequence.
  • the RNase H activating region comprises, for example, phosphodiester, phosphorothioate (preferably at least four of the nucleotides are phosphorothiote substitutions; morepreferably, 4-11 of the nucleotides are phosphorothiote substitutions); phosphorodithioate, 5'-thiophosphate, or methylphosphonate backbone chemistry or a combination thereof.
  • the RNase H activating region can also comprise a variety of sugar chemistries.
  • the RNase H activating region can comprise deoxyribose, arabino, fluoroarabino or a combination thereof, nucleotide sugar chemistry.
  • 2-5 A antisense chimera an antisense oligonucleotide containing a 5'- phosphorylated 2'-5'-linked adenylate residue. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5 A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al, 1993 Proc. Natl. Acad. Sci. USA 90, 1300; Silverman et al, 2000, Methods Enzymol, 313, 522-533; Player and Torrence, 1998, Pharmacol. Ther., 78, 55-113).
  • triplex forming oligonucleotides an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval- Valentin et al, 1992 Proc. Natl Acad. Sci. USA 89, 504; Fox, 2000, Curr. Med. Chem., 7, 17-37; Praseuth et. al, 2000, Biochim. Biophys. Acta, 1489, 181- 206).
  • RNA RNA sequences including but not limited to structural genes encoding a polypeptide.
  • “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another RNA sequence by either traditional Watson-Crick or other non-traditional types.
  • the binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., enzymatic nucleic acid cleavage, antisense or triple helix inhibition. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. Ill pp.123-133; Frier et al, 1986, Proc.
  • a percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • RNA is meant a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide or “2'-OH” is meant a nucleotide with a hydroxyl group at the 2' position of a ⁇ -D-ribo-furanose moiety.
  • decoy RNA is meant a RNA molecule that mimics the natural binding domain for a ligand. The decoy RNA therefore competes with natural binding target for the binding of a specific ligand.
  • TAR HIV trans- activation response
  • RNA can act as a "decoy” and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al., 1990, Cell, 63, 601-608). This is but a specific example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art.
  • enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.
  • RNA Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
  • a single ribozyme molecule is able to cleave many molecules of target RNA.
  • the ribozyme is a highly specific inhibitor of gene expression, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme.
  • the enzymatic nucleic acid molecule that cleave the specified sites in GRID-specific RNAs represent a novel therapeutic approach to treat a variety of pathologic indications, including but not limited to tissue/graft rejection and leukemia.
  • the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but can also be formed in the motif of a hepatitis delta virus, group I intron, group II intron or RNase P RNA (in association with an RNA guide sequence), Neurospora VS RNA, DNAzymes, NCH cleaving motifs, or G-cleavers.
  • hammerhead motifs are described by Dreyfus, supra, Rossi et al, 1992, AIDS Research and Human Retroviruses 8, 183.
  • hairpin motifs are described by Hampel et al, EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al, 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, Hampel et al, 1990 Nucleic Acids Res. 18, 299; and Chowrira & McSwiggen, US. Patent No. 5,631,359.
  • the hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16.
  • the RNase P motif is described by Guerrier- Takada et al, 1983 Cell 35, 849; Forster and Altaian, 1990, Science 249, 783; and Li and Altaian, 1996, Nucleic Acids Res. 24, 835.
  • the Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell, 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; and Guo and Collins, 1995, EMBO. J. 14, 363).
  • Group II introns are described by Griffin et al, 1995, Chem. Biol.
  • WO 98/58058 and G-cleavers are described in Kore et al, 1998, Nucleic Acids Research 26, 4116-4120 and Eckstein et al, Intemational PCT Publication No. WO 99/16871. Additional motifs include the Aptazyme (Breaker et al, WO 98/43993), Amberzyme (Class I motif; Figure 3; Beigelman et al, Intemational PCT publication No. WO 99/55857) and Zinzyme (Beigelman et al, Intemational PCT publication No. WO 99/55857), all these references are incorporated by reference herein in their totalities, including drawings and can also be used in the present invention.
  • a nucleic acid molecule of the instant invention can be between 13 and 100 nucleotides in length.
  • Exemplary enzymatic nucleic acid molecules of the invention are shown in Tables III-VIII and X.
  • enzymatic nucleic acid molecules of the invention are preferably between 15 and 50 nucleotides in length, more preferably between 25 and 40 nucleotides in length, e.g., 34, 36, or 38 nucleotides in length (for example see Jarvis et al., 1996, J. Biol. Chem., 271, 29107-29112).
  • Exemplary DNAzymes of the invention are preferably between 15 and 40 nucleotides in length, more preferably between 25 and 35 nucleotides in length, e.g., 29, 30, 31, or 32 nucleotides in length (see for example Santoro et al, 1998, Biochemistry, 37, 13330-13342; Chartrand et al, 1995, Nucleic Acids Research, 23, 4092-4096 and Cairns et al, 2000, Antisense & Nucleic Acid Drug Dev., 10, 323-332).
  • Exemplary antisense molecules of the invention are preferably between 15 and 75 nucleotides in length, more preferably between 20 and 35 nucleotides in length, e.g., 25, 26, 27, or 28 nucleotides in length (see for example Woolf et al, 1992, PNAS., 89, 7305-7309; Milner et al, 1997, Nature Biotechnology, 15, 537-541).
  • Exemplary triplex forming oligonucleotide molecules of the invention are preferably between 10 and 40 nucleotides in length, more preferably between 12 and 25 nucleotides in length, e.g., 18, 19, 20, or 21 nucleotides in length (see for example Maher et al, 1990, Biochemistry, 29, 8820-8826; Strobel and Dervan, 1990, Science, 249, 73-75).
  • Those skilled in the art will recognize that all that is required is for the nucleic acid molecule to be of length and conformation sufficient and suitable for the nucleic acid molecule to catalyze a reaction contemplated herein.
  • the length of the nucleic acid molecules of the instant invention are not limiting within the general limits stated.
  • a nucleic acid molecule that down regulates the replication of GRID or GRID-like gene comprises between 12 and 100 bases complementary to a GRID or GRID- like RNA. Even more preferably, a nucleic acid molecule that down regulates the replication of GRID or GRID-like gene comprises between 14 and 24 bases complementary to a GRID or GRID-like RNA.
  • the invention provides a method for producing a class of nucleic acid-based gene inhibiting agents which exhibit a high degree of specificity for the RNA of a desired target.
  • the enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of target RNAs encoding GRID or GRID- like proteins such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention.
  • Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required.
  • the nucleic acid molecules e.g., ribozymes and antisense
  • the invention features the use of nucleic acid-based inhibitors of the invention to specifically target genes that share homology with the GRID gene.
  • the invention describes the use of nucleic acid-based inhibitors to target the Grb2 (GenBank accession No. NM_002086) and GRAP (GenBank accession No. NM_006613) genes.
  • cell is used in its usual biological sense and does not refer to an entire multicellular organism.
  • the cell can be present in an organism which includes humans but is preferably a non-human multicellular organism, e.g., birds, plants and mammals such as cows, sheep, apes, monkeys, swine, dogs, and cats.
  • the cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).
  • GRID proteins is meant, a protein or a mutant protein derivative thereof, comprising an adapter-protein type of association to the activated CD28 co-stimulatory receptor, and to other signaling proteins including but not limited to SLP-76 and LAT.
  • highly conserved sequence region is meant a nucleotide sequence of one or more regions in a target gene that does not vary significantly from one generation to the other or from one biological system to the other.
  • nucleic acid-based inhibitors of GRID expression are useful for the prevention and/or treatment of diseases and conditions that are related to or will respond to the levels of GRID in a cell or tissue, alone or in combination with other therapies.
  • the nucleic acid-based inhibitors of GRID expressions are useful for the prevention and/or treatment of tissue/graft rejection and cancer, such as leukemia, among other conditions.
  • GRID expression specifically GRID gene
  • reduction in the level of the respective protein will relieve, to some extent, the symptoms of the disease or condition.
  • the invention features the use of nucleic acid-based inhibitors of the invention to specifically target regions of GRID gene that are not homologous to Grb2 gene.
  • the invention describes the use of nucleic acid- based inhibitors to target sequences that are unique to GRID gene.
  • nucleic acid-based inhibitors of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues using well-known methods described herein and generally known in the art.
  • the nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers.
  • the enzymatic nucleic acid inhibitors comprise sequences, which are complementary to the substrate sequences in Tables UI to X. Examples of such enzymatic nucleic acid molecules also are shown in Tables HI to VT ⁇ and X. Examples of such enzymatic nucleic acid molecules consist essentially of sequences defined in these Tables.
  • the invention features antisense nucleic acid molecules and 2-5A chimera including sequences complementary to the substrate sequences shown in Tables III to X.
  • nucleic acid molecules can include sequences as shown for the binding arms of the enzymatic nucleic acid molecules in Tables UI to VIII and X and sequences shown as GeneBlocTM sequences in Table X.
  • triplex molecules can be provided targeted to the corresponding DNA target regions, and containing the DNA equivalent of a target sequence or a sequence complementary to the specified target (substrate) sequence.
  • antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule.
  • an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop.
  • the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both.
  • the active nucleic acid molecule of the invention for example, an enzymatic nucleic acid molecule, contains an enzymatic center or core equivalent to those in the examples and binding arms able to bind RNA such that cleavage at the target site occurs.
  • a core region can, for example, include one or more loop, stem- loop structure, or linker which does not prevent enzymatic activity.
  • the underlined regions in the sequences in Tables III and IV can be such a loop, stem-loop, nucleotide linker, and/or non-nucleotide linker and can be represented generally as sequence "X".
  • a core sequence for a hammerhead enzymatic nucleic acid can comprise a conserved sequence, such as 5'-CUGAUGAG-3' and 5'-CGAA-3' connected by a sequence X, where X is 5'-GCCGUUAGGC-3' (SEQ ID NO 2236) or any other stem II region known in the art or a nucleotide and/or non-nucleotide linker.
  • sequence X can be a linker of > 2 nucleotides in length, preferably 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 26, 30, where the nucleotides can preferably be internally base-paired to form a stem of preferably > 2 base pairs.
  • sequence X can be a non- nucleotide linker.
  • the nucleotide linker X can be a nucleic acid aptamer, such as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and others (for a review see Gold et al, 1995, Annu. Rev. Biochem., 64, 763; and Szostak & Ellington, 1993, in The RNA World, ed. Gesteland and Atkins, pp. 511, CSH Laboratory Press).
  • RRE HIV Rev aptamer
  • TAR HIV Tat aptamer
  • a "nucleic acid aptamer" as used herein is meant to indicate a nucleic acid sequence capable of interacting with a ligand.
  • the ligand can be any natural or a synthetic molecule, including but not limited to a resin, metabolites, nucleosides, nucleotides, drugs, toxins, transition state analogs, peptides, lipids, proteins, amino acids, nucleic acid molecules, hormones, carbohydrates, receptors, cells, viruses, bacteria and others.
  • non-nucleotide linker X is as defined herein.
  • non-nucleotide linker include either abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds. Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 7 ⁇ :6353 and Nucleic Acids Res. 1987, 75:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 173:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 773:5109; Ma et al., Nucleic Acids Res.
  • non-nucleotide further refers to any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions and allows the remaining bases to exhibit their enzymatic activity.
  • the group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine.
  • the invention features an enzymatic nucleic acid molecule having one or more non-nucleotide moieties and having enzymatic activity to cleave an RNA or DNA molecule.
  • ribozymes or antisense molecules that interact with target RNA molecules and inhibit GRID activity are expressed from transcription units inserted into DNA or RNA vectors.
  • the recombinant vectors are preferably DNA plasmids or viral vectors.
  • Ribozyme or antisense expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retro virus, adeno virus, or alphavirus.
  • the recombinant vectors capable of expressing the ribozymes or antisense are delivered as described above, and persist in target cells.
  • viral vectors can be used that provide for transient expression of ribozymes or antisense.
  • Such vectors can be repeatedly administered as necessary. Once expressed, the ribozymes or antisense bind to the target RNA and inhibit its function or expression. Delivery of ribozyme or antisense expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell. Antisense DNA can be expressed endogenously via the use of a single stranded DNA intracellular expression vector.
  • vectors any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
  • patient is meant an organism, which is a donor or recipient of explanted cells or the cells themselves.
  • patient also refers to an organism to which the nucleic acid molecules of the invention can be administered.
  • a patient is a mammal or mammalian cells. More preferably, a patient is a human or human cells.
  • enhanced enzymatic activity is meant to include activity measured in cells and/or in vivo where the activity is a reflection of both the catalytic activity and the stability of the nucleic acid molecules of the invention.
  • the product of these properties can be increased in vivo compared to an all RNA enzymatic nucleic acid or all DNA enzyme.
  • the individual catalytic activity or stability of the nucleic acid molecule can be decreased (i.e., less than ten-fold), but the overall activity of the nucleic acid molecule is enhanced in vivo.
  • nucleic acid molecules of the instant invention can be used to treat diseases or conditions discussed above.
  • the patient can be treated, or other appropriate cells can be treated, as is evident to those skilled in the art, individually or in combination with one or more dmgs under conditions suitable for the treatment.
  • the described molecules can be used in combination with other known treatments to treat conditions or diseases discussed above.
  • the described molecules can be used in combination with one or more known therapeutic agents to treat tissue/graft rejection, leukemia and/or other disease states or conditions which respond to the modulation of GRID expression.
  • the invention features nucleic acid-based inhibitors (e.g., enzymatic nucleic acid molecules (ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups) and methods for their use to down regulate or inhibit the expression of genes (e.g., GRID) related to the progression and/or maintenance of tissue/graft rejection, leukemia and/or other disease states or conditions which respond to the modulation of GRID expression.
  • enzymatic nucleic acid molecules ribozymes
  • antisense nucleic acids 2-5A antisense chimeras
  • triplex DNA antisense nucleic acids containing RNA cleaving chemical groups
  • genes e.g., GRID
  • the invention provides mammalian cells containing one or more nucleic acid molecules and/or expression vectors of this invention.
  • the one or more nucleic acid molecules can independently be targeted to the same or different sites.
  • FIG. 1 shows the secondary structure model for seven different classes of enzymatic nucleic acid molecules. Arrow indicates the site of cleavage. indicate the target sequence. Lines interspersed with dots are meant to indicate tertiary interactions. - is meant to indicate base-paired interaction.
  • Group I Intron: P1-P9.0 represent various stem-loop structures (Cech et al, 1994, Nature Struc. Bio., 1, 273).
  • Group II Intron 5'SS means 5' splice site; 3'SS means 3 '-splice site; IBS means intron binding site; EBS means exon> binding site (Pyle et al, 1994, Biochemistry, 33, 2716).
  • VS RNA I-VI are meant to indicate six stem-loop structures; shaded regions are meant to indicate tertiary interaction (Collins, Intemational PCT Publication No. WO 96/19577).
  • HDV Ribozyme : I-IV are meant to indicate four stem-loop structures (Been et al, US Patent No. 5,625,047).
  • Hammerhead Ribozyme I-III are meant to indicate three stem-loop structures; stems I-III can be of any length and can be symmetrical or asymmetrical (Usman et al, 1996, Curr. Op. Struct. Bio., 1, 527).
  • Helix 2 and helix 5 can be covalently linked by one or more bases (i.e., r is > 1 base). Helix 1, 4 or 5 can also be extended by 2 or more base pairs (e.g., 4 - 20 base pairs) to stabilize the ribozyme stmcture, and preferably is a protein binding site.
  • each N and N' independently is any normal or modified base and each dash represents a potential base- pairing interaction.
  • These nucleotides can be modified at the sugar, base or phosphate. Complete base-pairing is not required in the helices, but is preferred.
  • Helix 1 and 4 can be of any size (i.e., o and p is each independently from 0 to any number, e.g., 20) as long as some base-pairing is maintained.
  • Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more can be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect.
  • Helix 4 can be formed from two separate molecules, i.e., without a connecting loop.
  • the connecting loop when present can be a ribonucleotide with or without modifications to its base, sugar or phosphate, "q" ⁇ is 2 bases.
  • the connecting loop can also be replaced with a non-nucleotide linker molecule.
  • H refers to bases A, U, or
  • Y refers to pyrimidine bases. " " refers to a covalent bond.
  • Figure 2 shows examples of chemically stabilized ribozyme motifs.
  • HH Rz represents hammerhead ribozyme motif (Usman et al, 1996, Curr. Op. Struct. Bio., 1, 527);
  • NCH Rz represents the NCH ribozyme motif (Ludwig & Sproat, Intemational PCT Publication No. WO 98/58058);
  • G-Cleaver represents G-cleaver ribozyme motif (Kore et al, 1998, Nucleic Acids Research 26, 4116-4120).
  • N or n represent independently a nucleotide which can be same or different and have complementarity to each other; rl, represents ribo-Inosine nucleotide; arrow indicates the site of cleavage within the target.
  • Position 4 of the HH Rz and the NCH Rz is shown as having 2'-C-allyl modification, but those skilled in the art will recognize that this position can be modified with other modifications well known in the art, so long as such modifications do not significantly inhibit the activity of the ribozyme.
  • FIG 3 shows an example of the Amberzyme ribozyme motif that is chemically stabilized (see, for example, Beigelman et al, Intemational PCT publication No. WO 99/55857, incorporated by reference herein; also referred to as Class I Motif).
  • the Amberzyme motif is a class of enzymatic nucleic molecules that do not require the presence of a ribonucleotide (2' -OH) group for its activity.
  • FIG 4 shows an example of the Zinzyme A ribozyme motif that is chemically stabilized (Beigelman et al, Intemational PCT publication No. WO 99/55857, incorporated by reference herein; also referred to as Class A or Class II Motif).
  • the Zinzyme motif is a class of enzymatic nucleic molecules that do not require the presence of a ribonucleotide (2' -OH) group for its activity.
  • Figure 5 shows an example of a DNAzyme motif described by Santoro et al, 1997
  • Figure 6 shows a graph of optimization of GeneBloc concentration.
  • a fluoresceinated randomized antisense GeneBloc (fGB) was used as a marker for uptake using a fixed concentration of lipid.
  • Cells were either untreated (A) or treated continuously for 24hrs with 10-200nM antisense GeneBloc (B-F). Following treatment, cells were analyzed by flow cytometry.
  • Gate Ml represents either untransfected cells or cells refractory to transfection.
  • Gate M2 represents the transfected cells.
  • Figure 7 shows a bar graph of a primary screen of twelve GRID GeneBlocs.
  • Taqman RNA assay was used to quantify the level of GRID transcript in Jurkat cells treated continuously for 24 hours with lOOnM antisense GeneBloc and 5-O ⁇ gm 1 cationic lipid. For comparison, all data was normalized to the level of ⁇ -actin. Error bars represent the standard error of the mean of triplicate points.
  • Figure 8 shows a graph demonstrating that flow cytometric sorting of transfected cells improves antisense GeneBloc mediated inhibition of GRID mRNA expression.
  • Jurkat cells were treated continuously for 24 and 72 hours with GB 14540 (75nM) or control GeneBloc GBC3.3 (75nM) spiked with 25nM fluorescent randomized GeneBloc (A) to facilitate the identification of transfected cells.
  • the 10% most and least fluorescent cells were sorted on a FACStar Plus.
  • Post-sort low transfecting (B) and high transfecting (C) fractions were re-analyzed for purity. Histograms A-D are representative of results obtained in all experiments and were taken from cells treated for 72 hours.
  • the GRID mRNA content of all samples was quantified by Taqman RNA assay and normalized to the ⁇ -actin content. For the purposes of inter- experiment comparison, all GB 14540 values were also normalized to the appropriate control GBC3.3 value.
  • D Normalized GRID mRNA levels in pre-sort samples;
  • E Normalized GRID mRNA levels in the post-sort low transfecting fraction;
  • F Normalized GRID mRNA levels in the post-sort high transfecting fraction. Error bars represent the range of duplicate points.
  • Figure 9 shows a graph representing the phenotypic analysis of antisense GeneBloc treated Jurkat cells following activation with anti-CD3 and anti-CD28 anti-sera.
  • Jurkat cells were treated continuously for 72 hours with the anti-GRID reagent GB 14540 (A, C) and the mismatch control reagent GB 17477 (B, D), activated for 22 hours (C, D) and stained for the surface activation marker CD69. Unactivated samples are shown in (A, B).
  • Antisense molecules can be modified or unmodified RNA, DNA, or mixed polymer oligonucleotides which primarily function by specifically binding to matching sequences resulting in inhibition of peptide synthesis (Wu-Pong, Nov 1994, BioPharm, 20-33).
  • the antisense oligonucleotide binds to target RNA by Watson Crick base-pairing and blocks gene expression by preventing ribosomal translation of the bound sequences either by steric blocking or by activating RNase H enzyme.
  • Antisense molecules can also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190).
  • antisense molecules have been described that utilize novel configurations of chemically modified nucleotides, secondary stmcture, and/or RNase H substrate domains (Woolf et al, Intemational PCT Publication No. WO 98/13526; Thompson et al, Intemational PCT Publication No. WO 99/54459; Hartmann et al, USSN 60/101,174 which was filed on September 21, 1998) all of these are incorporated by reference herein in their entirety.
  • antisense deoxyoligoribonucleotides can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex.
  • Antisense DNA can be expressed endogenously in vivo via the use of a single stranded DNA intracellular expression vector or equivalents and variations thereof.
  • TFO Triplex Forming Oligonucleotides
  • 2-5A Antisense Chimera The 2-5A system is an interferon mediated mechanism for RNA degradation found in higher vertebrates (Mitra et al, 1996, Proc Nat Acad Sci USA 93, 6780-6785). Two types of enzymes, 2-5A synthetase and RNase L, are required for RNA cleavage. The 2-5A synthetases require double stranded RNA to form 2'-5' oligoadenylates (2-5A). 2-5A then acts as an allosteric effector for utilizing RNase L which has the ability to cleave single stranded RNA. The ability to form 2-5A structures with double stranded RNA makes this system particularly useful for inhibition of viral replication.
  • (2 '-5') oligoadenylate structures can be covalently linked to antisense molecules to form chimeric oligonucleotides capable of RNA cleavage (Torrence, supra). These molecules putatively bind and activate a 2-5A dependent RNase, the oligonucleotide/enzyme complex then binds to a target RNA molecule which can then be cleaved by the RNase enzyme.
  • Enzymatic Nucleic Acid Several varieties of naturally occurring enzymatic RNAs are presently known. In addition, several in vitro selection (evolution) strategies (Orgel, 1979, Proc. R. Soc. London, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing cleavage and ligation of phosphodiester linkages (Joyce, 1989, Gene, 82, 83-87; Beaudry et al, 1992, Science 257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker et al, 1994, TIBTECH 12, 268; Barrel et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al, 1995, FASEB J., 9, 1183; Breaker, 1996, Curr.
  • Nucleic acid molecules of this invention can block to some extent GRID protein expression and can be used to treat disease or diagnose disease associated with levels of GRID.
  • the enzymatic nature of an enzymatic nucleic acid has significant advantages, such as the concentration of enzymatic nucleic acid necessary to affect a therapeutic treatment is lower. This advantage reflects the ability of the enzymatic nucleic acid to act enzymatically. Thus, a single enzymatic nucleic acid molecule is able to cleave many molecules of target RNA.
  • the enzymatic nucleic acid is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can be chosen to completely eliminate catalytic activity of an enzymatic nucleic acid molecule.
  • Nucleic acid molecules having an endonuclease enzymatic activity are able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence-specific manner. Such enzymatic nucleic acid molecules can be targeted to virtually any RNA transcript and achieve efficient cleavage in vitro (Zaug et al, 324, Nature 429 1986 ; Uhlenbeck, 1987 Nature 328, 596; Kim et al, 84 Proc. Natl. Acad. Sci. USA 8788, 1987; Dreyfus, 1988, Einstein Quart. J. Bio.
  • Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the RNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited (Warashina et al, 1999, Chemistry and Biology, 6, 237-250).
  • the nucleic acid molecules of the instant invention are also referred to as GeneBloc reagents, which are essentially nucleic acid molecules (e.g., ribozymes, antisense) capable of down-regulating gene expression.
  • GeneBlocs are modified oligonucleotides, including ribozymes and modified antisense oligonucleotides, that bind to and target specific mRNA molecules. Because GeneBlocs can be designed to target any specific mRNA, their potential applications are quite broad. Traditional antisense approaches have often relied heavily on the use of phosphorothioate modifications to enhance stability in biological samples, leading to a myriad of specificity problems stemming from non-specific protein binding and general cytotoxicity (Stein, 1995, Nature Medicine, 1, 1119).
  • GeneBlocs contain a number of modifications that confer nuclease resistance while making minimal use of phosphorothioate linkages, which reduces toxicity, increases binding affinity, and minimizes non-specific effects compared with traditional antisense oligonucleotides. Similar reagents have recently been utilized successfully in various cell culture systems (Vassar, et al, 1999, Science, 286, 735) and in vivo (Jarvis et al., manuscript in preparation). In addition, novel cationic lipids can be utilized to enhance cellular uptake in the presence of serum.
  • Targets for useful ribozymes and antisense nucleic acids can be determined as disclosed in Draper et al, WO 93/23569; Sullivan et al, WO 93/23057; Thompson et al, WO 94/02595; Draper et al, WO 95/04818; McSwiggen et al, US Patent No. 5,525,468. All of these publications are hereby incorporated by reference herein in their totality. Other examples include the following PCT applications, which concern inactivation of expression of disease-related genes: WO 95/23225, WO 95/13380, WO 94/02595, all of which are incorporated by reference herein.
  • Ribozymes and antisense to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described.
  • the sequences of human GRID RNAs were screened for optimal enzymatic nucleic acid and antisense target sites using a computer-folding algorithm. Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme, or G-Cleaver ribozyme binding/cleavage sites were identified.
  • Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver ribozyme binding/cleavage sites were identified.
  • the nucleic acid molecules were individually analyzed by computer folding (Jaeger et al, 1989 Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the sequences fold into the appropriate secondary structure. Those nucleic acid molecules with unfavorable intramolecular interactions, such as between the binding arms and the catalytic core, were eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity.
  • nucleic acid molecules were chemically synthesized. The method of synthesis used follows the procedure for normal DNA/RNA synthesis as described below and in Usman et al, 1987 J. Am. Chem. Soc, 109, 7845; Scaringe et al, 1990 Nucleic Acids Res., 18, 5433; Wincott et al, 1995 Nucleic Acids Res. 23, 2677-2684; and Caruthers et al, 1992, Methods in Enzymology 211,3-19. Synthesis of Nucleic acid Molecules
  • nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive.
  • small nucleic acid motifs (“small refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., antisense oligonucleotides, hammerhead or the NCH ribozymes) are preferably used for exogenous delivery.
  • the simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of RNA structure.
  • Exemplary molecules of the instant invention are chemically synthesized, and others can be similarly synthesized.
  • Oligonucleotides are synthesized using protocols known in the art as described in Caruthers et al, 1992, Methods in Enzymology 211, 3-19, Thompson et al, International PCT Publication No. WO 99/54459, Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al, 1997, Methods Mol. Bio., 74, 59, Brennan et al, 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, US patent No. 6,001,311. All of these references are inco ⁇ orated herein by reference.
  • oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
  • small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 ⁇ mol scale protocol with a 2.5 min coupling step for 2'-0- methylated nucleotides and a 45 sec coupling step for 2 '-deoxy nucleotides.
  • Table II outlines the amounts and the contact times of the reagents used in the synthesis cycle.
  • syntheses at the 0.2 ⁇ mol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with minimal modification to the cycle.
  • synthesizer include; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF (PERSEPTJVETM). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American Intemational Chemical, Inc.
  • Beaucage reagent (3H-l,2-Benzodithiol-3-one 1,1 -dioxide, 0.05 M in acetonitrile) is used.
  • Deprotection of the antisense oligonucleotides is performed as follows: the polymer- bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65 °C for 10 min. After cooling to -20 °C, the supematant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H20/3:l:l, vortexed and the supematant is then added to the first supematant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.
  • RNA including certain enzymatic nucleic acid molecules follows the procedure as described in Usman et al, 1987, J. Am. Chem. Soc, 109, 7845; Scaringe et al, 1990, Nucleic Acids Res., 18, 5433; Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684 and Wincott et al, 1997, Methods Mol Bio., 74, 59, and makes use of common ucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5 '-end, and phosphoramidites at the 3 '-end.
  • common ucleic acid protecting and coupling groups such as dimethoxytrityl at the 5 '-end, and phosphoramidites at the 3 '-end.
  • small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 ⁇ mol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2'-0-methylated nucleotides.
  • Table II outlines the amounts and the contact times of the reagents used in the synthesis cycle.
  • syntheses at the 0.2 ⁇ mol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with minimal modification to the cycle.
  • Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%.
  • synthesizer include; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM 12,
  • Deprotection of the RNA is performed using either a two-pot or one-pot protocol.
  • the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at
  • the supematant is removed from the polymer support.
  • the support is washed three times with 1.0 mL of EtOH:MeCN:H20/3:l:l, vortexed and the supematant is then added to the first supematant.
  • the combined supernatants, containing the oligoribonucleotide, are dried to a white powder.
  • the base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 ⁇ L of a solution of 1.5 mL N-methylpyrrolidinone, 750 ⁇ L TEA and 1 mL TEA»3HF to provide a 1.4 M HF concentration) and heated to 65 °C. After 1.5 h, the oligomer is quenched with 1.5 M NH4HCO3.
  • the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65 °C for 15 min.
  • the vial is brought to r.t. TEA « 3HF (0.1 mL) is added and the vial is heated at 65 °C for 15 min.
  • the sample is cooled at -20 °C and then quenched with 1.5 M NH4HCO3.
  • the quenched NH4HCO3 solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5%) TFA for 13 min. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.
  • Inactive hammerhead ribozymes or binding attenuated control (BAC) oligonucleotides are synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel, K. J., et al, 1992, Nucleic Acids Res_., 20, 3252). Similarly, one or more nucleotide substitutions can be introduced in other enzymatic nucleic acid molecules to inactivate the molecule and such molecules can serve as a negative control.
  • the average stepwise coupling yields are typically >98% (Wincott et al, 1995 Nucleic Acids Res. 23, 2677-2684).
  • the scale of synthesis can be adapted to be larger or smaller than the examples described above including but not limited to 96-well format, all that is important is the ratio of chemicals used in the reaction.
  • nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example by ligation (Moore et al, 1992, Science 256, 9923; Draper et al, Intemational PCT publication No. WO 93/23569;
  • nucleic acid molecules of the present invention are modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'- C-allyl, 2'-flouro, 2'-0-methyl, 2'-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser. 31, 163).
  • Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al, supra, the totality of which is hereby incorporated herein by reference) and are re-suspended in water.
  • the ribozyme and antisense construct sequences listed in Tables III to X can be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes with enzymatic activity are equivalent to the ribozymes described specifically in the Tables.
  • oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-0-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al, 1996, Biochemistry , 35, 14090).
  • nuclease resistant groups for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-0-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al, 1996, Biochemistry , 35, 14090).
  • nucleic acid-based molecules of the invention can lead to improved treatment of the disease progression by affording the possibility of combination therapies
  • nucleic acid molecules e.g., multiple antisense or enzymatic nucleic acid molecules targeted to different genes, nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of molecules (including different motifs) and/or other chemical or biological molecules).
  • the treatment of patients with nucleic acid molecules can also include combinations of different types of nucleic acid molecules.
  • Therapeutic nucleic acid molecules e.g., enzymatic nucleic acid molecules and antisense nucleic acid molecules
  • delivered exogenously should preferably be stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state.
  • the nucleic acid molecules should be resistant to nucleases in order to function as effective intracellular therapeutic agents when delivered exogenously.
  • nucleic acid catalysts having chemical modifications which maintain or enhance enzymatic activity are provided.
  • Such nucleic acid is also generally more resistant to nucleases than unmodified nucleic acid.
  • the activity may not be significantly lowered.
  • ribozymes are useful in a cell and/or in vivo even if activity over all is reduced 10 fold (Burgin et al, 1996, Biochemistry, 35, 14090).
  • Such ribozymes herein are said to "maintain” the enzymatic activity of an all RNA ribozyme.
  • nucleic acid molecules comprise a 5' and/or a 3'- cap stmcture.
  • cap stmcture is meant chemical modifications, which have been inco ⁇ orated at either terminus of the oligonucleotide (see, for example, Wincott et al, WO 97/26270, inco ⁇ orated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and can help in delivery and/or localization within a cell.
  • the cap can be present at the 5'-terminus (5'-cap) or at the 3'-terminus (3'-cap) or can be present on both termini.
  • the 5 '-cap is selected from the group consisting of inverted abasic residue (moiety), 4',5'-methylene nucleotide; l-(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide; 1 ,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; ⁇ reo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4- dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3 '-3 '-inverted nucleotide moiety; 3 '-3 '-inverted abasic moiety; 3
  • Suitable 3 '-caps include 4',5'-methylene nucleotide; l-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3- diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2- aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L- nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo- pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nu
  • non-nucleotide any group or compound which can be inco ⁇ orated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.
  • the group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine.
  • alkyl refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons.
  • the alkyl group can be substituted or unsubstituted.
  • alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched- chain, and cyclic groups.
  • the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
  • alkyl also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight- chain, branched-chain, and cyclic groups.
  • the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
  • alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups.
  • An "aryl” group refers to an aromatic group which has at least one ring having a conjugated ⁇ electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which can be optionally substituted.
  • the preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups.
  • alkylaryl refers to an alkyl group (as described above) covalently joined to an aryl group (as described above).
  • Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted.
  • Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms.
  • Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted.
  • An "amide” refers to an -C(0)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen.
  • An “ester” refers to an -C(0)-OR', where R is either alkyl, aryl, alkylaryl or hydrogen.
  • nucleotide is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar.
  • Nucleotides are recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group.
  • the nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., Intemational PCT Publication No. WO 92/07065; Usman et al., Intemational PCT Publication No. WO 93/15187; Uhlman & Peyman, supra all are hereby inco ⁇ orated by reference herein).
  • modified nucleic acid bases There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183.
  • Some of the non-limiting examples of chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6- trimethoxy benzene, 3 -methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
  • modified bases in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule.
  • nucleoside is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar.
  • Nucleosides are recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a nucleoside sugar moiety. Nucleosides generally comprise a base and sugar group.
  • the nucleosides can be unmodified or modified at the sugar, and/or base moiety, (also referred to interchangeably as nucleoside analogs, modified nucleosides, non-natural nucleosides, non-standard nucleosides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., Intemational PCT Publication No. WO 92/07065; Usman et al., Intemational PCT Publication No. WO 93/15187; Uhlman & Peyman, supra all are hereby inco ⁇ orated by reference herein).
  • modified nucleic acid bases There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183.
  • Some of the non-limiting examples of chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3 -methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
  • 6-methyluridine 6-methyluridine
  • propyne quesosine, 2- thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5- (carboxyhydroxymethyl)uridine, 5 '-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1- methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2- methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2- thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5- methyloxyuridine, 5-methyl-2 -thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D- mannosyl
  • modified bases in this aspect is meant nucleoside bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule.
  • the invention features modified ribozymes with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, mo ⁇ holino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions.
  • abasic sugar moieties lacking a base or having other chemical groups in place of a base at the 1' position, (for more details, see Wincott et al, Intemational PCT publication No. WO 97/26270).
  • unmodified nucleoside is meant one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1' carbon of ⁇ -D-ribo-furanose.
  • modified nucleoside any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.
  • amino 2'-NH 2 or 2'-0- NH 2 , which can be modified or unmodified.
  • modified groups are described, for example, in Eckstein et al, U.S. Patent 5,672,695 and
  • nucleic acid e.g., antisense and ribozyme
  • modifications can enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.
  • nucleic acid molecules can also include combinations of different types of nucleic acid molecules.
  • therapies can be devised which include a mixture of ribozymes (including different ribozyme motifs), antisense and/or 2-5A chimera molecules to one or more targets to alleviate symptoms of a disease.
  • nucleic acid molecules Methods for the delivery of nucleic acid molecules are described in Akhtar et al, 1992, Trends Cell Bio., 2, 139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995 which are both inco ⁇ orated herein by reference.
  • Sullivan et al, PCT WO 94/02595 further describes the general methods for delivery of enzymatic RNA molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule.
  • Nucleic acid molecules can may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by inco ⁇ oration into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.
  • nucleic acid molecules can be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles.
  • the nucleic acid/vehicle combination can be locally delivered by direct injection or by use of a catheter, infusion pump or stent.
  • routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of nucleic acid delivery and administration are provided in Sullivan et al, supra, Draper et al, PCT W093/23569, Beigelman et al, PCT WO99/05094, and Klimuk et al, PCT WO99/04819 all of which have been inco ⁇ orated by reference herein.
  • the molecules of the instant invention can be used as pharmaceutical agents.
  • Pharmaceutical agents prevent, inhibit the occurrence, or treat (i.e., alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a patient.
  • the negatively charged polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition.
  • standard protocols for formation of liposomes can be followed as described in the art.
  • the compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and other compositions known in the art.
  • the present invention also includes pharmaceutically acceptable formulations of the compounds described.
  • formulations include salts of the above compounds, e.g., acid addition salts, including salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
  • a pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or patient, preferably a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect.
  • systemic administration in vivo systemic abso ⁇ tion or accumulation of dmgs in the blood stream followed by distribution throughout the entire body.
  • Administration routes that lead to systemic abso ⁇ tion include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.
  • Each of these administration routes exposes the desired negatively charged polymers, e.g., nucleic acids, to an accessible diseased tissue.
  • the rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size.
  • a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES).
  • RES reticular endothelial system
  • a liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cancer cells.
  • pharmaceutically acceptable formulation is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity.
  • agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol, 13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc.
  • P-glycoprotein inhibitors such as Pluronic P85
  • biodegradable polymers such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc.
  • nanoparticles such as those made of polybutylcyanoacrylate, which can deliver dmgs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).
  • Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al, 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al, 1999, FEBS Lett, 421, 280-284; Pardridge et al, 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al, 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al, 1999, PNAS USA., 96, 7053-7058.
  • the invention also features the use of the composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes).
  • PEG-modified, or long-circulating liposomes or stealth liposomes These formulations offer a method for increasing the accumulation of drugs in target tissues.
  • This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al, Chem. Pharm. Bull. 1995, 43, 1005- 1011). All inco ⁇ orated by reference herein.
  • liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al, Science 1995, 267, 1275-1276; Oku et al, 1995, Biochim. Biophys. Ada, 1238, 86-90). All inco ⁇ orated by reference herein.
  • the long- circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al, J. Biol. Chem. 1995, 42, 24864-24870; Choi et al, Intemational PCT Publication No.
  • WO 96/10391 Ansell et al, Intemational PCT Publication No. WO 96/10390; Holland et al, Intemational PCT Publication No. WO 96/10392; all of which are inco ⁇ orated by reference herein).
  • Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.
  • compositions prepared for storage or administration which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985) hereby inco ⁇ orated by reference herein.
  • preservatives, stabilizers, dyes and flavoring agents may be provided. These include sodium benzoate, sorbic acid and esters of />-hydroxybenzoic acid.
  • antioxidants and suspending agents can be used.
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state.
  • the pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.
  • nucleic acid molecules of the present invention can also be administered to a patient in combination with other therapeutic compounds to increase the overall therapeutic effect.
  • the use of multiple compounds to treat an indication may increase the beneficial effects while reducing the presence of side effects.
  • nucleic acid molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985,
  • nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector.
  • the activity of such nucleic acids can be augmented by their release from the primary transcript by a ribozyme (Draper et al, PCT WO 93/23569, and Sullivan et al, PCT WO 94/02595; Ohkawa et al, 1992, Nucleic Acids Symp.
  • RNA molecules of the present invention are preferably expressed from transcription units (see, for example, Couture et al, 1996, TIG, 12, 510) inserted into DNA or RNA vectors.
  • the recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors can be constructed based on, but not limited to, adeno-associated vims, retrovims, adenovirus, or alphavirus.
  • the recombinant vectors capable of expressing the nucleic acid molecules are delivered as described above, and persist in target cells.
  • viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary.
  • Delivery of nucleic acid molecule expressing vectors can be systemic, such as by intravenous or intra-muscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allow for introduction into the desired target cell (for a review, see Couture et al, 1996, TIG., 12, 510).
  • the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules disclosed in the instant invention.
  • the nucleic acid sequence encoding the nucleic acid molecule of the instant invention is operable linked in a manner which allows expression of that nucleic acid molecule.
  • the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); c) a nucleic acid sequence encoding at least one of the nucleic acid catalyst of the instant invention; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • the vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5' side or the 3'-side of the sequence encoding the nucleic acid catalyst of the invention; and/or an intron (intervening sequences).
  • ORF open reading frame
  • RNA polymerase I RNA polymerase I
  • polymerase II RNA polymerase II
  • poly III RNA polymerase III
  • Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
  • Prokaryotic RNA polymerase promoters also can be used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci.
  • nucleic acid molecules such as ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani- Sabet et al, 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al, 1992, Proc. Natl. Acad. Sci. U S A, 89, 10802-6; Chen et al, 1992, Nucleic Acids Res., 20, 4581-9; Yu et al, 1993, Proc. Natl. Acad. Sci. U S A, 90, 6340-4; L'Huillier et al, 1992, EMBO , 11, 4411-8; Lisziewicz et al, 1993, Proc. Natl.
  • transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovims VA RNA are useful in generating high concentrations of desired RNA molecules such as ribozymes in cells (Thompson et al, supra; Couture and Stinchcomb, 1996, supra; Noonberg et al, 1994, Nucleic Acid Res., 22, 2830; Noonberg et al, US Patent No.
  • ribozyme transcription units can be inco ⁇ orated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovims or adeno-associated vims vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review, see Couture and Stinchcomb, 1996, supra).
  • plasmid DNA vectors such as adenovims or adeno-associated vims vectors
  • viral RNA vectors such as retroviral or alphavirus vectors
  • the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules of the invention, in a manner which allows expression of that nucleic acid molecule.
  • the expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; c) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3 '-end of said open reading frame; and wherein said sequence is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3 '-end of said open reading frame; and wherein said sequence is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • GeneBlocs to modulate the activity of GRID, a putative component of co- stimulatory signaling in T cells.
  • An array of GeneBlocs were designed and screened for their ability to reduce GRID mRNA levels whilst leaving transcripts from the closely related genes Grb2 and GRAP unaffected.
  • a series of experiments were conducted to optimize delivery of GeneBlocs to the Jurkat T cell line. Using these conditions, applicant has demonstrated the efficacy of these reagents at both the mRNA and protein level.
  • Anti-CD3/CD28 triggering of Jurkat cells pre-treated with the anti-GRTD GeneBloc results in an impairment of CD69 up-regulation consistent with an important role for GRID in transducing the co-stimulatory signal.
  • Example 1 Identification of Potential Target Sites in Human GRID RNA
  • the sequence of human GRID were screened for accessible sites using a computer- folding algorithm. Regions of the RNA were identified that do not form secondary folding structures. These regions contain potential ribozyme and/or antisense binding/cleavage sites. The sequences of these binding/cleavage sites are shown in Tables III-X.
  • Enzymatic nucleic acid target sites are chosen by analyzing sequences of Human GRID (for example, GenBank accession numbers: AJ011736 and Y18051) and prioritizing the sites on the basis of folding. Enzymatic nucleic acids are designed that bind each target and are individually analyzed by computer folding (Christoffersen et al, 1994 J. Mol Struc. Theochem, 311, 273; Jaeger et al, 1989, Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the enzymatic nucleic acid sequences fold into the appropriate secondary structure. Those enzymatic nucleic acids with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. As noted below, varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.
  • Example 3 Chemical Synthesis and Purification of Enzymatic nucleic acids and Antisense for Efficient Cleavage and/or blocking of GRID RNA
  • Enzymatic nucleic acids and antisense constructs are designed to anneal to various sites in the RNA message.
  • the binding arms of the enzymatic nucleic acids are complementary to the target site sequences described above, while the antisense constructs are fully complimentary to the target site sequences described above.
  • the enzymatic nucleic acids and antisense constructs were chemically synthesized. The method of synthesis used followed the procedure for normal RNA or DNA synthesis as described above and in Usman et al, (1987 J. Am. Chem.
  • Enzymatic nucleic acids and antisense constructs also can be synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Enzymatic nucleic acid and antisense constructs are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; see Wincott et al, supra; the totality of which is hereby inco ⁇ orated herein by reference) and are resuspended in water. The sequences of the chemically synthesized enzymatic nucleic acid and antisense constructs used in this study are shown below in Table III-X.
  • Enzymatic nucleic acids targeted to the human GRID RNA are designed and synthesized as described above. These enzymatic nucleic acids can be tested for cleavage activity in vitro, for example, using the following procedure.
  • the target sequences and the nucleotide location within the GRID RNA are given in Tables III-X.
  • Full-length or partially full-length, internally-labeled target RNA for enzymatic nucleic acid cleavage assay is prepared by in vitro transcription in the presence of [a- ⁇ 2p] CTP, passed over a G 50 Sephadex® column by spin chromatography and used as substrate RNA without further purification. Alternately, substrates are 5'-32p. end labeled using T4 polynucleotide kinase enzyme.
  • Assays are performed by pre- warming a 2X concentration of purified enzymatic nucleic acid in enzymatic nucleic acid cleavage buffer (50 mM Tris-HCl, pH 7.5 at 37°C, 10 mM MgCl2) and the cleavage reaction was initiated by adding the 2X enzymatic nucleic acid mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer. As an o initial screen, assays are carried out for 1 hour at 37 C using a final concentration of either
  • RNA or 1 mM ribozyme i.e., enzymatic nucleic acid excess.
  • the reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol o blue and 0.05% xylene cyanol after which the sample is heated to 95 C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel.
  • Substrate RNA and the specific RNA cleavage products generated by enzymatic nucleic acid cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.
  • Antisense nucleic acid molecules targeted to the human GRID RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure.
  • the target sequences and the nucleotide location within the GRID RNA are given in Tables III-X.
  • Grb2 and GRAP the closely related adapter proteins
  • twelve GeneBlocs (see Methods for details) targeting human GRID (GenBank accession number Y18051) were designed, each containing a minimum of six mismatches versus human Grb2 (M96995) and human GRAP (U52518).
  • M969952518 human GRAP
  • the most highly fluorescent cells represent the population of cells transfected with the highest concentration of active GeneBloc ('high transfecting'), whilst the cells that appear to be refractory to transfection should contain a significantly lower concentration active GeneBloc ('low transfecting').
  • GRID protein in cells treated continuously with active GeneBloc reagent GB 14540 and the mismatch control GB 17477 was assessed.
  • GB 14540 caused a substantial reduction in GRID protein levels as determined by the intensity of the GRID specific band whilst at earlier time-points (24 and 48 hrs) no reduction in protein was observed.
  • Cells treated with the mismatch control GB 17477 showed GRID levels comparable to the untreated sample.
  • Cells treated continuously with GB 14540 for periods up to 144 hours showed no further reduction in GRID protein levels, suggesting that the effect of the GeneBloc was maximal and sustained from 72 hours onwards. Whilst the effects of the anti-GRID GeneBloc on mRNA levels are seen at 24 hours, the reduction in GRID protein is delayed a further 48 hours indicating that GRID protein may have a relatively long half-life.
  • GB 14540 contains 6 and 7 mismatches respectively when aligned with the human Grb2 and GRAP sequences. Due to the presence of these mismatches, GB 14540 was not expected to inhibit Grb2 mRNA expression.
  • the Western blots used for the GRID assay were stripped and re-probed using an anti-Grb2 antibody. No difference in Grb2 protein levels was observed between the untreated sample and cells treated with either GB 14540 or the mismatch control reagent GB 17477, confirming that the GB 14540 was specific for GRID.
  • GRJD is a novel member of the Grb2 family of adapter proteins.
  • a role for GRID in T cell signaling has been postulated due to its association with known T cell signaling proteins [Law, 1999 #3296][Asada, 1999 #3243][Liu, 1999 #3245] and more recently the T cell co-stimulatory receptor CD28 following activation by cross-linking antibodies (Ellis et al.).
  • T cell signaling proteins [Law, 1999 #3296][Asada, 1999 #3243][Liu, 1999 #3245] and more recently the T cell co-stimulatory receptor CD28 following activation by cross-linking antibodies (Ellis et al.).
  • applicant studied the expression of early surface activation marker CD69 Jung et al, 1988, Cellular Immunology, 117, 352, Lanier et al, 1988, J. Exp.
  • a cationic lipid was found to be necessary to facilitate cellular uptake of oligonucleotide.
  • a lipid concentration of 2.5-5.0 ⁇ gml "1 was found to be optimal.
  • some cells are readily transfected by the GeneBloc, a sub-population of cells remained refractory to transfection (see Gate M2 vs. Ml in Figures 6D-6F).
  • the concentration of GeneBloc was varied between 10- 200nM. Transfection frequencies of up to 75% (as determined by fraction of cells in Gate M2) were observed in the 50-lOOnM range of GeneBloc concentration.
  • Human Jurkat cell lines E6.1 and J6 were maintained at 37°C in 5% CO z in flasks in RPMI 1641 (+ 25mM HEPES) supplemented with 10% fetal calf serum and glutamine. Cells were passaged at a density of 1 x 10 6 cells ml "1 . GeneBlocs were delivered to the cells using a modified centrifugation-based transfection protocol (Verma et al, 1998, BioTechniques, 25, 46). Cells were grown to a density of 1 x 10 6 cells ml "1 , harvested by centrifugation and re-suspended in fresh media at 0.75 x 10 6 cells ml "1 .
  • GeneBloc at 10X final concentration and cationic lipid (25 ⁇ gml "1 ) at 10X final concentration were prepared separately in RPMI media (no FCS or glutamine), mixed 1:1 and incubated at 37°C for 30 minutes. 1.6ml aliquots of the cell suspension was dispensed into a 6-well tissue-culture treated plate and 0.4ml of the GeneBloc rlipid mixture added drop-wise. The GeneBloc :lipid solution was evenly distributed by gentle agitation. Following centrifugation at lOOO ⁇ m for 60 minutes at room temperature, the 6-well plates were incubated for 24-72 hours at 37°C.
  • GRID primers forward, 5'-AGGATATGTGCCCAAGAATTTCATA-3') (SEQ ID NO 2239) and reverse, (5'-TGCCTGGTGTCGAGAGAGG-3') (SEQ ID NO 2240) and actin primers (forward, 5'-GCATGGGTCAGAAGGATTCCTAT-3') (SEQ ID NO 2241) and reverse, (5'-TGTAGAAGGTGTGGTGCCAGATT-3') (SEQ ID NO 2242) were purchased from Life Technologies.
  • the Taqman probes were labeled with a reporter dye (FAM or JOE) at the 5' termini and a quencher dye (TAMRA) at their 3' termini.
  • a combination RT-PCR and Taqman PCR was performed for each sample in triplicate on an ABI PRISM 7700 Sequence Detection System using the following program: 48°C for 30 minutes, 95°C for 10 minutes and then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.
  • the reaction was performed in a total volume of 40 ⁇ l with each tube containing 10U RNase inhibitor (Promega), 1.25U Amplitaq Gold (PE Biosystems), lOOnM of the GRID and Actin primers, lOOnM GRID FAM Taqman probe, lOOnM Actin JOE Taqman probe and 10U MuLV reverse transcriptase.
  • PCR Buffer PE Biosystems #4304441
  • dNTPs PE Biosystems #N808-0261
  • GRID expression modulation includes, but are not limited to. tissue/graft rejection and cancer, such as leukemia.
  • the present body of knowledge in GRID research indicates the need for methods to assay GRID activity and for compounds that can regulate GRID expression for research, diagnostic, and therapeutic use.
  • Radiotherapy, chemotherapeutic treatments, and Cyclosporin are non-limiting examples of compounds and/or methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) of the instant invention.
  • nucleic acid molecules e.g. ribozymes and antisense molecules
  • Those skilled in the art will recognize that other dmg compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. ribozymes and antisense molecules) are hence within the scope of the instant invention.
  • the nucleic acid molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of GRID RNA in a cell.
  • the close relationship between ribozyme activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA.
  • ribozymes described in this invention one can map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease.
  • ribozymes of this invention include detection of the presence of mRNAs associated with GRID-related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.
  • ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay.
  • the first ribozyme is used to identify wild-type RNA present in the sample and the second ribozyme is used to identify mutant RNA in the sample.
  • synthetic substrates of both wild-type and mutant RNA are cleaved by both ribozymes to demonstrate the relative ribozyme efficiencies in the reactions and the absence of cleavage of the "non-targeted" RNA species.
  • the cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population.
  • each analysis can require two ribozymes, two substrates and one unknown sample, which are combined into six reactions.
  • the presence of cleavage products is determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells.
  • the expression of mRNA whose protein product is implicated in the development of the phenotype i.e., GRID
  • GRID quantitative comparison of RNA levels is adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.
  • sequence-specific enzymatic nucleic acid molecules of the instant invention have many of the same applications for the study of RNA that DNA restriction endonucleases have for the study of DNA (Nathans et al, 1975 Ann. Rev. Biochem. 44:273).
  • the pattern of restriction fragments can be used to establish sequence relationships between two related RNAs, and large RNAs can be specifically cleaved to fragments of a size more useful for study.
  • the ability to engineer sequence specificity of the enzymatic nucleic acid molecule is ideal for cleavage of RNAs of unknown sequence.
  • Applicant describes the use of nucleic acid molecules to down- regulate gene expression of target genes in bacterial, microbial, fungal, viral, and eukaryotic systems including plant, or mammalian cells.
  • Reaction mechanism attack by the 3'-OH of guanosine to generate cleavage products with 3' -OH and 5' -guanosine.
  • the small (4-6 nt) binding site may make this ribozyme too non-specific for targeted RNA cleavage, however, the Tetrahymena group I intron has been used to repair a "defective" beta-galactosidase message by the ligation of new beta-galactosidase sequences onto the defective message
  • RNAse P RNA Ml RNA
  • RNA portion of a ubiquitous ribonucleoprotein enzyme • RNA portion of a ubiquitous ribonucleoprotein enzyme.
  • Reaction mechanism possible attack by M 2+ -OH to generate cleavage products with 3'-OH and 5'-phosphate.
  • RNAse P is found throughout the prokaryotes and eukaryotes.
  • the RNA subunit has been sequenced from bacteria, yeast, rodents, and primates.
  • Reaction mechanism 2'-OH of an internal adenosine generates cleavage products with 3'-OH and a "lariat" RNA containing a 3'-5' and a 2'-5' branch point.
  • Reaction mechanism attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2',3'-cyclic phosphate and 5' ⁇ OH ends.
  • Reaction mechanism attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2' ,3' -cyclic phosphate and 5'-OH ends.
  • Reaction mechanism attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2',3'-cyclic phosphate and 5'-OH ends.
  • RNA RNA as the infectious agent.
  • Ligation activity (in addition to cleavage activity) makes ribozyme amenable to engineering through in vitro selection [ xxxv ]
  • HDV Hepatitis Delta Virus
  • Folded ribozyme contains a pseudoknot structure [ x1 ].
  • Reaction mechanism attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2',3'-cyclic phosphate and 5'-OH ends.
  • xiv Forster, Anthony C; Altaian, Sidney. External guide sequences for an RNA enzyme.
  • RNA Lambowitz, Alan M.. A group II intron RNA is a catalytic component of a DNA endonuclease involved m tron mobility. Cell (Cambridge, Mass.) (1995), 83(4), 529-38. X 1 . Griffin, Edmund A., Jr.; Qm, Zhrfeng; Michels, Williams J., Jr.; Pyle, Anna Marie.
  • RNA model evidence for helixes and sequence requirement for substrate RNA.
  • AAAAGCUU C ACGUUACA 41 UGUAACGU CUGAUGAG GCCGUUAGGC CGAA AAGCUUUU 946
  • AAACUCUU C AGGGGACA 168 UGUCCCCU CUGAUGAG GCCGUUAGGC CGAA AAGAGUUU 1073
  • HSA011736 Homo sapiens mRNA for growth factor receptor binding protein (GRBLG) ; 1303 bp)
  • Underlined region can be any X sequence or linker as defined herein.
  • Table IV Human GRID NCH Ribozyme and Substrate Sequence
  • HSA011736 Homo sapiens mRNA for growth factor receptor binding protein (GRBLG) ; 1303 bp)
  • Underlined region can be any X sequence or linker as defined herein.
  • I Inosine Table V: Human GRID G-cleaver Ribozyme and Substrate Sequence
  • CAGCAGCU G CAGCAGCC 524 GGCUGCUG UGAUG GCAUGCACUAUGC GCG AGCUGCUG 1429
  • HSA011736 Homo sapiens mRNA for growth factor receptor binding protein (GRBLG) ; 1303 bp)

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Abstract

The present invention relates to nucleic acid molecules, including antisense and enzymatic nucleic acid molecules, such as hammerhead ribozymes, DNAzymes, and antisense, which modulate the expression of the GRID (Grb2-related with Insert Domain) gene.

Description

DESCRIPTION
METHOD AND REAGENT FOR THE INHIBITION OF GRID
Background Of The Invention
This invention claims priority from Jarvis et al, USSN (60/181,594), filed February 24, 2000, entitled "METHOD AND REAGENT FOR THE INHIBITION OF GRID". This application is hereby incorporated by reference herein in its entirety including the drawings.
The present invention concerns compounds, compositions, and methods for the study, diagnosis, and treatment of conditions and diseases related to the expression of the T-cell co-stimulatory adapter protein GRID (Grb2-related with Insert Domain).
The following is a brief description of the current understanding of GRID. The discussion is not meant to be complete and is provided only for understanding the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.
One of the emerging paradigms for signal transduction in lymphocytes is that receptors and other signaling molecules do not operate in isolation, but through the recruitment of a complex of other proteins (Pawson and Scott, 1997; Science, 278, 2075; Rudd, 1999, Cell, 96, 5). These other proteins serve to amplify and diversify the signal into a number of biochemical cascades. The archetypal adapter protein is Grb2, which serves to regulate downstream pathways such as Ras activation and Ca2+ mobilization (Lowenstein et al, 1992, Cell, 70, 431), and is ultimately responsible for modulating gene expression required for proliferation and differentiation. Grb2 is recruited to LAT and SLP-76 which are downstream targets in the signaling cascade initiated by ligation of the T-cell receptor by MHC-antigen. These functions are mediated by specialized domains which bind specific motifs and include the phosphotyrosine binding SH2 (Src homology) domain and SH3 domain which are associated with proline-rich PXXP motifs. Grb2, whose sole function appears to be the formation of bridges between other proteins, is entirely comprised of such domains having an SH3-SH2-SH3 structure (Peterson et al, 1998, Curr. Opin. Immunol, 10, 337; Koretzky, 1997, Immunol Today, 18, 401).
A novel member of the Grb2 family of adapter proteins termed GRID (Grb2-related with Insert Domain) has recently been identified (Asada et al, 1999, J. Exp. Med., 189, 1383; Liu et al, 1999, Curr. Biol, 9, 67; Liu et al, 1998, Oncogene, 17, 3073; Law et al, 1999, J. Exp. Med., 189, 1243; Qiu et al, 1998, Biochem. Biophys. Res. Commun., 253, 443; Bourette et al, 1998, Embo. J., 17, 7273). GRID is recruited to the T cell co- stimulatory receptor CD28 upon activation of this receptor by cross-linking antibodies. Although GRID shares significant similarity at the protein level with Grb2, possessing an SH3-SH2-SH3 domain structure, GRID also contains a unique proline-glutamine rich domain situated between the SH2 and C-terminal SH3 domain. The association of GRID with activated CD28 is absolutely dependent upon the integrity of the SH2 domain and phosphorylation of residue Y173 in the cytoplasmic tail of CD28. Although GRID has been shown to associate with other T cell signaling proteins including SLP-76 and LAT (Asada et al, supra; Liu et al, supra; Law et al, supra), it's role in T cell signaling pathways is not well defined.
Tari et al, 1999, Oncogene, 18(6), 1325-1332, describe the antisense inhibition of Grb2 in breast cancer cells in order to investigate the role of Grb2 in the proliferation of breast cancer cells. The resulting Grb2 inhibition led to MAP kinase inactivation in EGFR but not in ErbB2 expressing breast cancer cells.
Tari et al, 1998, J. Liposome Res., 8(2), 251-264, describe P-ethoxy antisense oligonucleotides targeting Bcr-Abl, Grb2, Crkl, and Bcl-2 mRNA. Delivery of these antisense oligonucleotides via liposome transfection results in the inhibition of corresponding proteins, thereby inducing growth inhibition in leukemia and lymphoma cell lines.
Lopez-Berestein et al, 1998, Intemational PCT publication No. WO 98/01547, describe inhibition of chronic myelogenous leukemic cell growth by liposomal-antisense oligodeoxynucleotides targeting Grb2 and Crkl .
Tari et al, 1997, Biochem. Biophys. Res. Commun., 235(2), 383-388, describe the antisense-based inhibition of Grb2 and Crkl proteins results in growth inhbition of Philadelphia chromosome positive leukemic cells.
Summary Of The Invention .
The invention features novel nucleic acid-based techniques [e.g., enzymatic nucleic acid molecules (for example, ribozymes or DNAzymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups] and methods for their use to modulate the expression of GRID (Grb2- related with Insert Domain).
The description below of the various aspects and embodiments is provided with reference to the exemplary gene GRID. However, the various aspects and embodiments are also directed to other genes which express GRID -like adapter proteins involved in T- cell co-activation. Those additional genes can be analyzed for target sites using the methods described for GRID. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.
In a preferred embodiment, the invention features the use of one or more of the nucleic acid-based techniques independently or in combination to inhibit the expression of the genes encoding GRID. For example, the nucleic acid-based techniques of the present invention can be used to inhibit the expression of GRID gene sequences found at GenBank Accession NOS. AJ011736, NM_004810, Y18051, AF121002, AF042380, AF129476, AF090456).
In another preferred embodiment, the invention features the use of an enzymatic nucleic acid molecule, preferably in the hammerhead, NCH (Inozyme), G-cleaver, amberzyme, zinzyme and/or DNAzyme motif, to inhibit the expression of GRID gene.
By "inhibit" it is meant that the activity of GRID or level of GRID RNAs or equivalent RNAs encoding one or more protein subunits of GRID or GRID-like proteins is reduced below that observed in the absence of the nucleic acid molecules of the invention. In one embodiment, the inhibition with enzymatic nucleic acid molecule preferably is below that level observed in the presence of an enzymatically inactive or attenuated molecule that is able to bind to the same site on the target RNA, but is unable to cleave that RNA. In another embodiment, inhibition with antisense oligonucleotides is preferably below that level observed in the presence of, for example, an oligonucleotide with scrambled sequence or with mismatches. In another embodiment, inhibition of GRID or GRID-like genes with the nucleic acid molecule of the instant invention is greater than in the presence of the nucleic acid molecule than in its absence.
By "enzymatic nucleic acid molecule" it is meant a nucleic acid molecule which has complementarity in a substrate-binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave target RNA. That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. These complementary regions allow sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA and thus pe mit cleavage. One hundred percent complementarity is preferred, but complementarity as low as 50-75% can also be useful in this invention (see for example Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al, 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). The nucleic acids can be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA? enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity. The specific enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving and/or ligation activity to the molecule (Cech et al., U.S. Patent No. 4,987,071; Cech et al, 1988, 260 JAMA 3030).
By "nucleic acid molecule" as used herein is meant a molecule having nucleotides. The nucleic acid can be single, double, or multiple stranded and may comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.
By "enzymatic portion" or "catalytic domain" is meant that portion or region of the enzymatic nucleic acid molecule essential for cleavage of a nucleic acid substrate (for example, see Figures 1-5).
By "substrate binding arm" or "substrate binding domain" is meant that portion or region of a enzymatic nucleic acid which is able to interact, for example, via complementarity (i.e., able to base-pair with), with a portion of its substrate. Preferably, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 can be base-paired (see for example Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al, 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). Examples of such arms are shown generally in Figures 1-5. That is, these arms contain sequences within an enzymatic nucleic acid which are intended to bring enzymatic nucleic acid and target RNA together through complementary base-pairing interactions. The enzymatic nucleic acid of the invention can have binding arms that are contiguous or non-contiguous and can be of varying lengths. The length of the binding arm(s) are preferably greater than or equal to four nucleotides and of sufficient length to stably interact with the target RNA. Preferably, the binding arm(s) are 12-100 nucleotides in length. More preferably, the binding arms are 14-24 nucleotides in length (see, for example, Werner and Uhlenbeck, supra; Hamman et al, supra; Hampel et al, EP0360257; Berzal-Herrance et al, 1993, EMBO J., 12, 2567-73). If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., five and five nucleotides, or six and six nucleotides, or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).
By "Inozyme" or "NCH" motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as NCH Rz in Figure 2. Inozymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCH/, where N is a nucleotide, C is cytidine and H is adenosine, uridine or cytidine, and / represents the cleavage site. H is used interchangeably with X. Inozymes can also possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCN/, where N is a nucleotide, C is cytidine, and / represents the cleavage site. "I" in Figure 2 represents an Inosine nucleotide, preferably a ribo-Inosine or xylo-Inosine nucleoside.
By "G-cleaver" motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as G-cleaver in Figure 2. G-cleavers possess endonuclease activity to cleave RNA substrates having a cleavage triplet NYN/, where N is a nucleotide,
Y is uridine or cytidine and / represents the cleavage site. G-cleavers may be chemically modified as is generally shown in Figure 2.
By "amberzyme" motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in Figure 3. Amberzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NG/N, where N is a nucleotide, G is guanosine, and / represents the cleavage site. Amberzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in Figure 3. In addition, differing nucleoside and/or non-nucleoside linkers can be used to substitute the 5'-gaaa-3' loops shown in the figure. Amberzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2' -OH) group within its own nucleic acid sequence for activity. By "zinzyme" motif is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in Figure 4. Zinzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet including but not limited to YG Y, where Y is uridine or cytidine, and G is guanosine and / represents the cleavage site. Zinzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in Figure 4, including substituting 2'-0-methyl guanosine nucleotides for guanosine nucleotides. In addition, differing nucleotide and/or non-nucleotide linkers can be used to substitute the 5'-gaaa-2' loop shown in the figure. Zinzymes represent a non- limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2' -OH) group within its own nucleic acid sequence for activity.
By 'DNAzyme' is meant, an enzymatic nucleic acid molecule that does not require the presence of a 2'-OH group for its activity. In particular embodiments the enzymatic nucleic acid molecule can have an attached linker(s) or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2' -OH groups. DNAzymes can be synthesized chemically or expressed endogenously in vivo, by means of a single stranded DNA vector or equivalent thereof. An example of a DNAzyme is shown in Figure 5 and is generally reviewed in Usman et al, International PCT Publication No. WO 95/11304; Chartrand et al, 1995, NAR 23, 4092; Breaker et al, 1995, Chem. Bio. 2, 655; Santoro et al, 1997, PNAS 94, 4262; Breaker, 1999, Nature Biotechnology, 17, 422-423; and Santoro et. al, 2000, J. Am. Chem. Soc, 122, 2433-39. Additional DNAzyme motifs can be selected for using techniques similar to those described in these references, and hence, are within the scope of the present invention.
By "sufficient length" is meant an oligonucleotide of greater than or equal to 3 nucleotides that is of a length great enough to provide the intended function under the expected condition. For example, for binding arms of enzymatic nucleic acid "sufficient length" means that the binding arm sequence is long enough to provide stable binding to a target site under the expected binding conditions. Preferably, the binding arms are not so long as to prevent useful turnover.
By "stably interact" is meant interaction of the oligonucleotides with target nucleic acid (e.g., by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions) that is sufficient to the intended purpose (e.g., cleavage of target RNA by an enzyme).
By "equivalent" RNA to GRID is meant to include those naturally occurring RNA molecules having homology (partial or complete) to GRID proteins or encoding for proteins with similar function as GRID in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites. The equivalent RNA sequence also includes in addition to the coding region, regions such as 5 '-untranslated region, 3 '-untranslated region, introns, intron-exon junction and the like.
By "homology" is meant the nucleotide sequence of two or more nucleic acid molecules is partially or completely identical.
By "antisense nucleic acid", it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al, 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al, US patent No. 5,849,902). Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both. For a review of current antisense strategies, see Schmajuk et al, 1999, J. Biol. Chem., 21 A, 21783-21789, Delihas et al, 1997, Nature, 15, 751-753, Stein et al, 1997, Antisense N. A. Drug Dev., 7, 151, Crooke, 2000, Methods Enzymol, 313, 3-45; Crooke, 1998, Biotech. Genet. Eng. Rev., 15, 121-157, Crooke, 1997, Ad. Pharmacol, 40, 1-49. In addition, antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex. The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA. Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof.
By "RNase H activating region" is meant a region (generally greater than or equal to 4-25 nucleotides in length, preferably from 5-11 nucleotides in length) of a nucleic acid molecule capable of binding to a target RNA to form a non-covalent complex that is recognized by cellular RNase H enzyme (see for example Arrow et al, US 5,849,902; Arrow et al, US 5,989,912). The RNase H enzyme binds to the nucleic acid molecule- target RNA complex and cleaves the target RNA sequence. The RNase H activating region comprises, for example, phosphodiester, phosphorothioate (preferably at least four of the nucleotides are phosphorothiote substitutions; morepreferably, 4-11 of the nucleotides are phosphorothiote substitutions); phosphorodithioate, 5'-thiophosphate, or methylphosphonate backbone chemistry or a combination thereof. In addition to one or more backbone chemistries described above, the RNase H activating region can also comprise a variety of sugar chemistries. For example, the RNase H activating region can comprise deoxyribose, arabino, fluoroarabino or a combination thereof, nucleotide sugar chemistry. Those skilled in the art will recognize that the foregoing are non-limiting examples and that any combination of phosphate, sugar and base chemistry of a nucleic acid that supports the activity of RNase H enzyme is within the scope of the definition of the RNase H activating region and the instant invention.
By "2-5 A antisense chimera" is meant an antisense oligonucleotide containing a 5'- phosphorylated 2'-5'-linked adenylate residue. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5 A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al, 1993 Proc. Natl. Acad. Sci. USA 90, 1300; Silverman et al, 2000, Methods Enzymol, 313, 522-533; Player and Torrence, 1998, Pharmacol. Ther., 78, 55-113).
By "triplex forming oligonucleotides" is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval- Valentin et al, 1992 Proc. Natl Acad. Sci. USA 89, 504; Fox, 2000, Curr. Med. Chem., 7, 17-37; Praseuth et. al, 2000, Biochim. Biophys. Acta, 1489, 181- 206).
By "gene" it is meant a nucleic acid that encodes RNA, for example, nucleic acid sequences including but not limited to structural genes encoding a polypeptide.
"Complementarity" refers to the ability of a nucleic acid to form hydrogen bond(s) with another RNA sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., enzymatic nucleic acid cleavage, antisense or triple helix inhibition. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. Ill pp.123-133; Frier et al, 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al, 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
By "RNA" is meant a molecule comprising at least one ribonucleotide residue. By "ribonucleotide" or "2'-OH" is meant a nucleotide with a hydroxyl group at the 2' position of a β-D-ribo-furanose moiety.
By "decoy RNA" is meant a RNA molecule that mimics the natural binding domain for a ligand. The decoy RNA therefore competes with natural binding target for the binding of a specific ligand. For example, it has been shown that over-expression of HIV trans- activation response (TAR) RNA can act as a "decoy" and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al., 1990, Cell, 63, 601-608). This is but a specific example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art.
Several varieties of naturally occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor of gene expression, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme.
The enzymatic nucleic acid molecule that cleave the specified sites in GRID-specific RNAs represent a novel therapeutic approach to treat a variety of pathologic indications, including but not limited to tissue/graft rejection and leukemia. In one of the preferred embodiments of the inventions described herein, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but can also be formed in the motif of a hepatitis delta virus, group I intron, group II intron or RNase P RNA (in association with an RNA guide sequence), Neurospora VS RNA, DNAzymes, NCH cleaving motifs, or G-cleavers. Examples of such hammerhead motifs are described by Dreyfus, supra, Rossi et al, 1992, AIDS Research and Human Retroviruses 8, 183. Examples of hairpin motifs are described by Hampel et al, EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al, 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, Hampel et al, 1990 Nucleic Acids Res. 18, 299; and Chowrira & McSwiggen, US. Patent No. 5,631,359. The hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16. The RNase P motif is described by Guerrier- Takada et al, 1983 Cell 35, 849; Forster and Altaian, 1990, Science 249, 783; and Li and Altaian, 1996, Nucleic Acids Res. 24, 835. The Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell, 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; and Guo and Collins, 1995, EMBO. J. 14, 363). Group II introns are described by Griffin et al, 1995, Chem. Biol. 2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; and Pyle et al, Intemational PCT Publication No. WO 96/22689. The Group I intron is described by Cech et al, U.S. Patent 4,987,071. DNAzymes are described by Usman et al, International PCT Publication No. WO 95/11304; Chartrand et al, 1995, NAR 23, 4092; Breaker et al, 1995, Chem. Bio. 2, 655; and Santoro et al, 1997, PNAS 94, 4262. NCH cleaving motifs are described in Ludwig & Sproat, Intemational PCT Publication No. WO 98/58058; and G-cleavers are described in Kore et al, 1998, Nucleic Acids Research 26, 4116-4120 and Eckstein et al, Intemational PCT Publication No. WO 99/16871. Additional motifs include the Aptazyme (Breaker et al, WO 98/43993), Amberzyme (Class I motif; Figure 3; Beigelman et al, Intemational PCT publication No. WO 99/55857) and Zinzyme (Beigelman et al, Intemational PCT publication No. WO 99/55857), all these references are incorporated by reference herein in their totalities, including drawings and can also be used in the present invention. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule (Cech et al, U.S. Patent No. 4,987,071).
In preferred embodiments of the present invention, a nucleic acid molecule of the instant invention can be between 13 and 100 nucleotides in length. Exemplary enzymatic nucleic acid molecules of the invention are shown in Tables III-VIII and X. For example, enzymatic nucleic acid molecules of the invention are preferably between 15 and 50 nucleotides in length, more preferably between 25 and 40 nucleotides in length, e.g., 34, 36, or 38 nucleotides in length (for example see Jarvis et al., 1996, J. Biol. Chem., 271, 29107-29112). Exemplary DNAzymes of the invention are preferably between 15 and 40 nucleotides in length, more preferably between 25 and 35 nucleotides in length, e.g., 29, 30, 31, or 32 nucleotides in length (see for example Santoro et al, 1998, Biochemistry, 37, 13330-13342; Chartrand et al, 1995, Nucleic Acids Research, 23, 4092-4096 and Cairns et al, 2000, Antisense & Nucleic Acid Drug Dev., 10, 323-332). Exemplary antisense molecules of the invention are preferably between 15 and 75 nucleotides in length, more preferably between 20 and 35 nucleotides in length, e.g., 25, 26, 27, or 28 nucleotides in length (see for example Woolf et al, 1992, PNAS., 89, 7305-7309; Milner et al, 1997, Nature Biotechnology, 15, 537-541). Exemplary triplex forming oligonucleotide molecules of the invention are preferably between 10 and 40 nucleotides in length, more preferably between 12 and 25 nucleotides in length, e.g., 18, 19, 20, or 21 nucleotides in length (see for example Maher et al, 1990, Biochemistry, 29, 8820-8826; Strobel and Dervan, 1990, Science, 249, 73-75). Those skilled in the art will recognize that all that is required is for the nucleic acid molecule to be of length and conformation sufficient and suitable for the nucleic acid molecule to catalyze a reaction contemplated herein. The length of the nucleic acid molecules of the instant invention are not limiting within the general limits stated.
Preferably, a nucleic acid molecule that down regulates the replication of GRID or GRID-like gene comprises between 12 and 100 bases complementary to a GRID or GRID- like RNA. Even more preferably, a nucleic acid molecule that down regulates the replication of GRID or GRID-like gene comprises between 14 and 24 bases complementary to a GRID or GRID-like RNA.
In a preferred embodiment, the invention provides a method for producing a class of nucleic acid-based gene inhibiting agents which exhibit a high degree of specificity for the RNA of a desired target. For example, the enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of target RNAs encoding GRID or GRID- like proteins such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention. Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required. Alternatively, the nucleic acid molecules (e.g., ribozymes and antisense) can be expressed from DNA and/or RNA vectors that are delivered to target cells. In a preferred embodiment, the invention features the use of nucleic acid-based inhibitors of the invention to specifically target genes that share homology with the GRID gene. For example, the invention describes the use of nucleic acid-based inhibitors to target the Grb2 (GenBank accession No. NM_002086) and GRAP (GenBank accession No. NM_006613) genes.
As used in herein "cell" is used in its usual biological sense and does not refer to an entire multicellular organism. The cell can be present in an organism which includes humans but is preferably a non-human multicellular organism, e.g., birds, plants and mammals such as cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).
By "GRID proteins" is meant, a protein or a mutant protein derivative thereof, comprising an adapter-protein type of association to the activated CD28 co-stimulatory receptor, and to other signaling proteins including but not limited to SLP-76 and LAT.
By "highly conserved sequence region" is meant a nucleotide sequence of one or more regions in a target gene that does not vary significantly from one generation to the other or from one biological system to the other.
The nucleic acid-based inhibitors of GRID expression are useful for the prevention and/or treatment of diseases and conditions that are related to or will respond to the levels of GRID in a cell or tissue, alone or in combination with other therapies. For example, the nucleic acid-based inhibitors of GRID expressions are useful for the prevention and/or treatment of tissue/graft rejection and cancer, such as leukemia, among other conditions.
By "related" is meant that the reduction of GRID expression (specifically GRID gene) RNA levels and thus reduction in the level of the respective protein will relieve, to some extent, the symptoms of the disease or condition.
In a preferred embodiment, the invention features the use of nucleic acid-based inhibitors of the invention to specifically target regions of GRID gene that are not homologous to Grb2 gene. Specifically, the invention describes the use of nucleic acid- based inhibitors to target sequences that are unique to GRID gene.
The nucleic acid-based inhibitors of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues using well-known methods described herein and generally known in the art. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers. In preferred embodiments, the enzymatic nucleic acid inhibitors comprise sequences, which are complementary to the substrate sequences in Tables UI to X. Examples of such enzymatic nucleic acid molecules also are shown in Tables HI to VTϋ and X. Examples of such enzymatic nucleic acid molecules consist essentially of sequences defined in these Tables.
In yet another embodiment, the invention features antisense nucleic acid molecules and 2-5A chimera including sequences complementary to the substrate sequences shown in Tables III to X. Such nucleic acid molecules can include sequences as shown for the binding arms of the enzymatic nucleic acid molecules in Tables UI to VIII and X and sequences shown as GeneBloc™ sequences in Table X. Similarly, triplex molecules can be provided targeted to the corresponding DNA target regions, and containing the DNA equivalent of a target sequence or a sequence complementary to the specified target (substrate) sequence. Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both.
By "consists essentially of is meant that the active nucleic acid molecule of the invention, for example, an enzymatic nucleic acid molecule, contains an enzymatic center or core equivalent to those in the examples and binding arms able to bind RNA such that cleavage at the target site occurs. Other sequences can be present which do not interfere with such cleavage. Thus, a core region can, for example, include one or more loop, stem- loop structure, or linker which does not prevent enzymatic activity. Thus, the underlined regions in the sequences in Tables III and IV can be such a loop, stem-loop, nucleotide linker, and/or non-nucleotide linker and can be represented generally as sequence "X". For example, a core sequence for a hammerhead enzymatic nucleic acid can comprise a conserved sequence, such as 5'-CUGAUGAG-3' and 5'-CGAA-3' connected by a sequence X, where X is 5'-GCCGUUAGGC-3' (SEQ ID NO 2236) or any other stem II region known in the art or a nucleotide and/or non-nucleotide linker. Similarly, for other nucleic acid molecules of the instant invention, such as Inozyme, G-cleaver, amberzyme, zinzyme, DNAzyme, antisensej 2-5A antisense, triplex forming nucleic acid, and decoy nucleic acids, other sequences or non-nucleotide linkers may be present that do not interfere with the function of the nucleic acid molecule. Sequence X can be a linker of > 2 nucleotides in length, preferably 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 26, 30, where the nucleotides can preferably be internally base-paired to form a stem of preferably > 2 base pairs. Alternatively or in addition, sequence X can be a non- nucleotide linker. In yet another embodiment, the nucleotide linker X can be a nucleic acid aptamer, such as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and others (for a review see Gold et al, 1995, Annu. Rev. Biochem., 64, 763; and Szostak & Ellington, 1993, in The RNA World, ed. Gesteland and Atkins, pp. 511, CSH Laboratory Press). A "nucleic acid aptamer" as used herein is meant to indicate a nucleic acid sequence capable of interacting with a ligand. The ligand can be any natural or a synthetic molecule, including but not limited to a resin, metabolites, nucleosides, nucleotides, drugs, toxins, transition state analogs, peptides, lipids, proteins, amino acids, nucleic acid molecules, hormones, carbohydrates, receptors, cells, viruses, bacteria and others.
In yet another embodiment, the non-nucleotide linker X is as defined herein. The term "non-nucleotide linker" as used herein include either abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds. Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 7§:6353 and Nucleic Acids Res. 1987, 75:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 173:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 773:5109; Ma et al., Nucleic Acids Res. 1993, 27:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990, 75:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 70:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al, Intemational Publication No. WO 89/02439; Usman et al, Intemational Publication No. WO 95/06731; Dudycz et al, Intemational Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 773:4000, all hereby incorporated by reference herein. The term "non-nucleotide" further refers to any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions and allows the remaining bases to exhibit their enzymatic activity. The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine. Thus, in a preferred embodiment, the invention features an enzymatic nucleic acid molecule having one or more non-nucleotide moieties and having enzymatic activity to cleave an RNA or DNA molecule.
In another aspect of the invention, ribozymes or antisense molecules that interact with target RNA molecules and inhibit GRID activity (e.g., inhibit GRID gene) are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme or antisense expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retro virus, adeno virus, or alphavirus. Preferably, the recombinant vectors capable of expressing the ribozymes or antisense are delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of ribozymes or antisense. Such vectors can be repeatedly administered as necessary. Once expressed, the ribozymes or antisense bind to the target RNA and inhibit its function or expression. Delivery of ribozyme or antisense expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell. Antisense DNA can be expressed endogenously via the use of a single stranded DNA intracellular expression vector.
By "vectors" is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
By "patient" is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. "Patient" also refers to an organism to which the nucleic acid molecules of the invention can be administered. Preferably, a patient is a mammal or mammalian cells. More preferably, a patient is a human or human cells.
By "enhanced enzymatic activity" is meant to include activity measured in cells and/or in vivo where the activity is a reflection of both the catalytic activity and the stability of the nucleic acid molecules of the invention. In this invention, the product of these properties can be increased in vivo compared to an all RNA enzymatic nucleic acid or all DNA enzyme. In some cases, the individual catalytic activity or stability of the nucleic acid molecule can be decreased (i.e., less than ten-fold), but the overall activity of the nucleic acid molecule is enhanced in vivo.
The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other dmgs, can be used to treat diseases or conditions discussed above. For example, to treat a disease or condition associated with the levels of GRID, the patient can be treated, or other appropriate cells can be treated, as is evident to those skilled in the art, individually or in combination with one or more dmgs under conditions suitable for the treatment.
In a further embodiment, the described molecules, such as antisense or ribozymes, can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described molecules can be used in combination with one or more known therapeutic agents to treat tissue/graft rejection, leukemia and/or other disease states or conditions which respond to the modulation of GRID expression.
In another preferred embodiment, the invention features nucleic acid-based inhibitors (e.g., enzymatic nucleic acid molecules (ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups) and methods for their use to down regulate or inhibit the expression of genes (e.g., GRID) related to the progression and/or maintenance of tissue/graft rejection, leukemia and/or other disease states or conditions which respond to the modulation of GRID expression.
In another aspect, the invention provides mammalian cells containing one or more nucleic acid molecules and/or expression vectors of this invention. The one or more nucleic acid molecules can independently be targeted to the same or different sites.
By "comprising" is meant including, but not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
Description Of The Preferred Embodiments
First the drawings will be described briefly.
Drawings Figure 1 shows the secondary structure model for seven different classes of enzymatic nucleic acid molecules. Arrow indicates the site of cleavage. indicate the target sequence. Lines interspersed with dots are meant to indicate tertiary interactions. - is meant to indicate base-paired interaction. Group I Intron: P1-P9.0 represent various stem-loop structures (Cech et al, 1994, Nature Struc. Bio., 1, 273). RNase P (M1RNA): EGS represents external guide sequence (Forster et al, 1990, Science, 249, 783; Pace et al, 1990, J. Biol. Chem., 265, 3587). Group II Intron: 5'SS means 5' splice site; 3'SS means 3 '-splice site; IBS means intron binding site; EBS means exon> binding site (Pyle et al, 1994, Biochemistry, 33, 2716). VS RNA: I-VI are meant to indicate six stem-loop structures; shaded regions are meant to indicate tertiary interaction (Collins, Intemational PCT Publication No. WO 96/19577). HDV Ribozyme: : I-IV are meant to indicate four stem-loop structures (Been et al, US Patent No. 5,625,047). Hammerhead Ribozyme: : I-III are meant to indicate three stem-loop structures; stems I-III can be of any length and can be symmetrical or asymmetrical (Usman et al, 1996, Curr. Op. Struct. Bio., 1, 527). Hairpin Ribozyme: Helix 1, 4 and 5 can be of any length; Helix 2 is between 3 and 8 base-pairs long; Y is a pyrimidine; Helix 2 (H2) is provided with a least 4 base pairs (i.e., n is 1, 2, 3 or 4) and helix 5 can be optionally provided of length 2 or more bases (preferably 3 - 20 bases, i.e., m is from 1 - 20 or more). Helix 2 and helix 5 can be covalently linked by one or more bases (i.e., r is > 1 base). Helix 1, 4 or 5 can also be extended by 2 or more base pairs (e.g., 4 - 20 base pairs) to stabilize the ribozyme stmcture, and preferably is a protein binding site. In each instance, each N and N' independently is any normal or modified base and each dash represents a potential base- pairing interaction. These nucleotides can be modified at the sugar, base or phosphate. Complete base-pairing is not required in the helices, but is preferred. Helix 1 and 4 can be of any size (i.e., o and p is each independently from 0 to any number, e.g., 20) as long as some base-pairing is maintained. Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more can be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect. Helix 4 can be formed from two separate molecules, i.e., without a connecting loop. The connecting loop when present can be a ribonucleotide with or without modifications to its base, sugar or phosphate, "q" ≥ is 2 bases. The connecting loop can also be replaced with a non-nucleotide linker molecule. H refers to bases A, U, or
C. Y refers to pyrimidine bases. " " refers to a covalent bond. (Burke et al, 1996,
Nucleic Acids & Mol Biol, 10, 129; Chowrira et al, US Patent No. 5,631,359).
Figure 2 shows examples of chemically stabilized ribozyme motifs. HH Rz, represents hammerhead ribozyme motif (Usman et al, 1996, Curr. Op. Struct. Bio., 1, 527); NCH Rz represents the NCH ribozyme motif (Ludwig & Sproat, Intemational PCT Publication No. WO 98/58058); G-Cleaver, represents G-cleaver ribozyme motif (Kore et al, 1998, Nucleic Acids Research 26, 4116-4120). N or n, represent independently a nucleotide which can be same or different and have complementarity to each other; rl, represents ribo-Inosine nucleotide; arrow indicates the site of cleavage within the target. Position 4 of the HH Rz and the NCH Rz is shown as having 2'-C-allyl modification, but those skilled in the art will recognize that this position can be modified with other modifications well known in the art, so long as such modifications do not significantly inhibit the activity of the ribozyme.
Figure 3 shows an example of the Amberzyme ribozyme motif that is chemically stabilized (see, for example, Beigelman et al, Intemational PCT publication No. WO 99/55857, incorporated by reference herein; also referred to as Class I Motif). The Amberzyme motif is a class of enzymatic nucleic molecules that do not require the presence of a ribonucleotide (2' -OH) group for its activity.
Figure 4 shows an example of the Zinzyme A ribozyme motif that is chemically stabilized (Beigelman et al, Intemational PCT publication No. WO 99/55857, incorporated by reference herein; also referred to as Class A or Class II Motif). The Zinzyme motif is a class of enzymatic nucleic molecules that do not require the presence of a ribonucleotide (2' -OH) group for its activity.
Figure 5 shows an example of a DNAzyme motif described by Santoro et al, 1997,
PNAS, 94, 4262.
Figure 6 shows a graph of optimization of GeneBloc concentration. A fluoresceinated randomized antisense GeneBloc (fGB) was used as a marker for uptake using a fixed concentration of lipid. Cells were either untreated (A) or treated continuously for 24hrs with 10-200nM antisense GeneBloc (B-F). Following treatment, cells were analyzed by flow cytometry. Gate Ml represents either untransfected cells or cells refractory to transfection. Gate M2 represents the transfected cells.
Figure 7 shows a bar graph of a primary screen of twelve GRID GeneBlocs. Taqman RNA assay was used to quantify the level of GRID transcript in Jurkat cells treated continuously for 24 hours with lOOnM antisense GeneBloc and 5-Oμgm 1 cationic lipid. For comparison, all data was normalized to the level of β-actin. Error bars represent the standard error of the mean of triplicate points.
Figure 8 shows a graph demonstrating that flow cytometric sorting of transfected cells improves antisense GeneBloc mediated inhibition of GRID mRNA expression. Jurkat cells were treated continuously for 24 and 72 hours with GB 14540 (75nM) or control GeneBloc GBC3.3 (75nM) spiked with 25nM fluorescent randomized GeneBloc (A) to facilitate the identification of transfected cells. After transfection, the 10% most and least fluorescent cells (gates M2 and Ml respectively) were sorted on a FACStar Plus. Post-sort low transfecting (B) and high transfecting (C) fractions were re-analyzed for purity. Histograms A-D are representative of results obtained in all experiments and were taken from cells treated for 72 hours. The GRID mRNA content of all samples was quantified by Taqman RNA assay and normalized to the β-actin content. For the purposes of inter- experiment comparison, all GB 14540 values were also normalized to the appropriate control GBC3.3 value. (D) Normalized GRID mRNA levels in pre-sort samples; (E) Normalized GRID mRNA levels in the post-sort low transfecting fraction; (F) Normalized GRID mRNA levels in the post-sort high transfecting fraction. Error bars represent the range of duplicate points.
Figure 9 shows a graph representing the phenotypic analysis of antisense GeneBloc treated Jurkat cells following activation with anti-CD3 and anti-CD28 anti-sera. Jurkat cells were treated continuously for 72 hours with the anti-GRID reagent GB 14540 (A, C) and the mismatch control reagent GB 17477 (B, D), activated for 22 hours (C, D) and stained for the surface activation marker CD69. Unactivated samples are shown in (A, B).
Mechanism of action of Nucleic Acid Molecules of the Invention
Antisense: Antisense molecules can be modified or unmodified RNA, DNA, or mixed polymer oligonucleotides which primarily function by specifically binding to matching sequences resulting in inhibition of peptide synthesis (Wu-Pong, Nov 1994, BioPharm, 20-33). The antisense oligonucleotide binds to target RNA by Watson Crick base-pairing and blocks gene expression by preventing ribosomal translation of the bound sequences either by steric blocking or by activating RNase H enzyme. Antisense molecules can also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190). In addition, binding of single stranded DNA to RNA can result in nuclease degradation of the heteroduplex (Wu-Pong, supra; Crooke, supra). To date, the only backbone modified DNA chemistry known to act as substrates for RNase H are phosphorothioates, phosphorodithioates, and borontrifluoridates. Recently it has been reported that 2'-arabino and 2'-fluoro arabino- containing oligos can also activate RNase H activity.
A number of antisense molecules have been described that utilize novel configurations of chemically modified nucleotides, secondary stmcture, and/or RNase H substrate domains (Woolf et al, Intemational PCT Publication No. WO 98/13526; Thompson et al, Intemational PCT Publication No. WO 99/54459; Hartmann et al, USSN 60/101,174 which was filed on September 21, 1998) all of these are incorporated by reference herein in their entirety.
In addition, antisense deoxyoligoribonucleotides can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex. Antisense DNA can be expressed endogenously in vivo via the use of a single stranded DNA intracellular expression vector or equivalents and variations thereof.
Triplex Forming Oligonucleotides (TFO): Single stranded DNA can be designed to bind to genomic DNA in a sequence specific manner. TFOs are comprised of pyrimidine- rich oligonucleotides which bind DNA helices through Hoogsteen Base-pairing (Wu-Pong, supra). The resulting triple helix composed of the DNA sense, DNA antisense, and TFO disrupts RNA synthesis by RNA polymerase. The TFO mechanism can result in gene expression or cell death since binding may be irreversible (Mukhopadhyay & Roth, supra).
2-5A Antisense Chimera: The 2-5A system is an interferon mediated mechanism for RNA degradation found in higher vertebrates (Mitra et al, 1996, Proc Nat Acad Sci USA 93, 6780-6785). Two types of enzymes, 2-5A synthetase and RNase L, are required for RNA cleavage. The 2-5A synthetases require double stranded RNA to form 2'-5' oligoadenylates (2-5A). 2-5A then acts as an allosteric effector for utilizing RNase L which has the ability to cleave single stranded RNA. The ability to form 2-5A structures with double stranded RNA makes this system particularly useful for inhibition of viral replication.
(2 '-5') oligoadenylate structures can be covalently linked to antisense molecules to form chimeric oligonucleotides capable of RNA cleavage (Torrence, supra). These molecules putatively bind and activate a 2-5A dependent RNase, the oligonucleotide/enzyme complex then binds to a target RNA molecule which can then be cleaved by the RNase enzyme.
Enzymatic Nucleic Acid: Several varieties of naturally occurring enzymatic RNAs are presently known. In addition, several in vitro selection (evolution) strategies (Orgel, 1979, Proc. R. Soc. London, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing cleavage and ligation of phosphodiester linkages (Joyce, 1989, Gene, 82, 83-87; Beaudry et al, 1992, Science 257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker et al, 1994, TIBTECH 12, 268; Barrel et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al, 1995, FASEB J., 9, 1183; Breaker, 1996, Curr. Op. Biotech., 7, 442; Santoro et al, 1997, Proc. Natl. Acad. Sci., 94, 4262; Tang et al, 1997, RNA 3, 914; Nakamaye & Eckstein, 1994, supra; Long & Uhlenbeck, 1994, supra; Ishizaka et al, 1995, supra; Vaish et al, 1997, Biochemistry 36, 6495; all of these are incorporated by reference herein). Each can catalyze a series of reactions including the hydrolysis of phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions.
Nucleic acid molecules of this invention can block to some extent GRID protein expression and can be used to treat disease or diagnose disease associated with levels of GRID.
The enzymatic nature of an enzymatic nucleic acid has significant advantages, such as the concentration of enzymatic nucleic acid necessary to affect a therapeutic treatment is lower. This advantage reflects the ability of the enzymatic nucleic acid to act enzymatically. Thus, a single enzymatic nucleic acid molecule is able to cleave many molecules of target RNA. In addition, the enzymatic nucleic acid is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can be chosen to completely eliminate catalytic activity of an enzymatic nucleic acid molecule.
Nucleic acid molecules having an endonuclease enzymatic activity are able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence-specific manner. Such enzymatic nucleic acid molecules can be targeted to virtually any RNA transcript and achieve efficient cleavage in vitro (Zaug et al, 324, Nature 429 1986 ; Uhlenbeck, 1987 Nature 328, 596; Kim et al, 84 Proc. Natl. Acad. Sci. USA 8788, 1987; Dreyfus, 1988, Einstein Quart. J. Bio. Med., 6, 92; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al, 17 Nucleic Acids Research 1371, 1989; Santoro et al, 1997 supra).
Because of their sequence specificity, ws-cleaving enzymatic nucleic acid molecules show promise as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the RNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited (Warashina et al, 1999, Chemistry and Biology, 6, 237-250).
The nucleic acid molecules of the instant invention are also referred to as GeneBloc reagents, which are essentially nucleic acid molecules (e.g., ribozymes, antisense) capable of down-regulating gene expression.
GeneBlocs are modified oligonucleotides, including ribozymes and modified antisense oligonucleotides, that bind to and target specific mRNA molecules. Because GeneBlocs can be designed to target any specific mRNA, their potential applications are quite broad. Traditional antisense approaches have often relied heavily on the use of phosphorothioate modifications to enhance stability in biological samples, leading to a myriad of specificity problems stemming from non-specific protein binding and general cytotoxicity (Stein, 1995, Nature Medicine, 1, 1119). In contrast, GeneBlocs contain a number of modifications that confer nuclease resistance while making minimal use of phosphorothioate linkages, which reduces toxicity, increases binding affinity, and minimizes non-specific effects compared with traditional antisense oligonucleotides. Similar reagents have recently been utilized successfully in various cell culture systems (Vassar, et al, 1999, Science, 286, 735) and in vivo (Jarvis et al., manuscript in preparation). In addition, novel cationic lipids can be utilized to enhance cellular uptake in the presence of serum. Since ribozymes and antisense oligonucleotides regulate gene expression at the RNA level, the ability to maintain a steady-state dose of GeneBloc over several days is important for target protein and phenotypic analysis. The advances in resistance to nuclease degradation and prolonged activity in vitro have supported the use of GeneBlocs in target validation applications.
Target sites
Targets for useful ribozymes and antisense nucleic acids can be determined as disclosed in Draper et al, WO 93/23569; Sullivan et al, WO 93/23057; Thompson et al, WO 94/02595; Draper et al, WO 95/04818; McSwiggen et al, US Patent No. 5,525,468. All of these publications are hereby incorporated by reference herein in their totality. Other examples include the following PCT applications, which concern inactivation of expression of disease-related genes: WO 95/23225, WO 95/13380, WO 94/02595, all of which are incorporated by reference herein. Rather than repeat the guidance provided in those documents here, specific examples of such methods are provided herein, not limiting to those in the art. Ribozymes and antisense to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described. The sequences of human GRID RNAs were screened for optimal enzymatic nucleic acid and antisense target sites using a computer-folding algorithm. Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme, or G-Cleaver ribozyme binding/cleavage sites were identified. These sites are shown in Tables III to VIII and X (all sequences are 5' to 3 ' in the tables; underlined regions can be any sequence or linker X as previously defined herein, the actual sequence is not relevant here). The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of enzymatic nucleic acid molecule. While human sequences can be screened and enzymatic nucleic acid molecule and/or antisense thereafter designed, as discussed in Stinchcomb et al, WO 95/23225, mouse targeted ribozymes are also useful to test efficacy of action of the enzymatic nucleic acid molecule and/or antisense prior to testing in humans.
Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver ribozyme binding/cleavage sites were identified. The nucleic acid molecules were individually analyzed by computer folding (Jaeger et al, 1989 Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the sequences fold into the appropriate secondary structure. Those nucleic acid molecules with unfavorable intramolecular interactions, such as between the binding arms and the catalytic core, were eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity.
Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver ribozyme binding/cleavage sites were identified and were designed to anneal to various sites in the RNA target. The binding arms are complementary to the target site sequences described above. The nucleic acid molecules were chemically synthesized. The method of synthesis used follows the procedure for normal DNA/RNA synthesis as described below and in Usman et al, 1987 J. Am. Chem. Soc, 109, 7845; Scaringe et al, 1990 Nucleic Acids Res., 18, 5433; Wincott et al, 1995 Nucleic Acids Res. 23, 2677-2684; and Caruthers et al, 1992, Methods in Enzymology 211,3-19. Synthesis of Nucleic acid Molecules
Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs ("small refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., antisense oligonucleotides, hammerhead or the NCH ribozymes) are preferably used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of RNA structure. Exemplary molecules of the instant invention are chemically synthesized, and others can be similarly synthesized.
Oligonucleotides (e.g.; antisense GeneBlocs) are synthesized using protocols known in the art as described in Caruthers et al, 1992, Methods in Enzymology 211, 3-19, Thompson et al, International PCT Publication No. WO 99/54459, Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al, 1997, Methods Mol. Bio., 74, 59, Brennan et al, 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, US patent No. 6,001,311. All of these references are incoφorated herein by reference. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 2.5 min coupling step for 2'-0- methylated nucleotides and a 45 sec coupling step for 2 '-deoxy nucleotides. Table II outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M = 6.6 μmol) of 2'-0-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 μL of 0.25 M = 15 μmol) can be used in each coupling cycle of 2'-0-methyl residues relative to polymer-bound 5'- hydroxyl. A 22-fold excess (40 μL of 0.11 M = 4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 μL of 0.25 M = 10 μmol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5 '-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF (PERSEPTJVE™). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American Intemational Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-l,2-Benzodithiol-3-one 1,1 -dioxide, 0.05 M in acetonitrile) is used.
Deprotection of the antisense oligonucleotides is performed as follows: the polymer- bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65 °C for 10 min. After cooling to -20 °C, the supematant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H20/3:l:l, vortexed and the supematant is then added to the first supematant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.
The method of synthesis used for normal RNA including certain enzymatic nucleic acid molecules follows the procedure as described in Usman et al, 1987, J. Am. Chem. Soc, 109, 7845; Scaringe et al, 1990, Nucleic Acids Res., 18, 5433; Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684 and Wincott et al, 1997, Methods Mol Bio., 74, 59, and makes use of common ucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5 '-end, and phosphoramidites at the 3 '-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2'-0-methylated nucleotides. Table II outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M = 6.6 μmol) of 2'-0-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M = 15 μmol) can be used in each coupling cycle of 2'-0-methyl residues relative to polymer-bound 5 '-hydroxyl. A 66-fold excess (120 μL of 0.11 M = 13.2 μmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 μL of 0.25 M = 30 μmol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5 '-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM 12,
49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American Intemational Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-l,2-Benzodithiol-3-one l,l-dioxide0.05 M in acetonitrile) is used.
Deprotection of the RNA is performed using either a two-pot or one-pot protocol.
For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at
65 °C for 10 min. After cooling to -20 °C, the supematant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H20/3:l:l, vortexed and the supematant is then added to the first supematant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder. The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mL TEA»3HF to provide a 1.4 M HF concentration) and heated to 65 °C. After 1.5 h, the oligomer is quenched with 1.5 M NH4HCO3.
Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65 °C for 15 min. The vial is brought to r.t. TEA«3HF (0.1 mL) is added and the vial is heated at 65 °C for 15 min. The sample is cooled at -20 °C and then quenched with 1.5 M NH4HCO3.
For purification of the trityl-on oligomers, the quenched NH4HCO3 solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5%) TFA for 13 min. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.
Inactive hammerhead ribozymes or binding attenuated control (BAC) oligonucleotides) are synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel, K. J., et al, 1992, Nucleic Acids Res_., 20, 3252). Similarly, one or more nucleotide substitutions can be introduced in other enzymatic nucleic acid molecules to inactivate the molecule and such molecules can serve as a negative control.
The average stepwise coupling yields are typically >98% (Wincott et al, 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the examples described above including but not limited to 96-well format, all that is important is the ratio of chemicals used in the reaction.
Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example by ligation (Moore et al, 1992, Science 256, 9923; Draper et al, Intemational PCT publication No. WO 93/23569;
Shabarova et al, 1991, Nucleic Acids Research 19, 4247; Bellon et al, 1997, Nucleosides
& Nucleotides, 16, 951; Bellon et al, 1997, Bioconjugate Chem. 8, 204).
The nucleic acid molecules of the present invention are modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'- C-allyl, 2'-flouro, 2'-0-methyl, 2'-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser. 31, 163). Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al, supra, the totality of which is hereby incorporated herein by reference) and are re-suspended in water. The sequences of the ribozymes and antisense constructs that are chemically synthesized, useful in this study, are shown in Tables III to X. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. The ribozyme and antisense construct sequences listed in Tables III to X can be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes with enzymatic activity are equivalent to the ribozymes described specifically in the Tables.
Optimizing Activity of the nucleic acid molecule of the invention.
Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) that prevent their degradation by semm ribonucleases can increase their potency (see e.g., Eckstein et al, Intemational Publication No. WO 92/07065; Perrault et al, 1990 Nature 344, 565; Pieken et al, 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al, Intemational Publication No. WO 93/15187; Rossi et al, Intemational Publication No. WO 91/03162; Sproat, US Patent No. 5,334,711; and Burgin et al, supra; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein). All these references are incoφorated by reference herein. Modifications which enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are preferably desired. There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-0-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al, 1996, Biochemistry , 35, 14090). Sugar modifications of nucleic acid molecules have been extensively described in the art (see Eckstein et al, International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci. , 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, US Patent No. 5,334,711 and Beigelman et al, 1995, J. Biol. Chem., 270, 25702; Beigelman et al, Intemational PCT publication No. WO 97/26270; Beigelman et al, US Patent No. 5,716,824; Usman et al, US patent No. 5,627,053; Woolf et al, Intemational PCT Publication No. WO 98/13526; Thompson et al, USSN 60/082,404 which was filed on April 20, 1998; Kaφeisky et al, 1998, Tetrahedron Lett., 39, 1131; Eamshaw and Gait, 1998, Biopolymers (Nucleic acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al, 1997, Bioorg. Med. Chem., 5, 1999-2010; all of the references are hereby incoφorated by reference herein in their totalities). Such publications describe general methods and strategies to determine the location of incoφoration of sugar, base and/or phosphate modifications and the like into ribozymes without inhibiting catalysis. In view of such teachings, similar modifications can be used as described herein to modify the nucleic acid molecules of the instant invention.
While chemical modification of oligonucleotide intemucleotide linkages with phosphorothioate, phosphorothioate, and/or 5'-methylphosphonate linkages improves stability, too many of these modifications may cause some toxicity. Therefore, when designing nucleic acid molecules the amount of these intemucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity resulting in increased efficacy and higher specificity of these molecules.
Use of the nucleic acid-based molecules of the invention can lead to improved treatment of the disease progression by affording the possibility of combination therapies
(e.g., multiple antisense or enzymatic nucleic acid molecules targeted to different genes, nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of molecules (including different motifs) and/or other chemical or biological molecules). The treatment of patients with nucleic acid molecules can also include combinations of different types of nucleic acid molecules.
Therapeutic nucleic acid molecules (e.g., enzymatic nucleic acid molecules and antisense nucleic acid molecules) delivered exogenously should preferably be stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. The nucleic acid molecules should be resistant to nucleases in order to function as effective intracellular therapeutic agents when delivered exogenously. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art (see, e.g., Wincott et al., 1995, Nucleic Acids Res., 23:2677; Carruthers, et al., 1992, Methods in Enzymology, 211:3-19, each incoφorated by reference herein) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.
In yet another preferred embodiment, nucleic acid catalysts having chemical modifications which maintain or enhance enzymatic activity are provided. Such nucleic acid is also generally more resistant to nucleases than unmodified nucleic acid. Thus, in a cell and/or in vivo the activity may not be significantly lowered. As exemplified herein such ribozymes are useful in a cell and/or in vivo even if activity over all is reduced 10 fold (Burgin et al, 1996, Biochemistry, 35, 14090). Such ribozymes herein are said to "maintain" the enzymatic activity of an all RNA ribozyme.
In another aspect the nucleic acid molecules comprise a 5' and/or a 3'- cap stmcture.
By "cap stmcture" is meant chemical modifications, which have been incoφorated at either terminus of the oligonucleotide (see, for example, Wincott et al, WO 97/26270, incoφorated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'-terminus (5'-cap) or at the 3'-terminus (3'-cap) or can be present on both termini. In non-limiting examples, the 5 '-cap is selected from the group consisting of inverted abasic residue (moiety), 4',5'-methylene nucleotide; l-(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide; 1 ,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; ^reo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4- dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3 '-3 '-inverted nucleotide moiety; 3 '-3 '-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3'-2'- inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3 '-phosphate; 3 '-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (for more details see Wincott et al, Intemational PCT publication No. WO 97/26270, incoφorated by reference herein).
Suitable 3 '-caps include 4',5'-methylene nucleotide; l-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3- diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2- aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L- nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo- pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5 '-5 '-inverted nucleotide moiety; 5'-5'-inverted abasic moiety; 5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate; 5'-amino; bridging and/or non-bridging 5'-ρhosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5'-mercapto moieties (for more details, see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incoφorated by reference herein).
By the term "non-nucleotide" is meant any group or compound which can be incoφorated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine.
An "alkyl" group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons.
More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
The alkyl group can be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, =0, =S, N02 or N(CH3)2, amino, or SH.
The term also includes alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched- chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group can be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, =0, =S, NO2, halogen, N(CH3)2, amino, or SH. The term "alkyl" also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight- chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group can be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, =0, =S, NO2 or N(CH3)2, amino or SH.
Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An "aryl" group refers to an aromatic group which has at least one ring having a conjugated π electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which can be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An "amide" refers to an -C(0)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to an -C(0)-OR', where R is either alkyl, aryl, alkylaryl or hydrogen.
By "nucleotide" is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotides are recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., Intemational PCT Publication No. WO 92/07065; Usman et al., Intemational PCT Publication No. WO 93/15187; Uhlman & Peyman, supra all are hereby incoφorated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6- trimethoxy benzene, 3 -methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6- methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5'- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D- galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3- methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7- methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5- methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio- N6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2- thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).
By "modified bases" in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule.
By "nucleoside" is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar. Nucleosides are recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a nucleoside sugar moiety. Nucleosides generally comprise a base and sugar group. The nucleosides can be unmodified or modified at the sugar, and/or base moiety, (also referred to interchangeably as nucleoside analogs, modified nucleosides, non-natural nucleosides, non-standard nucleosides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., Intemational PCT Publication No. WO 92/07065; Usman et al., Intemational PCT Publication No. WO 93/15187; Uhlman & Peyman, supra all are hereby incoφorated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3 -methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, quesosine, 2- thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5- (carboxyhydroxymethyl)uridine, 5 '-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1- methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2- methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2- thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5- methyloxyuridine, 5-methyl-2 -thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D- mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).
By "modified bases" in this aspect is meant nucleoside bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule.
In a preferred embodiment, the invention features modified ribozymes with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, moφholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al, 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39. These references are hereby incoφorated by reference herein.
By "abasic" is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1' position, (for more details, see Wincott et al, Intemational PCT publication No. WO 97/26270).
By "unmodified nucleoside" is meant one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1' carbon of β-D-ribo-furanose.
By "modified nucleoside" is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.
In connection with 2 '-modified nucleotides as described for the present invention, by "amino" is meant 2'-NH2 or 2'-0- NH2, which can be modified or unmodified. Such modified groups are described, for example, in Eckstein et al, U.S. Patent 5,672,695 and
Matulic-Adamic et al, WO 98/28317, respectively, which are both incoφorated by reference herein in their entireties.
Various modifications to nucleic acid (e.g., antisense and ribozyme) structure can be made to enhance the utility of these molecules. For example, modifications can enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.
Use of these molecules can lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes (including different ribozyme motifs) and/or other chemical or biological molecules). The treatment of patients with nucleic acid molecules can also include combinations of different types of nucleic acid molecules. Therapies can be devised which include a mixture of ribozymes (including different ribozyme motifs), antisense and/or 2-5A chimera molecules to one or more targets to alleviate symptoms of a disease.
Administration of Nucleic Acid Molecules
Methods for the delivery of nucleic acid molecules are described in Akhtar et al, 1992, Trends Cell Bio., 2, 139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995 which are both incoφorated herein by reference. Sullivan et al, PCT WO 94/02595, further describes the general methods for delivery of enzymatic RNA molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incoφoration into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, nucleic acid molecules can be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the nucleic acid/vehicle combination can be locally delivered by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of nucleic acid delivery and administration are provided in Sullivan et al, supra, Draper et al, PCT W093/23569, Beigelman et al, PCT WO99/05094, and Klimuk et al, PCT WO99/04819 all of which have been incoφorated by reference herein.
The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, inhibit the occurrence, or treat (i.e., alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a patient. The negatively charged polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed as described in the art. The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and other compositions known in the art.
The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, including salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or patient, preferably a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect.
By "systemic administration" is meant in vivo systemic absoφtion or accumulation of dmgs in the blood stream followed by distribution throughout the entire body. Administration routes that lead to systemic absoφtion include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes exposes the desired negatively charged polymers, e.g., nucleic acids, to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cancer cells. By pharmaceutically acceptable formulation is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol, 13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, MA; and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver dmgs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al, 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al, 1999, FEBS Lett, 421, 280-284; Pardridge et al, 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al, 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al, 1999, PNAS USA., 96, 7053-7058.
The invention also features the use of the composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al, Chem. Pharm. Bull. 1995, 43, 1005- 1011). All incoφorated by reference herein. Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al, Science 1995, 267, 1275-1276; Oku et al, 1995, Biochim. Biophys. Ada, 1238, 86-90). All incoφorated by reference herein. The long- circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al, J. Biol. Chem. 1995, 42, 24864-24870; Choi et al, Intemational PCT Publication No. WO 96/10391; Ansell et al, Intemational PCT Publication No. WO 96/10390; Holland et al, Intemational PCT Publication No. WO 96/10392; all of which are incoφorated by reference herein). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.
The present invention also includes compositions prepared for storage or administration which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985) hereby incoφorated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents may be provided. These include sodium benzoate, sorbic acid and esters of />-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.
A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.
The nucleic acid molecules of the present invention can also be administered to a patient in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication may increase the beneficial effects while reducing the presence of side effects.
Alternatively, certain of the nucleic acid molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985,
Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399;
Scanlon et al, 1991, Proc Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al, 1992,
Antisense Res. Dev., 2, 3-15; Dropulic et al, 1992, J. Virol, 66, 1432-41; Weerasinghe et al, 1991, J. Virol, 65, 5531-4; Ojwang et al, 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al, 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al, 1990 Science,
247, 1222-1225; Thompson et al, 1995, Nucleic Acids Res., 23, 2259; Good et al, 1997,
Gene Therapy, 4, 45; all of the references are hereby incoφorated in their totality by reference herein). Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a ribozyme (Draper et al, PCT WO 93/23569, and Sullivan et al, PCT WO 94/02595; Ohkawa et al, 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al, 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al, 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al, 1994, J. Biol Chem., 269, 25856; all of these references are hereby incoφorated in their totalities by reference herein).
In another aspect of the invention, RNA molecules of the present invention are preferably expressed from transcription units (see, for example, Couture et al, 1996, TIG, 12, 510) inserted into DNA or RNA vectors. The recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors can be constructed based on, but not limited to, adeno-associated vims, retrovims, adenovirus, or alphavirus. Preferably, the recombinant vectors capable of expressing the nucleic acid molecules are delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the nucleic acid molecule binds to the target mRNA. Delivery of nucleic acid molecule expressing vectors can be systemic, such as by intravenous or intra-muscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allow for introduction into the desired target cell (for a review, see Couture et al, 1996, TIG., 12, 510).
In one aspect, the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules disclosed in the instant invention. The nucleic acid sequence encoding the nucleic acid molecule of the instant invention is operable linked in a manner which allows expression of that nucleic acid molecule.
In another aspect, the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); c) a nucleic acid sequence encoding at least one of the nucleic acid catalyst of the instant invention; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5' side or the 3'-side of the sequence encoding the nucleic acid catalyst of the invention; and/or an intron (intervening sequences).
Transcription of the nucleic acid molecule sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters also can be used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. U S A, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res.., 21, 2867-72; Lieber et al, 1993, Methods Enzymol, 217, 47-66; Zhou et al, 1990, Mo/. Cell. Biol, 10, 4529-37). All of these references are incoφorated by reference herein.
Several investigators have demonstrated that nucleic acid molecules, such as ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani- Sabet et al, 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al, 1992, Proc. Natl. Acad. Sci. U S A, 89, 10802-6; Chen et al, 1992, Nucleic Acids Res., 20, 4581-9; Yu et al, 1993, Proc. Natl. Acad. Sci. U S A, 90, 6340-4; L'Huillier et al, 1992, EMBO , 11, 4411-8; Lisziewicz et al, 1993, Proc. Natl. Acad. Sci. U. S. A, 90, 8000-4; Thompson et al, 1995, Nucleic Acids Res., 23, 2259; and Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovims VA RNA are useful in generating high concentrations of desired RNA molecules such as ribozymes in cells (Thompson et al, supra; Couture and Stinchcomb, 1996, supra; Noonberg et al, 1994, Nucleic Acid Res., 22, 2830; Noonberg et al, US Patent No. 5,624,803; Good et al, 1997, Gene Ther., 4, 45; and Beigelman et al, International PCT Publication No. WO 96/18736; all of these publications are incoφorated by reference herein. The above ribozyme transcription units can be incoφorated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovims or adeno-associated vims vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review, see Couture and Stinchcomb, 1996, supra).
In yet another aspect, the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules of the invention, in a manner which allows expression of that nucleic acid molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; c) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
In another preferred embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3 '-end of said open reading frame; and wherein said sequence is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
In yet another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3 '-end of said open reading frame; and wherein said sequence is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
Examples.
The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.
The following examples demonstrate the selection and design of Antisense, hammerhead, DNAzyme, NCH, Amberzyme, Zinzyme, or G-Cleaver enzymatic nucleic acid molecules and binding/cleavage sites within GRID RNA.
Nucleic acid inhibition of GRID target RNA
The use of GeneBlocs to modulate the activity of GRID, a putative component of co- stimulatory signaling in T cells, is herein described. An array of GeneBlocs were designed and screened for their ability to reduce GRID mRNA levels whilst leaving transcripts from the closely related genes Grb2 and GRAP unaffected. A series of experiments were conducted to optimize delivery of GeneBlocs to the Jurkat T cell line. Using these conditions, applicant has demonstrated the efficacy of these reagents at both the mRNA and protein level. Anti-CD3/CD28 triggering of Jurkat cells pre-treated with the anti-GRTD GeneBloc results in an impairment of CD69 up-regulation consistent with an important role for GRID in transducing the co-stimulatory signal. Example 1: Identification of Potential Target Sites in Human GRID RNA
The sequence of human GRID were screened for accessible sites using a computer- folding algorithm. Regions of the RNA were identified that do not form secondary folding structures. These regions contain potential ribozyme and/or antisense binding/cleavage sites. The sequences of these binding/cleavage sites are shown in Tables III-X.
Example 2: Selection of Enzymatic Nucleic Acid Cleavage Sites in Human GRID RNA
Enzymatic nucleic acid target sites are chosen by analyzing sequences of Human GRID (for example, GenBank accession numbers: AJ011736 and Y18051) and prioritizing the sites on the basis of folding. Enzymatic nucleic acids are designed that bind each target and are individually analyzed by computer folding (Christoffersen et al, 1994 J. Mol Struc. Theochem, 311, 273; Jaeger et al, 1989, Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the enzymatic nucleic acid sequences fold into the appropriate secondary structure. Those enzymatic nucleic acids with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. As noted below, varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.
Example 3: Chemical Synthesis and Purification of Enzymatic nucleic acids and Antisense for Efficient Cleavage and/or blocking of GRID RNA
Enzymatic nucleic acids and antisense constructs are designed to anneal to various sites in the RNA message. The binding arms of the enzymatic nucleic acids are complementary to the target site sequences described above, while the antisense constructs are fully complimentary to the target site sequences described above. The enzymatic nucleic acids and antisense constructs were chemically synthesized. The method of synthesis used followed the procedure for normal RNA or DNA synthesis as described above and in Usman et al, (1987 J. Am. Chem. Soc, 109, 7845), Scaringe et al, (1990 Nucleic Acids Res., 18, 5433) and Wincott et al, supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5 '-end, and phosphoramidites at the 3'-end. The average stepwise coupling yields were typically >98%.
Enzymatic nucleic acids and antisense constructs also can be synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Enzymatic nucleic acid and antisense constructs are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; see Wincott et al, supra; the totality of which is hereby incoφorated herein by reference) and are resuspended in water. The sequences of the chemically synthesized enzymatic nucleic acid and antisense constructs used in this study are shown below in Table III-X.
Example 4: Enzymatic nucleic acid Cleavage of GRID RNA Target in vitro
Enzymatic nucleic acids targeted to the human GRID RNA are designed and synthesized as described above. These enzymatic nucleic acids can be tested for cleavage activity in vitro, for example, using the following procedure. The target sequences and the nucleotide location within the GRID RNA are given in Tables III-X.
Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for enzymatic nucleic acid cleavage assay is prepared by in vitro transcription in the presence of [a-^2p] CTP, passed over a G 50 Sephadex® column by spin chromatography and used as substrate RNA without further purification. Alternately, substrates are 5'-32p. end labeled using T4 polynucleotide kinase enzyme. Assays are performed by pre- warming a 2X concentration of purified enzymatic nucleic acid in enzymatic nucleic acid cleavage buffer (50 mM Tris-HCl, pH 7.5 at 37°C, 10 mM MgCl2) and the cleavage reaction was initiated by adding the 2X enzymatic nucleic acid mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer. As an o initial screen, assays are carried out for 1 hour at 37 C using a final concentration of either
40 nM or 1 mM ribozyme, i.e., enzymatic nucleic acid excess. The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol o blue and 0.05% xylene cyanol after which the sample is heated to 95 C for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific RNA cleavage products generated by enzymatic nucleic acid cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.
Example 5: Nucleic acid inhibition of GRID in vivo
Antisense nucleic acid molecules (GeneBlocs) targeted to the human GRID RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure. The target sequences and the nucleotide location within the GRID RNA are given in Tables III-X.
GRID shares 60.3% and 57.3% homology at the nucleotide level with the closely related adapter proteins Grb2 and GRAP. In order to discriminate between human GRID and other Grb2 family members, twelve GeneBlocs (see Methods for details) targeting human GRID (GenBank accession number Y18051) were designed, each containing a minimum of six mismatches versus human Grb2 (M96995) and human GRAP (U52518). In order to determine the optimal site for GeneBloc binding and inhibition of the target mRNA, the efficacy of the GeneBlocs was tested on Jurkat cells. A Taqman RNA assay was used to quantify the level of GRID transcript in cells treated continuously for 24hrs. The efficacy of the twelve GeneBlocs, normalized to the levels of a house-keeping gene (β- actin), is shown in Figure 7. The GeneBloc targeting site 152 (GeneBloc 14540) was the most efficacious, reducing GRID mRNA levels by up to 55% when compared with a randomized control GeneBloc (GBC3.3). To confirm that these effects were target specific, a four base-pair mismatch GeneBloc (GB 17477) was synthesized. GRID mRNA expression was unaffected in cells treated with the mismatch control GeneBloc compared to untreated cells.
Efficacy of the anti-GRID GeneBloc (GB 14540) in Jurkat cells
From the primary screen (Figure 7), the optimal GeneBloc, GB 14540, suppressed GRID mRNA levels by up to 55%. However, this represents the inhibition in a bulk population of cells, some of which are refractory to transfection (see Figure 6D-F). To investigate the correlation between dose and efficacy, GB 14540 was spiked with 25% fGB. Based on mixture experiments with active GeneBlocs in other systems, it was not expected that the presence of the fluorescent GeneBloc would interfere with anti-GRID activity of GB 14540. Thus, the most highly fluorescent cells represent the population of cells transfected with the highest concentration of active GeneBloc ('high transfecting'), whilst the cells that appear to be refractory to transfection should contain a significantly lower concentration active GeneBloc ('low transfecting').
Following transfection of a GB14540:fGB mixture, the high transfecting cells (Figure 8A, Gate M2, the 10% most fluorescent cells) and the low transfecting cells (Figure 8A, Gate Ml, the 10% least fluorescent cells) were purified by FACS sorting. Re- analysis of the sorted cell populations confirmed greater than 95% purity (Figure 8B-C). Taqman RNA analysis of the treated cells pre- and post-sort (Figure 8D-F) shows that although GB 14540 inhibition of GRID mRNA expression in an unsorted population is variable between experiments (0-30%, Figure 8D), the level of inhibition is significantly increased to 45-63% in the 'high transfecting' fraction (Figure 8F). In contrast, GRID mRNA levels in the 'low transfecting' fraction was similar to that of cells treated with control GBC3.3 (Figure 8E). These data suggest that the degree of GRID mRNA inhibition is dependent on the dose of GeneBloc delivered to the cells. To identify the optimal time-point for inhibition of GRID mRNA levels, samples were sorted as described above at 24 and 72 hours following continuous transfection. Analysis of pre- and post-sort samples at these time-points revealed that in pre-sort samples, inhibition of GRID transcript occurred within 24 hours and did not significantly increase throughout the time-course of the experiment (Figure 8D). In the 'high transfecting' fractions, reduction of GRID transcript was -45% at 24 hours and increased only fractionally at the 72 hour time-point (50-65%, Figure 8F). This suggests that GB 14540 reduced GRID mRNA levels rapidly following transfection and that inhibition was sustained in the continued presence of GB 14540. Analysis of GRID protein levels in GB 14540 treated cells
To determine whether the reduction in GRID transcript levels was associated with a loss of GRID protein, the level of GRID protein in cells treated continuously with active GeneBloc reagent GB 14540 and the mismatch control GB 17477 was assessed. When delivered continuously for 72 hours, GB 14540 caused a substantial reduction in GRID protein levels as determined by the intensity of the GRID specific band whilst at earlier time-points (24 and 48 hrs) no reduction in protein was observed. Cells treated with the mismatch control GB 17477 showed GRID levels comparable to the untreated sample. Cells treated continuously with GB 14540 for periods up to 144 hours showed no further reduction in GRID protein levels, suggesting that the effect of the GeneBloc was maximal and sustained from 72 hours onwards. Whilst the effects of the anti-GRID GeneBloc on mRNA levels are seen at 24 hours, the reduction in GRID protein is delayed a further 48 hours indicating that GRID protein may have a relatively long half-life.
The GeneBlocs were designed to target and discriminate GRID from the closely related adapter proteins Grb2 and GRAP. GB 14540 contains 6 and 7 mismatches respectively when aligned with the human Grb2 and GRAP sequences. Due to the presence of these mismatches, GB 14540 was not expected to inhibit Grb2 mRNA expression. The Western blots used for the GRID assay were stripped and re-probed using an anti-Grb2 antibody. No difference in Grb2 protein levels was observed between the untreated sample and cells treated with either GB 14540 or the mismatch control reagent GB 17477, confirming that the GB 14540 was specific for GRID.
Phenotypic effects of the anti-GRID GeneBloc on T cell activation
GRJD is a novel member of the Grb2 family of adapter proteins. A role for GRID in T cell signaling has been postulated due to its association with known T cell signaling proteins [Law, 1999 #3296][Asada, 1999 #3243][Liu, 1999 #3245] and more recently the T cell co-stimulatory receptor CD28 following activation by cross-linking antibodies (Ellis et al.). To further elucidate the role of GRID in T cell co-stimulatory pathways, applicant studied the expression of early surface activation marker CD69 (Jung et al, 1988, Cellular Immunology, 117, 352, Lanier et al, 1988, J. Exp. Med., 167, 1572) following activation of Jurkat cells treated with GB14540 and GB17477. Jurkat cells were activated by cross- linking anti-CD3 and anti-CD28 monoclonal antibodies using a sub-maximal stimulus to increase the sensitivity of the assay. In cells treated with the mismatch control GeneBloc, GB 17477, 5.7% stained CD69 positive following activation compared with 0.7% CD69 positive in unactivated cells (Figure 9D vs. 9B). In cells treated with the anti-GRJD reagent GB 14540, there was a marked reduction in the proportion of activated cells, with only 1.3% staining positive for CD69 (Figure 9C). Expression of CD69 in the unactivated sample remained unaltered at 0.6% (Figure 9A). As the activation stimulus was increased, the relative difference between the cells treated with GB 14540 and GB 17477 decreased even though the proportion of cells staining positive for CD69 increased. This can be attributed to the combination of residual GRID protein and supra-maximal activation stimulus. The latter component is particularly relevant to T cell activation since the dependency on co-stimulation is reduced as the strength of the CD3 signal increases (Geppert and Lipsky, 1988, J. Clin. Invest., 81, 1497, Geppert and Lipsky, 1987, Journal of Immunology, 138, 1660).
Taken together, these data suggest that the phenotypic effects described above can be attributed to GRID and not the closely related adapter protein Grb2. The inhibitory effects of GB 14540 on CD69 expression support a role for GRID in T cell co-stimulatory signaling.
Example 6: Delivery of GeneBloc reagents to Jurkat cells
As in many mammalian cell culture systems (Marcusson et al, 1998, Nuc Acids,
Res. 26, 2016), a cationic lipid was found to be necessary to facilitate cellular uptake of oligonucleotide. In preliminary experiments using a fluoresceinated randomized GeneBloc as a marker for uptake, a lipid concentration of 2.5-5.0 μgml"1 was found to be optimal. Although some cells are readily transfected by the GeneBloc, a sub-population of cells remained refractory to transfection (see Gate M2 vs. Ml in Figures 6D-6F). In order to minimize the refractory population, the concentration of GeneBloc was varied between 10- 200nM. Transfection frequencies of up to 75% (as determined by fraction of cells in Gate M2) were observed in the 50-lOOnM range of GeneBloc concentration. At lower concentrations (10-25nM), the transfection frequency dropped off very steeply whilst at higher concentrations, no further enhancement of transfection was observed. Cationic lipids however are not essential for the use of oligonucleotides in vivo (see McGraw et al, 1997, Anti-Cancer Drug Design, 12, 315-326; Henry et al, 1997, Anti-Cancer Drug Design, 12, 409-420).
Example 7: Flow Cytometry
Cultures were harvested, washed once and re-suspended in PBS containing 2% FCS. Cells were stained with a human anti-CD69 PE-conjugated antibody (Caltag) using an IgG2a PE-conjugate as an isotype control (Becton Dickinson). Cells were analyzed on a Becton Dickinson FACScan using CellQuest software. Cells were sorted on the basis of fluorescence in the FL1 channel using a Becton Dickinson FACStar Plus. In order to compare the efficiency of GeneBloc uptake using different transfection conditions, a coefficient of transfection was calculated by multiplying the proportion of control GeneBloc (as a fraction of total GeneBloc) and the transfection frequency.
Example 8: Protein Studies
Actively growing Jurkat cells (0.1-1.0 x 106) were harvested, washed once in PBS and re-suspended in 25 μl PBS. Cells were lysed by the addition of an equal volume of ice- cold 2x RIPA buffer (2% NP40, 1.0% sodium deoxycholate, 0.2% SDS in PBS with 2x protease and phosphatase inhibitors). Following a 30 minute incubation on ice, cell debris was removed by centrifugation and the supematant denatured at 100°C for 5 minutes following the addition of an equal volume of 2x SDS protein sample buffer. Prior to separation by SDS-PAGE electrophoresis, protein content was normalized using a Coomassie® Plus-200 protein assay reagent (Pierce). For Westem blotting, SDS-PAGE gels were transferred to PVDF membrane (Millipore). Antisera specific for GRID (rabbit polyclonal courtesy of Claire Ashman, GlaxoWellcome), p85 sub-unit of PI-3-kinase (#06- 195, Upstate Biotechnology) and Grb2 (sc-255, Santa Cmz) were used as primary antibodies with an anti-rabbit HRP conjugate as the secondary antibody. Bound antibody was visualized using the SuperSignal® West Dura chemiluminescent reagent. For re- probing, chemiluminescent substrate and bound antibody were removed with TBST (TBS + 0.5% Tween-20) and ImmunoPure® IgG Elution Buffer (Pierce) respectively.
Example 9: Cell Culture
Human Jurkat cell lines E6.1 and J6 were maintained at 37°C in 5% COz in flasks in RPMI 1641 (+ 25mM HEPES) supplemented with 10% fetal calf serum and glutamine. Cells were passaged at a density of 1 x 106 cells ml"1. GeneBlocs were delivered to the cells using a modified centrifugation-based transfection protocol (Verma et al, 1998, BioTechniques, 25, 46). Cells were grown to a density of 1 x 106 cells ml"1, harvested by centrifugation and re-suspended in fresh media at 0.75 x 106 cells ml"1. GeneBloc at 10X final concentration and cationic lipid (25μgml"1) at 10X final concentration were prepared separately in RPMI media (no FCS or glutamine), mixed 1:1 and incubated at 37°C for 30 minutes. 1.6ml aliquots of the cell suspension was dispensed into a 6-well tissue-culture treated plate and 0.4ml of the GeneBloc rlipid mixture added drop-wise. The GeneBloc :lipid solution was evenly distributed by gentle agitation. Following centrifugation at lOOOφm for 60 minutes at room temperature, the 6-well plates were incubated for 24-72 hours at 37°C.
Example 10: Real-time quantitative PCR (Taqman)
Human GRID oligonucleotide Taqman probe 6FAM-(5'- ACTCCAGTTTCCCAAATGGTTTCACGAA-3') (SEQ ID NO 2237) -TAMRA and human actin Taqman probe JOE-(5'-TCGAGCACGGCATCGTCACCAA-3') (SEQ ID NO 2238) -TAMRA were purchased from PE Applied Biosystems. GRID primers (forward, 5'-AGGATATGTGCCCAAGAATTTCATA-3') (SEQ ID NO 2239) and reverse, (5'-TGCCTGGTGTCGAGAGAGG-3') (SEQ ID NO 2240) and actin primers (forward, 5'-GCATGGGTCAGAAGGATTCCTAT-3') (SEQ ID NO 2241) and reverse, (5'-TGTAGAAGGTGTGGTGCCAGATT-3') (SEQ ID NO 2242) were purchased from Life Technologies. The Taqman probes were labeled with a reporter dye (FAM or JOE) at the 5' termini and a quencher dye (TAMRA) at their 3' termini. A combination RT-PCR and Taqman PCR was performed for each sample in triplicate on an ABI PRISM 7700 Sequence Detection System using the following program: 48°C for 30 minutes, 95°C for 10 minutes and then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The reaction was performed in a total volume of 40μl with each tube containing 10U RNase inhibitor (Promega), 1.25U Amplitaq Gold (PE Biosystems), lOOnM of the GRID and Actin primers, lOOnM GRID FAM Taqman probe, lOOnM Actin JOE Taqman probe and 10U MuLV reverse transcriptase. PCR Buffer (PE Biosystems #4304441) and dNTPs (PE Biosystems #N808-0261) were added according to the manufacturer's guidelines. A standard curve was generated using serially diluted purified RNA (300, 100, 33 and l ing) prepared from untreated Jurkat cells.
Example 11 : RNA isolation
Total RNA was isolated from Jurkat J6 or Jurkat E6.1 cells using the 96-well RNeasy kit (Qiagen) and a minor modification of their protocol. 90μl of RLT buffer was added to each sample, followed by an equal volume of 70% ethanol. Samples were mixed and transferred to a RNeasy-96-plate. A vacuum was applied for 15-60sec until the wells were dry. 80μl of lx DNase solution was added (40mM Tris-HCl pH 7.5, lOmM MgCl2, lOmM CaCl2, lOmM NaCl, 1.2U/μl RNase-free DNase I). Following incubation at room temperature for 15 minutes, 1ml of Buffer RW1 was added and incubated for a further 5 minutes. The buffer was removed by applying a vacuum. The wells were washed once in lml of RPE. A second 1ml aliquot of Buffer RPE was added and the RNeasy-96-plate centrifuged at 6000 rpm for 10 minutes. The RNA was eluted by the addition of 100ml of RNase-free water. Following incubation at room temperature for 1 minute, the RNA was recovered by centrifugation at 6000φm for 4 minutes and stored at -70°C.
Indications
Particular conditions and disease states that can be associated with GRID expression modulation include, but are not limited to. tissue/graft rejection and cancer, such as leukemia.
The present body of knowledge in GRID research indicates the need for methods to assay GRID activity and for compounds that can regulate GRID expression for research, diagnostic, and therapeutic use.
Radiation, chemotherapeutic treatments, and Cyclosporin are non-limiting examples of compounds and/or methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g. ribozymes and antisense molecules) of the instant invention. Those skilled in the art will recognize that other dmg compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. ribozymes and antisense molecules) are hence within the scope of the instant invention.
Diagnostic uses
The nucleic acid molecules of this invention (e.g., ribozymes) can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of GRID RNA in a cell. The close relationship between ribozyme activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple ribozymes described in this invention, one can map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments can lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules). Other in vitro uses of ribozymes of this invention include detection of the presence of mRNAs associated with GRID-related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.
In a specific example, ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay. The first ribozyme is used to identify wild-type RNA present in the sample and the second ribozyme is used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both ribozymes to demonstrate the relative ribozyme efficiencies in the reactions and the absence of cleavage of the "non-targeted" RNA species. The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus, each analysis can require two ribozymes, two substrates and one unknown sample, which are combined into six reactions. The presence of cleavage products is determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., GRID) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels is adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.
Additional Uses
Potential usefulness of sequence-specific enzymatic nucleic acid molecules of the instant invention have many of the same applications for the study of RNA that DNA restriction endonucleases have for the study of DNA (Nathans et al, 1975 Ann. Rev. Biochem. 44:273). For example, the pattern of restriction fragments can be used to establish sequence relationships between two related RNAs, and large RNAs can be specifically cleaved to fragments of a size more useful for study. The ability to engineer sequence specificity of the enzymatic nucleic acid molecule is ideal for cleavage of RNAs of unknown sequence. Applicant describes the use of nucleic acid molecules to down- regulate gene expression of target genes in bacterial, microbial, fungal, viral, and eukaryotic systems including plant, or mammalian cells.
All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incoφorated by reference to the same extent as if each reference had been incoφorated by reference in its entirety individually.
One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention, are defined by the scope of the claims.
It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims.
The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.
In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.
Other embodiments are within the following claims. TABLE I
Characteristics of naturally occurring ribozymes
Group I Introns
• Size: -150 to >1000 nucleotides.
• Requires a U in the target sequence immediately 5' of the cleavage site.
• Binds 4-6 nucleotides at the 5'-side of the cleavage site.
• Reaction mechanism: attack by the 3'-OH of guanosine to generate cleavage products with 3' -OH and 5' -guanosine.
• Additional protein cofactors required in some cases to help folding and maintenance of the active structure.
• Over 300 known members of this class. Found as an intervening sequence in Tetrahymena thermophila rRNA, fungal mitochondria, chloroplasts, phage T4, blue-green algae, and others.
• Major structural features largely established through phylogenetic comparisons, mutagenesis, and biochemical studies [v1]-
• Complete kinetic framework established for one ribozyme [ ,ivΛvi]-
• Studies of ribozyme folding and substrate docking underway [vii,vii7x]-
• Chemical modification investigation of important residues well established [*/>].
• The small (4-6 nt) binding site may make this ribozyme too non-specific for targeted RNA cleavage, however, the Tetrahymena group I intron has been used to repair a "defective" beta-galactosidase message by the ligation of new beta-galactosidase sequences onto the defective message
RNAse P RNA (Ml RNA)
• Size: -290 to 400 nucleotides.
• RNA portion of a ubiquitous ribonucleoprotein enzyme.
• Cleaves tRNA precursors to form mature tRNA [xiii].
• Reaction mechanism: possible attack by M 2+ -OH to generate cleavage products with 3'-OH and 5'-phosphate.
• RNAse P is found throughout the prokaryotes and eukaryotes. The RNA subunit has been sequenced from bacteria, yeast, rodents, and primates.
• Recruitment of endogenous RNAse P for therapeutic applications is possible through hybridization of an External Guide Sequence (EGS) to the target RNA [χ*vv]
• Important phosphate and 2' OH contacts recently identified [xvi viij Group II Introns
• Size: >1000 nucleotides.
• Trans cleavage of target RNAs recently demonstrated [x ui^ix].
• Sequence requirements not fully determined.
• Reaction mechanism: 2'-OH of an internal adenosine generates cleavage products with 3'-OH and a "lariat" RNA containing a 3'-5' and a 2'-5' branch point.
• Only natural ribozyme with demonstrated participation in DNA cleavage [xx,xxi] i addition to RNA cleavage and ligation.
• Major structural features largely estabHshed through phylogenetic comparisons [xxii].
• Important 2' OH contacts beginning to be identified [xxiii]
• Kinetic framework under development [xxiv] Neurospora VS RNA
Size: -144 nucleotides.
Trans cleavage of hairpin target RNAs recently demonstrated [ xv].
Sequence requirements not fully determined.
Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2',3'-cyclic phosphate and 5'~OH ends.
Binding sites and structural requirements not fully determined.
Only 1 known member of this class. Found in Neurospora VS RNA.
Hammerhead Ribozyme
(see text for references)
• Size: -13 to 40 nucleotides.
• Requires the target sequence UH immediately 5' of the cleavage site.
• Binds a variable number nucleotides on both sides of the cleavage site.
• Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2' ,3' -cyclic phosphate and 5'-OH ends.
• 14 known members of this class. Found in a number of plant pathogens (virusoids) that use RNA as the infectious agent.
• Essential structural features largely defined, including 2 crystal structures [x vi xvϋ]
• Minimal ligation activity demonstrated (for engineering through in vitro selection) [xxviii]
• Complete kinetic framework established for two or more ribozymes rxxixl
• Chemical modification investigation of important residues well established [xxx]. Hairpin Ribozyme
• Size: -50 nucleotides.
• Requires the target sequence GUC immediately 3' of the cleavage site.
• Binds 4-6 nucleotides at the 5'-side of the cleavage site and a variable number to the 3'-side of the cleavage site.
• Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2',3'-cyclic phosphate and 5'-OH ends.
• 3 known members of this class. Found in three plant pathogen (satellite RNAs of the tobacco ringspot virus, arabis mosaic virus and chicory yellow mottle virus) which uses RNA as the infectious agent.
• Essential structural features largely defined [χχχi,χχχii,χχχiii,χχχiv]
• Ligation activity (in addition to cleavage activity) makes ribozyme amenable to engineering through in vitro selection [xxxv]
• Complete kinetic framework established for one ribozyme [ xxvfj.
• Chemical modification investigation of important residues begun
[xxxvii xxxviiil
Hepatitis Delta Virus (HDV) Ribozyme
• Size: -60 nucleotides.
• Trans cleavage of target RNAs demonstrated [χχχiχ].
• Binding sites and structural requirements not fully determined, although no sequences 5' of cleavage site are required. Folded ribozyme contains a pseudoknot structure [x1].
• Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2',3'-cyclic phosphate and 5'-OH ends.
• Only 2 known members of this class. Found in human HDV. • xliCircular form of HDV isxlii active and shows increased nuclease stability [xi ]
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Table II:
Figure imgf000059_0001
Wait time does not include contact time during delivery. Table III: Human GRID Hammerhead Ribozyme and Substrate Sequence
Pos Substrate SeqID Ribozyme Seq ID
13 GGCACAGU U AAUGGAUC 1 GAUCCAUU CUGAUGAG GCCGUUAGGC CGAA ACUGUGCC 906
14 GCACAGUU A AUGGAUCU 2 AGAUCCAU CUGAUGAG GCCGUUAGGC CGAA AACUGUGC 907
21' UAAUGGAU C UGUAAACU 3 AGUUUACA CUGAUGAG GCCGUUAGGC CGAA AUCCAUUA 908
25 GGAUCUGU A AACUUGCA 4 UGCAAGUU CUGAUGAG GCCGUUAGGC CGAA ACAGAUCC 909
30 UGUAAACU U GCACCCUC 5 GAGGGUGC CUGAUGAG GCCGUUAGGC CGAA AGUUUACA 910
38 UGCACCCU C UUUCAGAG 6 CUCUGAAA CUGAUGAG GCCGUUAGGC CGAA AGGGUGCA 911
40 CACCCUCU U UCAGAGUG 7 CACUCUGA CUGAUGAG GCCGUUAGGC CGAA AGAGGGUG 912
41" ACCCUCUU U CAGAGUGG 8 CCACUCUG CUGAUGAG GCCGUUAGGC CGAA AAGAGGGU 913
42 CCCUCUUU C AGAGUGGU 9 ACCACUCU CUGAUGAG GCCGUUAGGC CGAA AAAGAGGG 914
51 AGAGUGGU A CAUGGAAG 10 CUUCCAUG CUGAUGAG GCCGUUAGGC CGAA ACCACUCU 915
76 AAGUGGAU C CAUACUCU 11 AGAGUAUG CUGAUGAG GCCGUUAGGC CGAA AUCCACUU 916
80 GGAUCCAU A CUCUGAAA 12 UUUCAGAG CUGAUGAG GCCGUUAGGC CGAA AUGGAUCC 917
83 UCCAUACU C UGAAAUGC 13 GCAUUUCA CUGAUGAG GCCGUUAGGC CGAA AGUAUGGA 918
95 AAUGCAGU A ACUCUGAU 14 AUCAGAGU CUGAUGAG GCCGUUAGGC CGAA ACUGCAUU 919
99 CAGUAACU C UGAUGCUU 15 AAGCAUCA CUGAUGAG GCCGUUAGGC CGAA AGUUACUG 920
107 CUGAUGCU U GAAUUUGU 16 ACAAAUUC CUGAUGAG GCCGUUAGGC CGAA AGCAUCAG 921
112 GCUUGAAU U UGUUCUCC 17 GGAGAACA CUGAUGAG GCCGUUAGGC CGAA AUUCAAGC 922
113 CUUGAAUU U GUUCUCCC 18 GGGAGAAC CUGAUGAG GCCGUUAGGC CGAA AAUUCAAG 923
116 GAAUUUGU U CUCCCUUC 19 GAAGGGAG CUGAUGAG GCCGUUAGGC CGAA ACAAAUUC 924
117 AAUUUGUU c UCCCUUCU 20 AGAAGGGA CUGAUGAG GCCGUUAGGC CGAA AACAAAUU 925
119 UUUGUUCU c CCUUCUUG 21 CAAGAAGG CUGAUGAG GCCGUUAGGC CGAA AGAACAAA 926
123 UUCUCCCU u CUUGCCAG 22 CUGGCAAG CUGAUGAG GCCGUUAGGC CGAA AGGGAGAA 927
124 UCUCCCUU c UUGCCAGA 23 UCUGGCAA CUGAUGAG GCCGUUAGGC CGAA AAGGGAGA 928
126 UCCCUUCU u GCCAGAAA 24 UUUCUGGC CUGAUGAG GCCGUUAGGC CGAA AGAAGGGA 929
139 GAAAGGAU u CUAAUAAC 25 GUUAUUAG CUGAUGAG GCCGUUAGGC CGAA AUCCUUUC 930
140 AAAGGAUU c UAAUAACU 26 AGUUAUUA CUGAUGAG GCCGUUAGGC CGAA AAUCCUUU 931
142 AGGAUUCU A AUAACUCG 27 CGAGUUAU CUGAUGAG GCCGUUAGGC CGAA AGAAUCCU 932
145 AUUCUAAU A ACUCGGUG 28 CACCGAGU CUGAUGAG GCCGUUAGGC CGAA AUUAGAAU 933
149 UAAUAACU C GGUGUCAA 29 UUGACACC CUGAUGAG GCCGUUAGGC CGAA AGUUAUUA 934
155 CUCGGUGU C AAAGCCAA 30 UUGGCUUU CUGAUGAG GCCGUUAGGC CGAA ACACCGAG 935
169 CAAGACAU A AACUCAAU 31 AUUGAGUU CUGAUGAG GCCGUUAGGC CGAA AUGUCUUG 936
174 CAUAAACϋ C AAUCUCUU 32 AAGAGAUU CUGAUGAG GCCGUUAGGC CGAA AGUUUAUG 937
178 AACUCAAU C UCUUCUCU 33 AGAGAAGA CUGAUGAG GCCGUUAGGC CGAA AUUGAGUU 938
180 CUCAAUCU C UUCUCUUC 34 GAAGAGAA CUGAUGAG GCCGUUAGGC CGAA AGAUUGAG 939
182 CAAUCUCU U CUCUUCCA 35 UGGAAGAG CUGAUGAG GCCGUUAGGC CGAA AGAGAUUG 940
183 AAUCUCUU C UCUUCCAA 36 UUGGAAGA CUGAUGAG GCCGUUAGGC CGAA AAGAGAUU 941
185 UCUCUUCU C UUCCAAAA 37 UUUUGGAA CUGAUGAG GCCGUUAGGC CGAA AGAAGAGA 942
187 UCUUCUCU u CCAAAAGC 38 GCUUUUGG CUGAUGAG GCCGUUAGGC CGAA AGAGAAGA 943
188 CUUCUCUU C CAAAAGCU 39 AGCUUUUG CUGAUGAG GCCGUUAGGC CGAA AAGAGAAG 944
197 CAAAAGCU u CACGUUAC 40 GUAACGUG CUGAUGAG GCCGUUAGGC CGAA AGCUUUUG 945
198 AAAAGCUU C ACGUUACA 41 UGUAACGU CUGAUGAG GCCGUUAGGC CGAA AAGCUUUU 946
203 CUUCACGU u ACAGCAUG 42 CAUGCUGU CUGAUGAG GCCGUUAGGC CGAA ACGUGAAG 947
204 UUCACGUU A CAGCAUGG 43 CCAUGCUG CUGAUGAG GCCGUUAGGC CGAA AACGUGAA 948
220 GAAGCUGU u GCCAAGUU 44 AACUUGGC CUGAUGAG GCCGUUAGGC CGAA ACAGCUUC 949
228 UGCCAAGU u UGAUUUCA 45 UGAAAUCA CUGAUGAG GCCGUUAGGC CGAA ACUUGGCA 950 229 GCCAAGUU U GAUUUCAC 46 GUGAAAUC CUGAUGAG GCCGUUAGGC CGAA AACUUGGC 951
233 AGUUUGAU U UCACUGCU 47 AGCAGUGA CUGAUGAG GCCGUUAGGC CGAA AUCAAACU 952
234 GUUUGAUU U CACUGCUU 48 AAGCAGUG CUGAUGAG GCCGUUAGGC CGAA AAUCAAAC 953
235 UUUGAUUU C ACUGCUUC 49 GAAGCAGU CUGAUGAG GCCGUUAGGC CGAA AAAUCAAA 954
242 UCACUGCU U CAGGUGAG 50 CUCACCUG CUGAUGAG GCCGUUAGGC CGAA AGCAGUGA 955
243 CACUGCUU C AGGUGAGG 51 CCUCACCU CUGAUGAG GCCGUUAGGC CGAA AAGCAGUG 956
264 ACUGAGCU U UCACACUG 52 CAGUGUGA CUGAUGAG GCCGUUAGGC CGAA AGCUCAGU 957
265 CUGAGCUU U CACACUGG 53 CCAGUGUG CUGAUGAG GCCGUUAGGC CGAA AAGCUCAG 958
266 UGAGCUUU C ACACUGGA 54 UCCAGUGU CUGAUGAG GCCGUUAGGC CGAA AAAGCUCA 959
280 GGAGAUGU U UUGAAGAU 55 AUCUUCAA CUGAUGAG GCCGUUAGGC CGAA ACAUCUCC 960
281 GAGAUGUU U UGAAGAUU 56 AAUCUUCA CUGAUGAG GCCGUUAGGC CGAA AACAUCUC 961
282 AGAUGUUU U GAAGAUUU 57 AAAUCUUC CUGAUGAG GCCGUUAGGC CGAA AAACAUCU 962
289 UUGAAGAU U UUAAGUAA 58 UUACUUAA CUGAUGAG GCCGUUAGGC CGAA AUCUUCAA 963
290 UGAAGAUU U UAAGUAAC 59 GUUACUUA CUGAUGAG GCCGUUAGGC CGAA AAUCUUCA 964
291 GAAGAUUU U AAGUAACC 60 GGUUACUU CUGAUGAG GCCGUUAGGC CGAA AAAUCUUC 965
292 AAGAUUUU A AGUAACCA 61 UGGUUACU CUGAUGAG GCCGUUAGGC CGAA AAAAUCUU 966
296 UUUUAAGU A ACCAAGAG 62 CUCUUGGU CUGAUGAG GCCGUUAGGC CGAA ACUUAAAA 967
312 GGAGUGGU U UAAGGCGG 63 CCGCCUUA CUGAUGAG GCCGUUAGGC CGAA ACCACUCC 968
313 GAGUGGUU U AAGGCGGA 64 UCCGCCUU CUGAUGAG GCCGUUAGGC CGAA AACCACUC 969
314 AGUGGUUU A AGGCGGAG 65 CUCCGCCU CUGAUGAG GCCGUUAGGC CGAA AAACCACU 970
325 GCGGAGCU U GGGAGCCA 66 UGGCUCCC CUGAUGAG GCCGUUAGGC CGAA AGCUCCGC 971
342 GGAAGGAU A UGUGCCCA 67 UGGGCACA CUGAUGAG GCCGUUAGGC CGAA AUCCUUCC 972
356 CCAAGAAU U UCAUAGAC 68 GUCUAUGA CUGAUGAG GCCGUUAGGC CGAA AUUCUUGG 973
357 CAAGAAUU U CAUAGACA 69 UGUCUAUG CUGAUGAG GCCGUUAGGC CGAA AAUUCUUG 974
358 AAGAAUUU C AUAGACAU 70 AUGUCUAU CUGAUGAG GCCGUUAGGC CGAA AAAUUCUU 975
361 AAUUUCAU A GACAUCCA 71 UGGAUGUC CUGAUGAG GCCGUUAGGC CGAA AUGAAAUU 976
367 AUAGACAU C CAGUUUCC 72 GGAAACUG CUGAUGAG GCCGUUAGGC CGAA AUGUCUAU 977
372 CAUCCAGU U UCCCAAAU 73 AUUUGGGA CUGAUGAG GCCGUUAGGC CGAA ACUGGAUG 978
373 AUCCAGUU U CCCAAAUG 74 CAUUUGGG CUGAUGAG GCCGUUAGGC CGAA AACUGGAU 979
374 UCCAGUUU C CCAAAUGG 75 CCAUUUGG CUGAUGAG GCCGUUAGGC CGAA AAACUGGA 980
384 CAAAUGGU U UCACGAAG 76 CUUCGUGA CUGAUGAG GCCGUUAGGC CGAA ACCAUUUG 981
385 AAAUGGUU u CACGAAGG 77 CCUUCGUG CUGAUGAG GCCGUUAGGC CGAA AACCAUUU 982
386 AAUGGUUU C ACGAAGGC 78 GCCUUCGU CUGAUGAG GCCGUUAGGC CGAA AAACCAUU 983
397 GAAGGCCU c UCUCGACA 79 UGUCGAGA CUGAUGAG GCCGUUAGGC CGAA AGGCCUUC 984
399 AGGCCUCU c UCGACACC 80 GGUGUCGA CUGAUGAG GCCGUUAGGC CGAA AGAGGCCU 985
401 GCCUCUCU c GACACCAG 81 CUGGUGUC CUGAUGAG GCCGUUAGGC CGAA AGAGAGGC 986
420 AGAGAACU u ACUCAUGG 82 CCAUGAGU CUGAUGAG GCCGUUAGGC CGAA AGUUCUCU 987
421 GAGAACUU A CUCAUGGG 83 CCCAUGAG CUGAUGAG GCCGUUAGGC CGAA AAGUUCUC 988
424 AACUUACU c AUGGGCAA 84 UUGCCCAU CUGAUGAG GCCGUUAGGC CGAA AGUAAGUU 989
439 AAGGAGGU U GGCUUCUU 85 AAGAAGCC CUGAUGAG GCCGUUAGGC CGAA ACCUCCUU 990
444 GGUUGGCU U CUUCAUCA 86 UGAUGAAG CUGAUGAG GCCGUUAGGC CGAA AGCCAACC 991
445 GUUGGCUU c UUCAUCAU 87 AUGAUGAA CUGAUGAG GCCGUUAGGC CGAA AAGCCAAC 992
447 UGGCUUCU u CAUCAUCC 88 GGAUGAUG CUGAUGAG GCCGUUAGGC CGAA AGAAGCCA 993
448 GGCUUCUU c AUCAUCCG 89 CGGAUGAU CUGAUGAG GCCGUUAGGC CGAA AAGAAGCC 994
451 UUCUUCAU c AUCCGGGC 90 GCCCGGAU CUGAUGAG GCCGUUAGGC CGAA AUGAAGAA 995
454 UUCAUCAU c CGGGCCAG 91 CUGGCCCG CUGAUGAG GCCGUUAGGC CGAA AUGAUGAA 996
471 CCAGAGCU c CCCAGGGG 92 CCCCUGGG CUGAUGAG GCCGUUAGGC CGAA AGCUCUGG 997
483 AGGGGACU u CUCCAUCU 93 AGAUGGAG CUGAUGAG GCCGUUAGGC CGAA AGUCCCCU 998
484 GGGGACUU c UCCAUCUC 94 GAGAUGGA CUGAUGAG GCCGUUAGGC CGAA AAGUCCCC 999 486 GGACUUCU C CAUCUCUG 95 CAGAGAUG CUGAUGAG GCCGUUAGGC CGAA AGAAGUCC 1000
490 UUCUCCAU C UCUGUCAG 96 CUGACAGA CUGAUGAG GCCGUUAGGC CGAA AUGGAGAA 1001
492 CUCCAUCU C UGUCAGGC 97 GCCUGACA CUGAUGAG GCCGUUAGGC CGAA AGAUGGAG 1002
496 AUCUCUGU C AGGCAUGA 98 UCAUGCCU CUGAUGAG GCCGUUAGGC CGAA ACAGAGAU 1003
514 GAUGACGU U CAACACUU 99 AAGUGUUG CUGAUGAG GCCGUUAGGC CGAA ACGUCAUC 1004
515 AUGACGUU C AACACUUC 100 GAAGUGUU CUGAUGAG GCCGUUAGGC CGAA AACGUCAU 1005
522 UCAACACU U CAAGGUCA 101 UGACCUUG CUGAUGAG GCCGUUAGGC CGAA AGUGUUGA 1006
523 CAACACUU C AAGGUCAU 102 AUGACCUU CUGAUGAG GCCGUUAGGC CGAA AAGUGUUG 1007
529 UUCAAGGU C AUGCGAGA 103 UCUCGCAU CUGAUGAG GCCGUUAGGC CGAA ACCUUGAA 1008
548 ACAAGGGU A AUUACUUU 104 AAAGUAAU CUGAUGAG GCCGUUAGGC CGAA ACCCUUGU 1009
551 AGGGUAAU U ACUUUCUG 105 CAGAAAGU CUGAUGAG GCCGUUAGGC CGAA AUUACCCU 1010
552 GGGUAAUU A CUUUCUGU 106 ACAGAAAG CUGAUGAG GCCGUUAGGC CGAA AAUUACCC 1011
555 UAAUUACU U UCUGUGGA 107 UCCACAGA CUGAUGAG GCCGUUAGGC CGAA AGUAAUUA 1012
556 AAUUACUU U CUGUGGAC 108 GUCCACAG CUGAUGAG GCCGUUAGGC CGAA AAGUAAUU 1013
557 AUUACUUU C UGUGGACU 109 AGUCCACA CUGAUGAG GCCGUUAGGC CGAA AAAGUAAU 1014
573 UGAGAAGU U UCCAUCCC 110 GGGAUGGA CUGAUGAG GCCGUUAGGC CGAA ACUUCUCA 1015
574 GAGAAGUU U CCAUCCCU 111 AGGGAUGG CUGAUGAG GCCGUUAGGC CGAA AACUUCUC 1016
575 AGAAGUUU C CAUCCCUA 112 UAGGGAUG CUGAUGAG GCCGUUAGGC CGAA AAACUUCU 1017
579 GUUUCCAU C CCUAAAUA 113 UAUUUAGG CUGAUGAG GCCGUUAGGC CGAA AUGGAAAC 1018
583 CCAUCCCU A AAUAAGCU 114 AGCUUAUU CUGAUGAG GCCGUUAGGC CGAA AGGGAUGG 1019
587 CCCUAAAU A AGCUGGUA 115 UACCAGCU CUGAUGAG GCCGUUAGGC CGAA AUUUAGGG 1020
595 AAGCUGGU A GACUACUA 116 UAGUAGUC CUGAUGAG GCCGUUAGGC CGAA ACCAGCUU 1021
600 GGUAGACU A CUACAGGA 117 UCCUGUAG CUGAUGAG GCCGUUAGGC CGAA AGUCUACC 1022
603 AGACUACU A CAGGACAA 118 UUGUCCUG CUGAUGAG GCCGUUAGGC CGAA AGUAGUCU 1023
614 GGACAAAU U CCAUCUCC 119 GGAGAUGG CUGAUGAG GCCGUUAGGC CGAA AUUUGUCC 1024
615 GACAAAUU C CAUCUCCA 120 UGGAGAUG CUGAUGAG GCCGUUAGGC CGAA AAUUUGUC 1025
619 AAUUCCAU C UCCAGACA 121 UGUCUGGA CUGAUGAG GCCGUUAGGC CGAA AUGGAAUU 1026
621 UUCCAUCU C CAGACAGA 122 UCUGUCUG CUGAUGAG GCCGUUAGGC CGAA AGAUGGAA 1027
637 AAGCAGAU C UUCCUUAG 123 CUAAGGAA CUGAUGAG GCCGUUAGGC CGAA AUCUGCUU 1028
639 GCAGAUCU U CCUUAGAG 124 CUCUAAGG CUGAUGAG GCCGUUAGGC CGAA AGAUCUGC 1029
640 CAGAUCUU C CUUAGAGA 125 UCUCUAAG CUGAUGAG GCCGUUAGGC CGAA AAGAUCUG 1030
643 AUCUUCCU u AGAGACAG 126 CUGUCUCU CUGAUGAG GCCGUUAGGC CGAA AGGAAGAU 1031
644 UCUUCCUU A GAGACAGA 127 UCUGUCUC CUGAUGAG GCCGUUAGGC CGAA AAGGAAGA 1032
671 ACCAGGGU C ACCGGGGC 128 GCCCCGGU CUGAUGAG GCCGUUAGGC CGAA ACCCUGGU 1033
699 CCGGAGGU c CCAGGGAG 129 CUCCCUGG CUGAUGAG GCCGUUAGGC CGAA ACCUCCGG 1034
718 CCACACCU c AGUGGGGC 130 GCCCCACU CUGAUGAG GCCGUUAGGC CGAA AGGUGUGG 1035
742 GAAGAAAU c CGACCUUC 131 GAAGGUCG CUGAUGAG GCCGUUAGGC CGAA AUUUCUUC 1036
749 UCCGACCU u CGAUGAAC 132 GUUCAUCG CUGAUGAG GCCGUUAGGC CGAA AGGUCGGA 1037
750 CCGACCUU c GAUGAACC 133 GGUUCAUC CUGAUGAG GCCGUUAGGC CGAA AAGGUCGG 1038
768 GAAGCUGU c GGAUCACC 134 GGUGAUCC CUGAUGAG GCCGUUAGGC CGAA ACAGCUUC 1039
773 UGUCGGAU c ACCCCCCG 135 CGGGGGGU CUGAUGAG GCCGUUAGGC CGAA AUCCGACA 1040
787 CCGACCCU u CCCCUGCA 136 UGCAGGGG CUGAUGAG GCCGUUAGGC CGAA AGGGUCGG 1041
788 CGACCCUU c CCCUGCAG 137 CUGCAGGG CUGAUGAG GCCGUUAGGC CGAA AAGGGUCG 1042
821 CACAGCCU c CGCAAUAU 138 AUAUUGCG CUGAUGAG GCCGUUAGGC CGAA AGGCUGUG 1043
828 UCCGCAAU A UGCCCCAG 139 CUGGGGCA CUGAUGAG GCCGUUAGGC CGAA AUUGCGGA 1044
873 GCAGCGAU A UCUGCAGC 140 GCUGCAGA CUGAUGAG GCCGUUAGGC CGAA AUCGCUGC 1045
875 AGCGAUAU c UGCAGCAC 141 GUGCUGCA CUGAUGAG GCCGUUAGGC CGAA AUAUCGCU 1046
890 ACCACCAU u UCCACCAG 142 CUGGUGGA CUGAUGAG GCCGUUAGGC CGAA AUGGUGGU 1047
891 CCACCAUU u CCACCAGG 143 CCUGGUGG CUGAUGAG GCCGUUAGGC CGAA AAUGGUGG 1048 892 CACCAUUU C CACCAGGA 144 UCCUGGUG CUGAUGAG GCCGUUAGGC CGAA AAAUGGUG 1049
919 GGCAGCCU U GACAUAAA 145 UUUAUGUC CUGAUGAG GCCGUUAGGC CGAA AGGCUGCC 1050
925 CUUGACAU A AAUGAUGG 146 CCAUCAUU CUGAUGAG GCCGUUAGGC CGAA AUGUCAAG 1051
938 AUGGGCAU U GUGGCACC 147 GGUGCCAC CUGAUGAG GCCGUUAGGC CGAA AUGCCCAU 1052
951 CACCGGCU U GGGCAGUG 148 CACUGCCC CUGAUGAG GCCGUUAGGC CGAA AGCCGGUG 1053
976 GCGGCCCU C AUGCAUCG 149 CGAUGCAU CUGAUGAG GCCGUUAGGC CGAA AGGGCCGC 1054
983 UCAUGCAU C GGAGACAC 150 GUGUCUCC CUGAUGAG GCCGUUAGGC CGAA AUGCAUGA 1055
1009 GUGCAGCϋ C CAGGCGGC 151 GCCGCCUG CUGAUGAG GCCGUUAGGC CGAA AGCUGCAC 1056
1047 GGCGCUGU A UGACUUUG 152 CAAAGUCA CUGAUGAG GCCGUUAGGC CGAA ACAGCGCC 1057
1053 GUAUGACU U UGAGGCCC 153 GGGCCUCA CUGAUGAG GCCGUUAGGC CGAA AGUCAUAC 1058
1054 UAUGACUU U GAGGCCCU 154 AGGGCCUC CUGAUGAG GCCGUUAGGC CGAA AAGUCAUA 1059
1083 GCUGGGGU U CCACAGCG 155 CGCUGUGG CUGAUGAG GCCGUUAGGC CGAA ACCCCAGC 1060
1084 CUGGGGUU C CACAGCGG 156 CCGCUGUG CUGAUGAG GCCGUUAGGC CGAA AACCCCAG 1061
1108 GUGGAGGU C CUGGAUAG 157 CUAUCCAG CUGAUGAG GCCGUUAGGC CGAA ACCUCCAC 1062
1115 UCCUGGAU A GCUCCAAC 158 GUUGGAGC CUGAUGAG GCCGUUAGGC CGAA AUCCAGGA 1063
1119 GGAUAGCU C CAACCCAU 159 AUGGGUUG CUGAUGAG GCCGUUAGGC CGAA AGCUAUCC 1064
1128 CAACCCAU C CUGGUGGA 160 UCCACCAG CUGAUGAG GCCGUUAGGC CGAA AUGGGUUG 1065
1165 CUGGGCCU C UUCCCUGC 161 GCAGGGAA CUGAUGAG GCCGUUAGGC CGAA AGGCCCAG 1066
1167 GGGCCUCU U CCCUGCCA 162 UGGCAGGG CUGAUGAG GCCGUUAGGC CGAA AGAGGCCC 1067
1168 GGCCUCUU c CCUGCCAA 163 UUGGCAGG CUGAUGAG GCCGUUAGGC CGAA AAGAGGCC 1068
1179 UGCCAACU A CGUGGCAC 164 GUGCCACG CUGAUGAG GCCGUUAGGC CGAA AGUUGGCA 1069
1200 GACCCGAU A AACUCUUC 165 GAAGAGUU CUGAUGAG GCCGUUAGGC CGAA AUCGGGUC 1070
1205 GAUAAACU C UUCAGGGG 166 CCCCUGAA CUGAUGAG GCCGUUAGGC CGAA AGUUUAUC 1071
1207 UAAACUCU U CAGGGGAC 167 GUCCCCUG CUGAUGAG GCCGUUAGGC CGAA AGAGUUUA 1072
1208 AAACUCUU C AGGGGACA 168 UGUCCCCU CUGAUGAG GCCGUUAGGC CGAA AAGAGUUU 1073
1223 CAGAAGCU U UUUGUCUG 169 CAGACAAA CUGAUGAG GCCGUUAGGC CGAA AGCUUCUG 1074
1224 AGAAGCUU U UUGUCUGG 170 CCAGACAA CUGAUGAG GCCGUUAGGC CGAA AAGCUUCU 1075
1225 GAAGCUUU U UGUCUGGA 171 UCCAGACA CUGAUGAG GCCGUUAGGC CGAA AAAGCUUC 1076
1226 AAGCUUUU U GUCUGGAG 172 CUCCAGAC CUGAUGAG GCCGUUAGGC CGAA AAAAGCUU 1077
1229 CUUUUUGU C UGGAGCUG 173 CAGCUCCA CUGAUGAG GCCGUUAGGC CGAA ACAAAAAG 1078
1274 GCUGGACU C CAUGACUA 174 UAGUCAUG CUGAUGAG GCCGUUAGGC CGAA AGUCCAGC 1079
1282 CCAUGACU A UAUAUACA 175 UGUAUAUA CUGAUGAG GCCGUUAGGC CGAA AGUCAUGG 1080
1284 AUGACUAU A UAUACAUA 176 UAUGUAUA CUGAUGAG GCCGUUAGGC CGAA AUAGUCAU 1081
1286 GACUAUAU A UACAUACA 177 UGUAUGUA CUGAUGAG GCCGUUAGGC CGAA AUAUAGUC 1082
1288 CUAUAUAU A CAUACAUC 178 GAUGUAUG CUGAUGAG GCCGUUAGGC CGAA AUAUAUAG 1083
1292 AUAUACAU A CAUCUAUC 179 GAUAGAUG CUGAUGAG GCCGUUAGGC CGAA AUGUAUAU 1084
Input Sequence = HSA011736. Cut Site = UH/ .
Stem Length = 8 . Core Sequence = CUGAUGAG GCCGUUAGGC CGAA
HSA011736 (Homo sapiens mRNA for growth factor receptor binding protein (GRBLG) ; 1303 bp)
Underlined region can be any X sequence or linker as defined herein. Table IV: Human GRID NCH Ribozyme and Substrate Sequence
Pos Substrate Se ID Ribozyme SeqID
10 GGAGGCAC A GUUAAUGG 180 CCAUUAAC CUGAUGAG GCCGUUAGGC CGAA IUGCCUCC 1085
22 AAUGGAUC U GUAAACUU 181 AAGUUUAC CUGAUGAG GCCGUUAGGC CGAA IAUCCAUU 1086
29 CUGUAAAC U UGCACCCU 182 AGGGUGCA CUGAUGAG GCCGUUAGGC CGAA IUUUACAG 1087
33 AAACUUGC A CCCUCUUU 183 AAAGAGGG CUGAUGAG GCCGUUAGGC CGAA ICAAGUUU 1088
35 ACUUGCAC C CUCUUUCA 184 UGAAAGAG CUGAUGAG GCCGUUAGGC CGAA IUGCAAGU 1089
36 CUUGCACC C UCUUUCAG 185 CUGAAAGA CUGAUGAG GCCGUUAGGC CGAA IGUGCAAG 1090
37 UUGCACCC U CUUUCAGA 186 UCUGAAAG CUGAUGAG GCCGUUAGGC CGAA IGGUGCAA 1091
39 GCACCCUC U UUCAGAGU 187 ACUCUGAA CUGAUGAG GCCGUUAGGC CGAA IAGGGUGC 1092
43 CCUCUUUC A GAGUGGUA 188 UACCACUC CUGAUGAG GCCGUUAGGC CGAA. IAAAGAGG 1093
53 AGUGGUAC A UGGAAGAC 189 GUCUUCCA CUGAUGAG GCCGUUAGGC CGAA IUACCACU 1094
62 UGGAAGAC A GCACAAAG 190 CUUUGUGC CUGAUGAG GCCGUUAGGC CGAA IUCUUCCA 1095
65 AAGACAGC A CAAAGUGG 191 CCACUUUG CUGAUGAG GCCGUUAGGC CGAA ICUGUCUU 1096
67 GACAGCAC A AAGUGGAU 192 AUCCACUU CUGAUGAG GCCGUUAGGC CGAA IUGCUGUC 1097
77 AGUGGAUC C AUACUCUG 193 CAGAGUAU CUGAUGAG GCCGUUAGGC CGAA IAUCCACU 1098
78 GUGGAUCC A UACUCUGA 194 UCAGAGUA CUGAUGAG GCCGUUAGGC CGAA IGAUCCAC 1099
82 AUCCAUAC U CUGAAAUG 195 CAUUUCAG CUGAUGAG GCCGUUAGGC CGAA IUAUGGAU 1100
84 CCAUACUC U GAAAUGCA 196 UGCAUUUC CUGAUGAG GCCGUUAGGC CGAA IAGUAUGG 1101
92 UGAAAUGC A GUAACUCU 197 AGAGUUAC CUGAUGAG GCCGUUAGGC CGAA ICAUUUCA 1102
98 GCAGUAAC U CUGAUGCU 198 AGCAUCAG CUGAUGAG GCCGUUAGGC CGAA IUUACUGC 1103
100 AGUAACUC U GAUGCUUG 199 CAAGCAUC CUGAUGAG GCCGUUAGGC CGAA IAGUUACU 1104
106 UCUGAUGC u UGAAUUUG 200 CAAAUUCA CUGAUGAG GCCGUUAGGC CGAA ICAUCAGA 1105
118 AUUUGUUC u CCCUUCUU 201 AAGAAGGG CUGAUGAG GCCGUUAGGC CGAA lAACAAAU 1106
120 UUGUUCUC c CUUCUUGC 202 GCAAGAAG CUGAUGAG GCCGUUAGGC CGAA lAGAACAA 1107
121 UGUUCUCC c UUCUUGCC 203 GGCAAGAA CUGAUGAG GCCGUUAGGC CGAA IGAGAACA 1108
122 GUUCUCCC u UCUUGCCA 204 UGGCAAGA CUGAUGAG GCCGUUAGGC CGAA IGGAGAAC 1109
125 CUCCCUUC u UGCCAGAA 205 UUCUGGCA CUGAUGAG GCCGUUAGGC CGAA IAAGGGAG 1110
129 CUUCUUGC c AGAAAGGA 206 UCCUUUCU CUGAUGAG GCCGUUAGGC CGAA ICAAGAAG 1111
130 UUCUUGCC A GAAAGGAU 207 AUCCUUUC CUGAUGAG GCCGUUAGGC CGAA IGCAAGAA 1112
141 AAGGAUUC U AAUAACUC 208 GAGUUAUU CUGAUGAG GCCGUUAGGC CGAA IAAUCCUU 1113
148 CUAAUAAC U CGGUGUCA 209 UGACACCG CUGAUGAG GCCGUUAGGC CGAA IUUAUUAG 1114
156 UCGGUGUC A AAGCCAAG 210 CUUGGCUU CUGAUGAG GCCGUUAGGC CGAA IACACCGA 1115
161 GUCAAAGC C AAGACAUA 211 UAUGUCUU CUGAUGAG GCCGUUAGGC CGAA ICUUUGAC 1116
162 UCAAAGCC A AGACAUAA 212 UUAUGUCU CUGAUGAG GCCGUUAGGC CGAA IGCUUUGA 1117
167 GCCAAGAC A UAAACUCA 213 UGAGUUUA CUGAUGAG GCCGUUAGGC CGAA IUCUUGGC 1118
173 ACAUAAAC U CAAUCUCU 214 AGAGAUUG' CUGAUGAG GCCGUUAGGC CGAA lUUUAUGU 1119
175 AUAAACUC A AUCUCUUC 215 GAAGAGAU CUGAUGAG GCCGUUAGGC CGAA IAGUUUAU 1120
179 ACUCAAUC U CUUCUCUU 216 AAGAGAAG CUGAUGAG GCCGUUAGGC CGAA IAUUGAGU 1121
181 UCAAUCUC U UCUCUUCC 217 GGAAGAGA CUGAUGAG GCCGUUAGGC CGAA IAGAUUGA 1122
184 AUCUCUUC u CUUCCAAA 218 UUUGGAAG CUGAUGAG GCCGUUAGGC CGAA IAAGAGAU 1123
186 CUCUUCUC u UCCAAAAG 219 CUUUUGGA CUGAUGAG GCCGUUAGGC CGAA IAGAAGAG 1124
189 UUCUCUUC c AAAAGCUU 220 AAGCUUUU CUGAUGAG GCCGUUAGGC CGAA IAAGAGAA 1125
190 UCUCUUCC A AAAGCUUC 221 GAAGCUUU CUGAUGAG GCCGUUAGGC CGAA IGAAGAGA 1126
196 CCAAAAGC U UCACGUUA 222 UAACGUGA CUGAUGAG GCCGUUAGGC CGAA ICUUUUGG 1127
199 AAAGCUUC A CGUUACAG 223 CUGUAACG CUGAUGAG GCCGUUAGGC CGAA IAAGCUUU 1128
206 CACGUUAC A GCAUGGAA 224 UUCCAUGC CUGAUGAG GCCGUUAGGC CGAA IUAACGUG 1129
209 GUUACAGC A UGGAAGCU 225 AGCUUCCA CUGAUGAG GCCGUUAGGC CGAA ICUGUAAC 1130
217 AUGGAAGC U GUUGCCAA 226 UUGGCAAC CUGAUGAG GCCGUUAGGC CGAA ICUUCCAU 1131
223 GCUGUUGC c AAGUUUGA 227 UCAAACUU CUGAUGAG GCCGUUAGGC CGAA ICAACAGC 1132
224 CUGUUGCC A AGUUUGAU 228 AUCAAACU CUGAUGAG GCCGUUAGGC CGAA IGCAACAG 1133
236 UUGAUUUC A CUGCUUCA 229 UGAAGCAG CUGAUGAG GCCGUUAGGC CGAA IAAAUCAA 1134 238 GAUUUCAC U GCUUCAGG 230 CCUGAAGC CUGAUGAG GCCGUUAGGC CGAA IUGAAAUC 1135
241 UUCACUGC u UCAGGUGA 231 UCACCUGA CUGAUGAG GCCGUUAGGC CGAA ICAGUGAA 1136
244 ACUGCUUC A GGUGAGGA 232 UCCUCACC CUGAUGAG GCCGUUAGGC CGAA IAAGCAGU 1137
258 GGAUGAAC U GAGCUUUC 233 GAAAGCUC CUGAUGAG GCCGUUAGGC CGAA IUUCAUCC 1138
263 AACUGAGC U UUCACACU 234 AGUGUGAA CUGAUGAG GCCGUUAGGC CGAA ICUCAGUU 1139
267 GAGCUUUC A CACUGGAG 235 CUCCAGUG CUGAUGAG GCCGUUAGGC CGAA IAAAGCUC 1140
269 GCUUUCAC A CUGGAGAU 236 AUCUCCAG CUGAUGAG GCCGUUAGGC CGAA lUGAAAGC 1141
271 UUUCACAC U GGAGAUGU 237 ACAUCUCC CUGAUGAG GCCGUUAGGC CGAA lUGUGAAA 1142
299 UAAGUAAC C AAGAGGAG 238 CUCCUCUU CUGAUGAG GCCGUUAGGC CGAA IUUACUUA 1143
300 AAGUAACC A AGAGGAGU 239 ACUCCUCU CUGAUGAG GCCGUUAGGC CGAA IGUUACUU 1144
324 GGCGGAGC U UGGGAGCC 240 GGCUCCCA CUGAUGAG GCCGUUAGGC CGAA ICUCCGCC 1145
332 UUGGGAGC C AGGAAGGA 241 UCCUUCCU CUGAUGAG GCCGUUAGGC CGAA ICUCCCAA 1146
333 UGGGAGCC A GGAAGGAU 242 AUCCUUCC CUGAUGAG GCCGUUAGGC CGAA IGCUCCCA 1147
348 AUAUGUGC C CAAGAAUU 243 AAUUCUUG CUGAUGAG GCCGUUAGGC CGAA ICAC UAU 1148
349 UAUGUGCC C AAGAAUUU 244 AAAUUCUU CUGAUGAG GCCGUUAGGC CGAA IGCACAUA 1149
350 AUGUGCCC A AGAAUUUC 245 GAAAUUCU CUGAUGAG GCCGUUAGGC CGAA IGGCACAU 1150
359 AGAAUUUC A UAGACAUC 246 GAUGUCUA CUGAUGAG GCCGUUAGGC CGAA IAAAUUCU 1151
365 UCAUAGAC A UCCAGUUU 247 AAACUGGA CUGAUGAG GCCGUUAGGC CGAA IUCUAUGA 1152
368 UAGACAUC C AGUUUCCC 248 GGGAAACU CUGAUGAG GCCGUUAGGC CGAA IAUGUCUA 1153
369 AGACAUCC A GUUUCCCA 249 UGGGAAAC CUGAUGAG GCCGUUAGGC CGAA IGAUGUCU 1154
375 CCAGUUUC C CAAAUGGU 250 ACCAUUUG CUGAUGAG GCCGUUAGGC CGAA IAAACUGG 1155
376 CAGUUUCC C AAAUGGUU 251 AACCAUUU CUGAUGAG GCCGUUAGGC CGAA IGAAACUG 1156
377 AGUUUCCC A AAUGGUUU 252 AAACCAUU CUGAUGAG GCCGUUAGGC CGAA IGGAAACU 1157
387 AUGGUUUC A CGAAGGCC 253 GGCCUUCG CUGAUGAG GCCGUUAGGC CGAA IAAACCAU 1158
395 ACGAAGGC C UCUCUCGA 254 UCGAGAGA CUGAUGAG GCCGUUAGGC CGAA ICCUUCGU 1159
396 CGAAGGCC U CUCUCGAC 255 GUCGAGAG CUGAUGAG GCCGUUAGGC CGAA IGCCUUCG 1160
398 AAGGCCUC U CUCGACAC 256 GUGUCGAG CUGAUGAG GCCGUUAGGC CGAA IAGGCCUU 1161
400 GGCCUCUC U CGACACCA 257 UGGUGUCG CUGAUGAG GCCGUUAGGC CGAA IAGAGGCC 1162
405 CUCUCGAC A CCAGGCAG 258 CUGCCUGG CUGAUGAG GCCGUUAGGC CGAA IUCGAGAG 1163
407 CUCGACAC C AGGCAGAG 259 CUCUGCCU CUGAUGAG GCCGUUAGGC CGAA IUGUCGAG 1164
408 UCGACACC A GGCAGAGA 260 UCUCUGCC CUGAUGAG GCCGUUAGGC CGAA IGUGUCGA 1165
412 CACCAGGC A GAGAACUU 261 AAGUUCUC CUGAUGAG GCCGUUAGGC CGAA ICCUGGUG 1166
419 CAGAGAAC U UACUCAUG 262 CAUGAGUA CUGAUGAG GCCGUUAGGC CGAA IUUCUCUG 1167
423 GAACUUAC u CAUGGGCA 263 UGCCCAUG CUGAUGAG GCCGUUAGGC CGAA IUAAGUUC 1168
425 ACUUACUC A UGGGCAAG 264 CUUGCCCA CUGAUGAG GCCGUUAGGC CGAA IAGUAAGU 1169
431 UCAUGGGC A AGGAGGUU 265 AACCUCCU CUGAUGAG GCCGUUAGGC CGAA ICCCAUGA 1170
443 AGGUUGGC U UCUUCAUC 266 GAUGAAGA CUGAUGAG GCCGUUAGGC CGAA ICCAACCU 1171
446 UUGGCUUC U UCAUCAUC 267 GAUGAUGA CUGAUGAG GCCGUUAGGC CGAA lAAGCCAA 1172
449 GCUUCUUC A UCAUCCGG 268 CCGGAUGA CUGAUGAG GCCGUUAGGC CGAA IAAGAAGC 1173
452 UCUUCAUC A UCCGGGCC 269 GGCCCGGA CUGAUGAG GCCGUUAGGC CGAA IAUGAAGA 1174
455 UCAUCAUC C GGGCCAGC 270 GCUGGCCC CUGAUGAG GCCGUUAGGC CGAA IAUGAUGA 1175
460 AUCCGGGC C AGCCAGAG 271 CUCUGGCU CUGAUGAG GCCGUUAGGC CGAA ICCCGGAU 1176
461 UCCGGGCC A GCCAGAGC 272 GCUCUGGC CUGAUGAG GCCGUUAGGC CGAA IGCCCGGA 1177
464 GGGCCAGC C AGAGCUCC 273 GGAGCUCU CUGAUGAG GCCGUUAGGC CGAA ICUGGCCC 1178
465 GGCCAGCC A GAGCUCCC 274 GGGAGCUC CUGAUGAG GCCGUUAGGC CGAA IGCUGGCC 1179
470 GCCAGAGC U CCCCAGGG 275 CCCUGGGG CUGAUGAG GCCGUUAGGC CGAA ICUCUGGC 1180
472 CAGAGCUC C CCAGGGGA 276 UCCCCUGG CUGAUGAG GCCGUUAGGC CGAA IAGCUCUG 1181
473 AGAGCUCC C CAGGGGAC 277 GUCCCCUG CUGAUGAG GCCGUUAGGC CGAA IGAGCUCU 1182
474 GAGCUCCC C AGGGGACU 278 AGUCCCCU CUGAUGAG GCCGUUAGGC CGAA IGGAGCUC 1183
475 AGCUCCCC A GGGGACUU 279 AAGUCCCC CUGAUGAG GCCGUUAGGC CGAA IGGGAGCU 1184
482 CAGGGGAC U UCUCCAUC 280 GAUGGAGA CUGAUGAG GCCGUUAGGC CGAA IUCCCCUG 1185
485 GGGACUUC U CCAUCUCU 281 AGAGAUGG CUGAUGAG GCCGUUAGGC CGAA IAAGUCCC 1186
487 GACUUCUC c AUCUCUGU 282 ACAGAGAU CUGAUGAG GCCGUUAGGC CGAA IAGAAGUC 1187
488 ACUUCUCC A UCUCUGUC 283 GACAGAGA CUGAUGAG GCCGUUAGGC CGAA IGAGAAGU 1188 491 UCUCCAUC U CUGUCAGG 284 CCUGACAG CUGAUGAG GCCGUUAGGC CGAA IAUGGAGA 1189
493 UCCAUCUC U GUCAGGCA 285 UGCCUGAC CUGAUGAG GCCGUUAGGC CGAA IAGAUGGA 1190
497 UCUCUGUC A GGCAUGAG 286 CUCAUGCC CUGAUGAG GCCGUUAGGC CGAA IACAGAGA 1191
501 UGUCAGGC A UGAGGAUG 287 CAUCCUCA CUGAUGAG GCCGUUAGGC CGAA ICCUGACA 1192
516 UGACGUUC A ACACUUCA 288 UGAAGUGU CUGAUGAG GCCGUUAGGC CGAA lAACGUCA 1193
519 CGUUCAAC A CUUCAAGG 289 CCUUGAAG CUGAUGAG GCCGUUAGGC CGAA IUUGAACG 1194
521 UUCAACAC U UCAAGGUC 290 GACCUUGA CUGAUGAG GCCGUUAGGC CGAA IUGUUGAA 1195
524 AACACUUC A AGGUCAUG 291 CAUGACCU CUGAUGAG GCCGUUAGGC CGAA IAAGUGUU 1196
530 UCAAGGUC A UGCGAGAC 292 GUCUCGCA CUGAUGAG GCCGUUAGGC CGAA IACCUUGA 1197
539 UGCGAGAC A ACAAGGGU 293 ACCCUUGU CUGAUGAG GCCGUUAGGC CGAA lUCUCGCA 1198
542 GAGACAAC A AGGGUAAU 294 AUUACCCU CUGAUGAG GCCGUUAGGC CGAA IUUGUCUC 1199
554 GUAAUUAC U UUCUGUGG 295 CCACAGAA CUGAUGAG GCCGUUAGGC CGAA IUAAUUAC 1200
558 UUACUUUC U GUGGACUG 296 CAGUCCAC CUGAUGAG GCCGUUAGGC CGAA IAAAGUAA 1201
565 CUGUGGAC U GAGAAGUU 297 AACUUCUC CUGAUGAG GCCGUUAGGC CGAA IUCCACAG 1202
576 GAAGUUUC C AUCCCUAA 298 UUAGGGAU CUGAUGAG GCCGUUAGGC CGAA IAAACUUC 1203
577 AAGUUUCC A UCCCUAAA 299 UUUAGGGA CUGAUGAG GCCGUUAGGC CGAA IGAAACUU 1204
580 UUUCCAUC C CUAAAUAA 300 UUAUUUAG CUGAUGAG GCCGUUAGGC CGAA lAUGGAAA 1205
581 UUCCAUCC C UAAAUAAG 301 CUUAUUUA CUGAUGAG GCCGUUAGGC CGAA IGAUGGAA 1206
582 UCCAUCCC U AAAUAAGC 302 GCUUAUUU CUGAUGAG GCCGUUAGGC CGAA IGGAUGGA 1207
591 AAAUAAGC U GGUAGACU 303 AGUCUACC CUGAUGAG GCCGUUAGGC CGAA ICUUAUUU 1208
599 UGGUAGAC U ACUACAGG 304 CCUGUAGU CUGAUGAG GCCGUUAGGC CGAA IUCUACCA 1209
602 UAGACUAC U ACAGGACA 305 UGUCCUGU CUGAUGAG GCCGUUAGGC CGAA IUAGUCUA 1210
605 ACUACUAC A GGACAAAU 306 AUUUGUCC CUGAUGAG GCCGUUAGGC CGAA IUAGUAGU 1211
610 UACAGGAC A AAUUCCAU 307 AUGGAAUU CUGAUGAG GCCGUUAGGC CGAA IUCCUGUA 1212
616 ACAAAUUC C AUCUCCAG 308 CUGGAGAU CUGAUGAG GCCGUUAGGC CGAA IAAUUUGU 1213
617 CAAAUUCC A UCUCCAGA 309 UCUGGAGA CUGAUGAG GCCGUUAGGC CGAA IGAAUUUG 1214
620 AUUCCAUC U CCAGACAG 310 CUGUCUGG CUGAUGAG GCCGUUAGGC CGAA IAUGGAAU 1215
622 UCCAUCUC C AGACAGAA 311 UUCUGUCU CUGAUGAG GCCGUUAGGC CGAA IAGAUGGA 1216
623 CCAUCUCC A GACAGAAG 312 CUUCUGUC CUGAUGAG GCCGUUAGGC CGAA IGAGAUGG 1217
627 CUCCAGAC A GAAGCAGA 313 UCUGCUUC CUGAUGAG GCCGUUAGGC CGAA IUCUGGAG 1218
633 ACAGAAGC A GAUCUUCC 314 GGAAGAUC CUGAUGAG GCCGUUAGGC CGAA ICUUCUGU 1219
638 AGCAGAUC U UCCUUAGA 315 UCUAAGGA CUGAUGAG GCCGUUAGGC CGAA IAUCUGCU 1220
641 AGAUCUUC C UUAGAGAC 316 GUCUCUAA CUGAUGAG GCCGUUAGGC CGAA IAAGAUCU 1221
642 GAUCUUCC U UAGAGACA 317 UGUCUCUA CUGAUGAG GCCGUUAGGC CGAA IGAAGAUC 1222
650 UUAGAGAC A GAACCCGA 318 UCGGGUUC CUGAUGAG GCCGUUAGGC CGAA IUCUCUAA 1223
655 GACAGAAC C CGAGAAGA 319 UCUUCUCG CUGAUGAG GCCGUUAGGC CGAA IUUCUGUC 1224
656 ACAGAACC C GAGAAGAC 320 GUCUUCUC CUGAUGAG GCCGUUAGGC CGAA IGUUCUGU 1225
665 GAGAAGAC C AGGGUCAC 321 GUGACCCU CUGAUGAG GCCGUUAGGC CGAA IUCUUCUC 1226
666 AGAAGACC A GGGUCACC 322 GGUGACCC CUGAUGAG GCCGUUAGGC CGAA IGUCUUCU 1227
672 CCAGGGUC A CCGGGGCA 323 UGCCCCGG CUGAUGAG GCCGUUAGGC CGAA IACCCUGG 1228
674 AGGGUCAC C GGGGCAAC 324 GUUGCCCC CUGAUGAG GCCGUUAGGC CGAA IUGACCCU 1229
680 ACCGGGGC A ACAGCCUG 325 CAGGCUGU CUGAUGAG GCCGUUAGGC CGAA ICCCCGGU 1230
683 GGGGCAAC A GCCUGGAC 326 GUCCAGGC CUGAUGAG GCCGUUAGGC CGAA IUUGCCCC 1231
686 GCAACAGC C UGGACCGG 327 CCGGUCCA CUGAUGAG GCCGUUAGGC CGAA ICUGUUGC 1232
687 CAACAGCC U GGACCGGA 328 UCCGGUCC CUGAUGAG GCCGUUAGGC CGAA IGCUGUUG 1233
692 GCCUGGAC C GGAGGUCC 329 GGACCUCC CUGAUGAG GCCGUUAGGC CGAA IUCCAGGC 1234
700 CGGAGGUC C CAGGGAGG 330 CCUCCCUG CUGAUGAG GCCGUUAGGC CGAA IACCUCCG 1235
701 GGAGGUCC C AGGGAGGC 331 GCCUCCCU CUGAUGAG GCCGUUAGGC CGAA IGACCUCC 1236
702 GAGGUCCC A GGGAGGCC 332 GGCCUCCC CUGAUGAG GCCGUUAGGC CGAA IGGACCUC 1237
710 AGGGAGGC C CACACCUC 333 GAGGUGUG CUGAUGAG GCCGUUAGGC CGAA ICCUCCCU 1238
711 GGGAGGCC C ACACCUCA 334 UGAGGUGU CUGAUGAG GCCGUUAGGC CGAA IGCCUCCC 1239
712 GGAGGCCC A CACCUCAG 335 CUGAGGUG CUGAUGAG GCCGUUAGGC CGAA IGGCCUCC 1240
714 AGGCCCAC A CCUCAGUG 336 CACUGAGG CUGAUGAG GCCGUUAGGC CGAA IUGGGCCU 1241
716 GCCCACAC C UCAGUGGG 337 CCCACUGA CUGAUGAG GCCGUUAGGC CGAA IUGUGGGC 1242 717 CCCACACC U CAGUGGGG 338 CCCCACUG CUGAUGAG GCCGUUAGGC CGAA IGUGUGGG 1243
719 CACACCUC A GUGGGGCU 339 AGCCCCAC CUGAUGAG GCCGUUAGGC CGAA lAGGUGUG 1244
727 AGUGGGGC U GUGGGAGA 340 UCUCCCAC CUGAUGAG GCCGUUAGGC CGAA ICCCCACU 1245
743 AAGAAAUC C GACCUUCG 341 CGAAGGUC CUGAUGAG GCCGUUAGGC CGAA IAUUUCUU 1246
747 AAUCCGAC C UUCGAUGA 342 UCAUCGAA CUGAUGAG GCCGUUAGGC CGAA lUCGGAUU 1247
748 AUCCGACC U UCGAUGAA 343 UUCAUCGA CUGAUGAG GCCGUUAGGC CGAA IGUCGGAU 1248
758 CGAUGAAC C GGAAGCUG 344 CAGCUUCC CUGAUGAG GCCGUUAGGC CGAA IUUCAUCG 1249
765 CCGGAAGC U GUCGGAUC 345 GAUCCGAC CUGAUGAG GCCGUUAGGC CGAA ICUUCCGG 1250
774 GUCGGAUC A CCCCCCGA 346 UCGGGGGG CUGAUGAG GCCGUUAGGC CGAA IAUCCGAC 1251
776 CGGAUCAC C CCCCGACC 347 GGUCGGGG CUGAUGAG GCCGUUAGGC CGAA IUGAUCCG 1252
111 GGAUCACC C CCCGACCC 348 GGGUCGGG CUGAUGAG GCCGUUAGGC CGAA IGUGAUCC 1253
778 GAUCACCC C CCGACCCU 349 AGGGUCGG CUGAUGAG GCCGUUAGGC CGAA IGGUGAUC 1254
779 AUCACCCC C CGACCCUU 350 AAGGGUCG CUGAUGAG GCCGUUAGGC CGAA IGGGUGAU 1255
780 UCACCCCC C GACCCUUC 351 GAAGGGUC CUGAUGAG GCCGUUAGGC CGAA IGGGGUGA 1256
784 CCCCCGAC C CUUCCCCU 352 AGGGGAAG CUGAUGAG GCCGUUAGGC CGAA IUCGGGGG 1257
785 CCCCGACC C UUCCCCUG 353 CAGGGGAA CUGAUGAG GCCGUUAGGC CGAA IGUCGGGG 1258
786 CCCGACCC u UCCCCUGC 354 GCAGGGGA CUGAUGAG GCCGUUAGGC CGAA IGGUCGGG 1259
789 GACCCUUC c CCUGCAGC 355 GCUGCAGG CUGAUGAG GCCGUUAGGC CGAA IAAGGGUC 1260
790 ACCCUUCC c CUGCAGCA 356 UGCUGCAG CUGAUGAG GCCGUUAGGC CGAA IGAAGGGU 1261
791 CCCUUCCC c UGCAGCAG 357 CUGCUGCA CUGAUGAG GCCGUUAGGC CGAA IGGAAGGG 1262
792 CCUUCCCC u GCAGCAGC 358 GCUGCUGC CUGAUGAG GCCGUUAGGC CGAA IGGGAAGG 1263
795 UCCCCUGC A GCAGCACC 359 GGUGCUGC CUGAUGAG GCCGUUAGGC CGAA ICAGGGGA 1264
798 CCUGCAGC A GCACCAGC 360 GCUGGUGC CUGAUGAG GCCGUUAGGC CGAA ICUGCAGG 1265
801 GCAGCAGC A CCAGCACC 361 GGUGCUGG CUGAUGAG GCCGUUAGGC CGAA ICUGCUGC 1266
803 AGCAGCAC C AGCACCAG 362 CUGGUGCU CUGAUGAG GCCGUUAGGC CGAA IUGCUGCU 1267
804 GCAGCACC A GCACCAGC 363 GCUGGUGC CUGAUGAG GCCGUUAGGC CGAA IGUGCUGC 1268
807 GCACCAGC A CCAGCCAC 364 GUGGCUGG CUGAUGAG GCCGUUAGGC CGAA ICUGGUGC 1269
809 ACCAGCAC C AGCCACAG 365 CUGUGGCU CUGAUGAG GCCGUUAGGC CGAA IUGCUGGU 1270
810 CCAGCACC A GCCACAGC 366 GCUGUGGC CUGAUGAG GCCGUUAGGC CGAA IGUGCUGG 1271
813 GCACCAGC C ACAGCCUC 367 GAGGCUGU CUGAUGAG GCCGUUAGGC CGAA ICUGGUGC 1272
814 CACCAGCC A CAGCCUCC 368 GGAGGCUG CUGAUGAG GCCGUUAGGC CGAA IGCUGGUG 1273
816 CCAGCCAC A GCCUCCGC 369 GCGGAGGC CUGAUGAG GCCGUUAGGC CGAA IUGGCUGG 1274
819 GCCACAGC C UCCGCAAU 370 AUUGCGGA CUGAUGAG GCCGUUAGGC CGAA ICUGUGGC 1275
820 CCACAGCC U CCGCAAUA 371 UAUUGCGG CUGAUGAG GCCGUUAGGC CGAA IGCUGUGG 1276
822 ACAGCCUC C GCAAUAUG 372 CAUAUUGC CUGAUGAG GCCGUUAGGC CGAA IAGGCUGU 1277
825 GCCUCCGC A AUAUGCCC 373 GGGCAUAU CUGAUGAG GCCGUUAGGC CGAA ICGGAGGC 1278
832 CAAUAUGC C CCAGCGCC 374 GGCGCUGG CUGAUGAG GCCGUUAGGC CGAA ICAUAUUG 1279
833 AAUAUGCC C CAGCGCCC 375 GGGCGCUG CUGAUGAG GCCGUUAGGC CGAA IGCAUAUU 1280
834 AUAUGCCC C AGCGCCCC 376 GGGGCGCU CUGAUGAG GCCGUUAGGC CGAA IGGCAUAU 1281
835 UAUGCCCC A GCGCCCCA 377 UGGGGCGC CUGAUGAG GCCGUUAGGC CGAA IGGGCAUA 1282
840 CCCAGCGC C CCAGCAGC 378 GCUGCUGG CUGAUGAG GCCGUUAGGC CGAA ICGCUGGG 1283
841 CCAGCGCC C CAGCAGCU 379 AGCUGCUG CUGAUGAG GCCGUUAGGC CGAA IGCGCUGG 1284
842 CAGCGCCC C AGCAGCUG 380 CAGCUGCU CUGAUGAG GCCGUUAGGC CGAA IGGCGCUG 1285
843 AGCGCCCC A GCAGCUGC 381 GCAGCUGC CUGAUGAG GCCGUUAGGC CGAA IGGGCGCU 1286
846 GCCCCAGC A GCUGCAGC 382 GCUGCAGC CUGAUGAG GCCGUUAGGC CGAA ICUGGGGC 1287
849 GCAGCAGC U GCAGCAGC 383 GCUGCUGC CUGAUGAG GCCGUUAGGC CGAA ICUGCUGG 1288
852 GCAGCUGC A GCAGCCCC 384 GGGGCUGC CUGAUGAG GCCGUUAGGC CGAA ICAGCUGC 1289
855 GCUGCAGC A GCCCCCAC 385 GUGGGGGC CUGAUGAG GCCGUUAGGC CGAA ICUGCAGC 1290
858 GCAGCAGC C CCCACAGC 386 GCUGUGGG CUGAUGAG GCCGUUAGGC CGAA ICUGCUGC 1291
859 CAGCAGCC C CCACAGCA 387 UGCUGUGG CUGAUGAG GCCGUUAGGC CGAA IGCUGCUG 1292
860 AGCAGCCC C CACAGCAG 388 CUGCUGUG CUGAUGAG GCCGUUAGGC CGAA IGGCUGCU 1293
861 GCAGCCCC C ACAGCAGC 389 GCUGCUGU CUGAUGAG GCCGUUAGGC CGAA IGGGCUGC 1294
862 CAGCCCCC A CAGCAGCG 390 CGCUGCUG CUGAUGAG GCCGUUAGGC CGAA IGGGGCUG 1295
1 864 GCCCCCAC A GCAGCGAU 391 AUCGCUGC CUGAUGAG GCCGUUAGGC CGAA IUGGGGGC 1296 867 CCCACAGC A GCGAUAUC 392 GAUAUCGC CUGAUGAG GCCGUUAGGC CGAA ICUGUGGG 1297
876 GCGAUAUC U GCAGCACC 393 GGUGCUGC CUGAUGAG GCCGUUAGGC CGAA IAUAUCGC 1298
879 AUAUCUGC A GCACCACC 394 GGUGGUGC CUGAUGAG GCCGUUAGGC CGAA ICAGAUAU 1299
882 UCUGCAGC A CCACCAUU 395 AAUGGUGG CUGAUGAG GCCGUUAGGC CGAA ICUGCAGA 1300
884 UGCAGCAC C ACCAUUUC 396 GAAAUGGU CUGAUGAG GCCGUUAGGC CGAA IUGCUGCA 1301
885 GCAGCACC A CCAUUUCC 397 GGAAAUGG CUGAUGAG GCCGUUAGGC CGAA IGUGCUGC 1302
887 AGCACCAC C AUUUCCAC 398 GUGGAAAU CUGAUGAG GCCGUUAGGC CGAA IUGGUGCU 1303
888 GCACCACC A UUUCCACC 399 GGUGGAAA CUGAUGAG GCCGUUAGGC CGAA IGUGGUGC 1304
893 ACCAUUUC C ACCAGGAA 400 UUCCUGGU CUGAUGAG GCCGUUAGGC CGAA IAAAUGGU 1305
894 CCAUUUCC A CCAGGAAC 401 GUUCCUGG CUGAUGAG GCCGUUAGGC CGAA IGAAAUGG 1306
896 AUUUCCAC C AGGAACGC 402 GCGUUCCU CUGAUGAG GCCGUUAGGC CGAA IUGGAAAU 1307
897 UUUCCACC A GGAACGCC 403 GGCGUUCC CUGAUGAG GCCGUUAGGC CGAA IGUGGAAA 1308
905 AGGAACGC C GAGGAGGC 404 GCCUCCUC CUGAUGAG GCCGUUAGGC CGAA ICGUUCCU 1309
914 GAGGAGGC A GCCUUGAC 405 GUCAAGGC CUGAUGAG GCCGUUAGGC CGAA ICCUCCUC 1310
917 GAGGCAGC C UUGACAUA 406 UAUGUCAA CUGAUGAG GCCGUUAGGC CGAA ICUGCCUC 1311
918 AGGCAGCC U UGACAUAA 407 UUAUGUCA CUGAUGAG GCCGUUAGGC CGAA IGCUGCCU 1312
923 GCCUUGAC A UAAAUGAU 408 AUCAUUUA CUGAUGAG GCCGUUAGGC CGAA IUCAAGGC 1313
936 UGAUGGGC A UUGUGGCA 409 UGCCACAA CUGAUGAG GCCGUUAGGC CGAA ICCCAUCA 1314
944 AUUGUGGC A CCGGCUUG 410 CAAGCCGG CUGAUGAG GCCGUUAGGC CGAA ICCACAAU 1315
946 UGUGGCAC C GGCUUGGG 411 CCCAAGCC CUGAUGAG GCCGUUAGGC CGAA IUGCCACA 1316
950 GCACCGGC U UGGGCAGU 412 ACUGCCCA CUGAUGAG GCCGUUAGGC CGAA ICCGGUGC 1317
956 GCUUGGGC A GUGAAAUG 413 CAUUUCAC CUGAUGAG GCCGUUAGGC CGAA ICCCAAGC 1318
973 AAUGCGGC C CUCAUGCA 414 UGCAUGAG CUGAUGAG GCCGUUAGGC CGAA ICCGCAUU 1319
974 AUGCGGCC C UCAUGCAU 415 AUGCAUGA CUGAUGAG GCCGUUAGGC CGAA IGCCGCAU 1320
975 UGCGGCCC U CAUGCAUC 416 GAUGCAUG CUGAUGAG GCCGUUAGGC CGAA IGGCCGCA 1321
977 CGGCCCUC A UGCAUCGG 417 CCGAUGCA CUGAUGAG GCCGUUAGGC CGAA IAGGGCCG 1322
981 CCUCAUGC A UCGGAGAC 418 GUCUCCGA CUGAUGAG GCCGUUAGGC CGAA ICAUGAGG 1323
990 UCGGAGAC A CACAGACC 419 GGUCUGUG CUGAUGAG GCCGUUAGGC CGAA IUCUCCGA 1324
992 GGAGACAC A CAGACCCA 420 UGGGUCUG CUGAUGAG GCCGUUAGGC CGAA IUGUCUCC 1325
994 AGACACAC A GACCCAGU 421 ACUGGGUC CUGAUGAG GCCGUUAGGC CGAA lUGUGUCU 1326
998 ACACAGAC C CAGUGCAG 422 CUGCACUG CUGAUGAG GCCGUUAGGC CGAA IUCUGUGU 1327
999 CACAGACC C AGUGCAGC 423 GCUGCACU CUGAUGAG GCCGUUAGGC CGAA IGUCUGUG 1328
1000 ACAGACCC A GUGCAGCU .424 AGCUGCAC CUGAUGAG GCCGUUAGGC CGAA IGGUCUGU 1329
1005 CCCAGUGC A GCUCCAGG 425 CCUGGAGC CUGAUGAG GCCGUUAGGC CGAA ICACUGGG 1330
1008 AGUGCAGC U CCAGGCGG 426 CCGCCUGG CUGAUGAG GCCGUUAGGC CGAA ICUGCACU 1331
1010 UGCAGCUC C AGGCGGCA 427 UGCCGCCU CUGAUGAG GCCGUUAGGC CGAA IAGCUGCA 1332
1011 GCAGCUCC A GGCGGCAG 428 CUGCCGCC CUGAUGAG GCCGUUAGGC CGAA IGAGCUGC 1333
1018 CAGGCGGC A GGGCGAGU 429 ACUCGCCC CUGAUGAG GCCGUUAGGC CGAA ICCGCCUG 1334
1036 CGGUGGGC C CGGGCGCU 430 AGCGCCCG CUGAUGAG GCCGUUAGGC CGAA ICCCACCG 1335
1037 GGUGGGCC C GGGCGCUG 431 CAGCGCCC CUGAUGAG GCCGUUAGGC CGAA IGCCCACC 1336
1044 CCGGGCGC U GUAUGACU 432 AGUCAUAC CUGAUGAG GCCGUUAGGC CGAA ICGCCCGG 1337
1052 UGUAUGAC U UUGAGGCC 433 GGCCUCAA CUGAUGAG GCCGUUAGGC CGAA IUCAUACA 1338
1060 UUUGAGGC C CUGGAGGA 434 UCCUCCAG CUGAUGAG GCCGUUAGGC CGAA ICCUCAAA 1339
1061 UUGAGGCC C UGGAGGAU 435 AUCCUCCA CUGAUGAG GCCGUUAGGC CGAA IGCCUCAA 1340
1062 UGAGGCCC U GGAGGAUG 436 CAUCCUCC CUGAUGAG GCCGUUAGGC CGAA IGGCCUCA 1341
1077 UGACGAGC U GGGGUUCC 437 GGAACCCC CUGAUGAG GCCGUUAGGC CGAA ICUCGUCA 1342
1085 UGGGGUUC C ACAGCGGG 438 CCCGCUGU CUGAUGAG GCCGUUAGGC CGAA IAACCCCA 1343
1086 GGGGUUCC A CAGCGGGG 439 CCCCGCUG CUGAUGAG GCCGUUAGGC CGAA IGAACCCC 1344
1088 GGUUCCAC A GCGGGGAG 440 CUCCCCGC CUGAUGAG GCCGUUAGGC CGAA IUGGAACC 1345
1109 UGGAGGUC C UGGAUAGC 441 GCUAUCCA CUGAUGAG GCCGUUAGGC CGAA IACCUCCA 1346
1110 GGAGGUCC U GGAUAGCU 442 AGCUAUCC CUGAUGAG GCCGUUAGGC CGAA IGACCUCC 1347
1118 UGGAUAGC U CCAACCCA 443 UGGGUUGG CUGAUGAG GCCGUUAGGC CGAA ICUAUCCA 1348
1120 GAUAGCUC C AACCCAUC 444 •GAUGGGUU CUGAUGAG GCCGUUAGGC CGAA IAGCUAUC 1349
1121 AUAGCUCC A ACCCAUCC 445 GGAUGGGU CUGAUGAG GCCGUUAGGC CGAA IGAGCUAU 1350 1124 GCUCCAAC C CAUCCUGG 446 CCAGGAUG CUGAUGAG GCCGUUAGGC CGAA IUUGGAGC 1351
1125 CUCCAACC C AUCCUGGU 447 ACCAGGAU CUGAUGAG GCCGUUAGGC CGAA IGUUGGAG 1352
1126 UCCAACCC A UCCUGGUG 448 CACCAGGA CUGAUGAG GCCGUUAGGC CGAA IGGUUGGA 1353
1129 AACCCAUC C UGGUGGAC 449 GUCCACCA CUGAUGAG GCCGUUAGGC CGAA IAUGGGUU 1354
1130 ACCCAUCC U GGUGGACC 450 GGUCCACC CUGAUGAG GCCGUUAGGC CGAA IGAUGGGU 1355
1138 UGGUGGAC C GGCCGCCU 451 AGGCGGCC CUGAUGAG GCCGUUAGGC CGAA IUCCACCA 1356
1142 GGACCGGC C GCCUGCAC 452 GUGCAGGC CUGAUGAG GCCGUUAGGC CGAA ICCGGUCC 1357
1145 CCGGCCGC C UGCACAAC 453 GUUGUGCA CUGAUGAG GCCGUUAGGC CGAA ICGGCCGG 1358
1146 CGGCCGCC U GCACAACA 454 UGUUGUGC CUGAUGAG GCCGUUAGGC CGAA IGCGGCCG 1359
1149 CCGCCUGC A CAACAAGC 455 GCUUGUUG CUGAUGAG GCCGUUAGGC CGAA ICAGGCGG 1360
1151 GCCUGGAC A ACAAGCUG 456 CAGCUUGU CUGAUGAG GCCGUUAGGC CGAA IUGCAGGC 1361
1154 UGCACAAC A AGCUGGGC 457 GCCCAGCU CUGAUGAG GCCGUUAGGC CGAA IUUGUGCA 1362
1158 CAACAAGC U GGGCCUCU 458 AGAGGCCC CUGAUGAG GCCGUUAGGC CGAA ICUUGUUG 1363
1163 AGCUGGGC C UCUUCCCU 459 AGGGAAGA CUGAUGAG GCCGUUAGGC CGAA ICCCAGCU 1364
1164 GCUGGGCC U CUUCCCUG 460 CAGGGAAG CUGAUGAG GCCGUUAGGC CGAA IGCCCAGC 1365
1166 UGGGCCUC U UCCCUGCC 461 GGCAGGGA CUGAUGAG GCCGUUAGGC CGAA IAGGCCCA 1366
1169 GCCUCUUC C CUGCCAAC 462 GUUGGCAG CUGAUGAG GCCGUUAGGC CGAA IAAGAGGC 1367
1170 CCUCUUCC C UGCCAACU 463 AGUUGGCA CUGAUGAG GCCGUUAGGC CGAA IGAAGAGG 1368
1171 CUCUUCCC U GCCAACUA 464 UAGUUGGC CUGAUGAG GCCGUUAGGC CGAA IGGAAGAG 1369
1174 UUCCCUGC C AACUACGU 465 ACGUAGUU CUGAUGAG GCCGUUAGGC CGAA ICAGGGAA 1370
1175 UCCCUGCC A ACUACGUG 466 CACGUAGU CUGAUGAG GCCGUUAGGC CGAA IGCAGGGA 1371
1178 CUGCCAAC U ACGUGGCA 467 UGCCACGU CUGAUGAG GCCGUUAGGC CGAA IUUGGCAG 1372
1186 UACGUGGC A CCCAUGAC 468 GUCAUGGG CUGAUGAG GCCGUUAGGC CGAA ICCACGUA 1373
1188 CGUGGCAC C CAUGACCC 469 GGGUCAUG CUGAUGAG GCCGUUAGGC CGAA IUGCCACG 1374
1189 GUGGCACC C AUGACCCG 470 CGGGUCAU CUGAUGAG GCCGUUAGGC CGAA IGUGCCAC 1375
1190 UGGCACCC A UGACCCGA 471 UCGGGUCA CUGAUGAG GCCGUUAGGC CGAA IGGUGCCA 1376
1195 CCCAUGAC C CGAUAAAC 472 GUUUAUCG CUGAUGAG GCCGUUAGGC CGAA IUCAUGGG 1377
1196 CCAUGACC C GAUAAACU 473 AGUUUAUC CUGAUGAG GCCGUUAGGC CGAA IGUCAUGG 1378
1204 CGAUAAAC u CUUCAGGG 474 CCCUGAAG CUGAUGAG GCCGUUAGGC CGAA IUUUAUCG 1379
1206 AUAAACUC u UCAGGGGA 475 UCCCCUGA CUGAUGAG GCCGUUAGGC CGAA IAGUUUAU 1380
1209 AACUCUUC A GGGGACAG 476 CUGUCCCC CUGAUGAG GCCGUUAGGC CGAA IAAGAGUU 1381
1216 CAGGGGAC A GAAGCUUU 477 AAAGCUUC CUGAUGAG GCCGUUAGGC CGAA IUCCCCUG 1382
1222 ACAGAAGC U UUUUGUCU 478 AGACAAAA CUGAUGAG GCCGUUAGGC CGAA ICUUCUGU 1383
1230 UUUUUGUC U GGAGCUGC 479 GCAGCUCC CUGAUGAG GCCGUUAGGC CGAA IACAAAAA 1384
1236 UCUGGAGC U GCCCACAA 480 UUGUGGGC CUGAUGAG GCCGUUAGGC CGAA ICUCCAGA 1385
1239 GGAGCUGC C CACAAGAA 481 UUCUUGUG CUGAUGAG GCCGUUAGGC CGAA ICAGCUCC 1386
1240 GAGCUGCC C ACAAGAAA 482 UUUCUUGU CUGAUGAG GCCGUUAGGC CGAA IGCAGCUC 1387
1241 AGCUGCCC A CAAGAAAG 483 CUUUCUUG CUGAUGAG GCCGUUAGGC CGAA IGGCAGCU 1388
1243 CUGCCCAC A AGAAAGAG 484 CUCUUUCU CUGAUGAG GCCGUUAGGC CGAA IUGGGCAG 1389
1255 AAGAGGGC A AGGAAAAA 485 UUUUUCCU CUGAUGAG GCCGUUAGGC CGAA ICCCUCUU 1390
1268 AAAAAGGC U GGACUCCA 486 UGGAGUCC CUGAUGAG GCCGUUAGGC CGAA ICCUUUUU 1391
1273 GGCUGGAC U CCAUGACU 487 AGUCAUGG CUGAUGAG GCCGUUAGGC CGAA IUCCAGCC 1392
1275 CUGGACUC c AUGACUAU 488 AUAGUCAU CUGAUGAG GCCGUUAGGC CGAA IAGUCCAG 1393
1276 UGGACUCC A UGACUAUA 489 UAUAGUCA CUGAUGAG GCCGUUAGGC CGAA IGAGUCCA 1394
1281 UCCAUGAC U AUAUAUAC 490 GUAUAUAU CUGAUGAG GCCGUUAGGC CGAA IUCAUGGA 1395
1290 AUAUAUAC A UACAUCUA 491 UAGAUGUA CUGAUGAG GCCGUUAGGC CGAA IUAUAUAU 1396
11294 AUACAUAC A UCUAUCUA 492 UAGAUAGA CUGAUGAG GCCGUUAGGC CGAA I AUGUAU 1397
Input Sequence = HSA011736. Cut Site = CH/ . Stem Length = 8 . Core Sequence = CUGAUGAG GCCGUUAGGC CGAA
HSA011736 (Homo sapiens mRNA for growth factor receptor binding protein (GRBLG) ; 1303 bp)
Underlined region can be any X sequence or linker as defined herein.
I = Inosine Table V: Human GRID G-cleaver Ribozyme and Substrate Sequence
Pos Substrate SeqID Ribozyme SeqID
31 GUAAACUU G CACCCUCU 493 AGAGGGUG UGAUG GCAUGCACUAUGC GCG AAGUUUAC 1398
85 CAUACUCU G AAAUGCAG 494 CUGCAUUU UGAUG GCAUGCACUAUGC GCG AGAGUAUG 1399
90 UCUGAAAU G CAGUAACU 495 AGUUACUG UGAUG GCAUGCACUAUGC GCG AUUUCAGA 1400
101 GUAACUCU G AUGCUUGA 496 UCAAGCAU UGAUG GCAUGCACUAUGC GCG AGAGUUAC 1401
104 ACUCUGAU G CUUGAAUU 497 AAUUCAAG UGAUG GCAUGCACUAUGC GCG AUCAGAGU 1402
108 UGAUGCUU G AAUUUGUU 498 AACAAAUU UGAUG GCAUGCACUAUGC GCG AA'GCAUCA 1403
127 CCCUUCUU G CCAGAAAG 499 CUUUCUGG UGAUG GCAUGCACUAUGC GCG AAGAAGGG 1404
221 AAGCUGUU G CCAAGUUU 500 AAACUUGG UGAUG GCAUGCACUAUGC GCG AACAGCUU 1405
230 CCAAGUUU G AUUUCACU 501 AGUGAAAU UGAUG GCAUGCACUAUGC GCG AAACUUGG 1406
239 AUUUCACU G CUUCAGGU 502 ACCUGAAG UGAUG GCAUGCACUAUGC GCG AGUGAAAU 1407
248 CUUCAGGU G AGGAUGAA 503 UUCAUCCU UGAUG GCAUGCACUAUGC GCG ACCUGAAG 1408
254 GUGAGGAU G AACUGAGC 504 GCUCAGUU UGAUG GCAUGCACUAUGC GCG AUCCUCAC 1409
259 GAUGAACU G AGCUUUCA 505 UGAAAGCU UGAUG GCAUGCACUAUGC GCG AGUUCAUC 1410
283 GAUGUUUU G AAGAUUUU 506 AAAAUCUU UGAUG GCAUGCACUAUGC GCG AAAACAUC 1411
346 GGAUAUGU G CCCAAGAA 507 UUCUUGGG UGAUG GCAUGCACUAUGC GCG ACAUAUCC 1412
389 GGUUUCAC G AAGGCCUC 508 GAGGCCUU UGAUG GCAUGCACUAUGC GCG GUGAAACC 1413
402 CCUCUCUC G ACACCAGG 509 CCUGGUGU UGAUG GCAUGCACUAUGC GCG GAGAGAGG 1414
503 UCAGGCAU G AGGAUGAC 510 GUCAUCCU UGAUG GCAUGCACUAUGC GCG AUGCCUGA 1415
509 AUGAGGAU G ACGUUCAA 511 UUGAACGU UGAUG GCAUGCACUAUGC GCG AUCCUCAU 1416
532 AAGGUCAU G CGAGACAA 512 UUGUCUCG UGAUG GCAUGCACUAUGC GCG AUGACCUU 1417
534 GGUCAUGC G AGACAACA 513 UGUUGUCU UGAUG GCAUGCACUAUGC GCG GCAUGACC 1418
566 UGUGGACU G AGAAGUUU 514 AAACUUCU UGAUG GCAUGCACUAUGC GCG AGUCCACA 1419
657 CAGAACCC G AGAAGACC 515 GGUCUUCU UGAUG GCAUGCACUAUGC GCG GGGUUCUG 1420
744 AGAAAUCC G ACCUUCGA 516 UCGAAGGU UGAUG GCAUGCACUAUGC GCG GGAUUUCU 1421
751 CGACCUUC G AUGAACCG 517 CGGUUCAU UGAUG GCAUGCACUAUGC GCG GAAGGUCG 1422
754 CCUUCGAU G AACCGGAA 518 UUCCGGUU UGAUG GCAUGCACUAUGC GCG AUCGAAGG 1423
781 CACCCCCC G ACCCUUCC 519 GGAAGGGU UGAUG GCAUGCACUAUGC GCG GGGGGGUG 1424
793 CUUCCCCU G CAGCAGCA 520 UGCUGCUG UGAUG GCAUGCACUAUGC GCG AGGGGAAG 1425
823 CAGCCUCC G CAAUAUGC 521 GCAUAUUG UGAUG GCAUGCACUAUGC GCG GGAGGCUG 1426
830 CGCAAUAU G CCCCAGCG 522 CGCUGGGG UGAUG GCAUGCACUAUGC GCG AUAUUGCG 1427
838 GCCCCAGC G CCCCAGCA 523 UGCUGGGG UGAUG GCAUGCACUAUGC GCG GCUGGGGC 1428
850 CAGCAGCU G CAGCAGCC 524 GGCUGCUG UGAUG GCAUGCACUAUGC GCG AGCUGCUG 1429
870 ACAGCAGC G AUAUCUGC 525 GCAGAUAU UGAUG GCAUGCACUAUGC GCG GCUGCUGU 1430
877 CGAUAUCU G CAGCACCA 526 UGGUGCUG UGAUG GCAUGCACUAUGC GCG AGAUAUCG 1431
903 CCAGGAAC G CCGAGGAG 527 CUCCUCGG UGAUG GCAUGCACUAUGC GCG GUUCCUGG 1432
906 GGAACGCC G AGGAGGCA 528 UGCCUCCU UGAUG GCAUGCACUAUGC GCG GGCGUUCC 1433
920 GCAGCCUU G ACAUAAAU 529 AUUUAUGU UGAUG GCAUGCACUAUGC GCG AAGGCUGC 1434
929 ACAUAAAU G AUGGGCAU 530 AUGCCCAU UGAUG GCAUGCACUAUGC GCG AUUUAUGU 1435
959 UGGGCAGU G AAAUGAAU 531 AUUCAUUU UGAUG GCAUGCACUAUGC GCG ACUGCCCA 1436
964 AGUGAAAU G AAUGCGGC 532 GCCGCAUU UGAUG GCAUGCACUAUGC GCG AUUUCACU 1437
968 AAAUGAAU G CGGCCCUC 533 GAGGGCCG UGAUG GCAUGCACUAUGC GCG AUUCAUUU 1438
979 GCCCUCAU G CAUCGGAG 534 CUCCGAUG UGAUG GCAUGCACUAUGC GCG AUGAGGGC 1439
1003 GACCCAGU G CAGCUCCA 535 UGGAGCUG UGAUG GCAUGCACUAUGC GCG ACUGGGUC 1440
1023 GGCAGGGC G AGUGCGGU 536 ACCGCACU UGAUG GCAUGCACUAUGC GCG GCCCUGCC 1441
1027 GGGCGAGU G CGGUGGGC 537 GCCCACCG UGAUG GCAUGCACUAUGC GCG ACUCGCCC 1442
1042 GCCCGGGC G CUGUAUGA 538 UCAUACAG UGAUG GCAUGCACUAUGC GCG GCCCGGGC 1443
1049 CGCUGUAU G ACUUUGAG 539 CUCAAAGU UGAUG GCAUGCACUAUGC GCG AUACAGCG 1444
1055 AUGACUUU G AGGCCCUG 540 CAGGGCCU UGAUG GCAUGCACUAUGC GCG AAAGUCAU 1445
1070 UGGAGGAU G ACGAGCUG 541 CAGCUCGU UGAUG GCAUGCACUAUGC GCG AUCCUCCA 1446
1073 AGGAUGAC G AGCUGGGG 542 CCCCAGCU UGAUG GCAUGCACUAUGC GCG GUCAUCCU 1447
Figure imgf000071_0001
Input Sequence = HSA011736. Cut Site = YG/M or UG/U. Stem Length = 8. Core Sequence = UGAUG GCAUGCACUAUGC GCG
HSA011736 (Homo sapiens mRNA for growth factor receptor binding protein (GRBLG) ; 1303 bp)
Table VI: Human GRID Zinzyme and Substrate Sequence
Pos Substrate SeqID Zinzyme SeqID
11 GAGGCACA G UUAAUGGA 550 UCCAUUAA GCCGAAAGGCGAGUCAAGGUCU UGUGCCUC 1455
23 AUGGAUCU G UAAACUUG 551 CAAGUUUA GCCGAAAGGCGAGUCAAGGUCU AGAUCCAU 1456
31 GUAAACUU G CACCCUCU 493 AGAGGGUG GCCGAAAGGCGAGUCAAGGUCU AAGUUUAC 1457
46 CUUUCAGA G UGGUACAU 552 AUGUACC GCCGAAAGGCGAGUCAAGGUCU UCUGAAAG 1458
49 UCAGAGUG G UACAUGGA 553 UCCAUGUA GCCGAAAGGCGAGUCAAGGUCU CACUCUGA 1459
63 GGAAGACA G CACAAAGU 554 ACUUUGUG GCCGAAAGGCGAGUCAAGGUCU UGUCUUCC 1460
70 AGCACAAA G UGGAUCCA 555 UGGAUCCA GCCGAAAGGCGAGUCAAGGUCU UUUGUGCU 1461
90 UCUGAAAU G CAGUAACU 495 AGUUACUG GCCGAAAGGCGAGUCAAGGUCU AUUUCAGA 1462
93 GAAAUGCA G UAACUCUG 556 CAGAGUUA GCCGAAAGGCGAGUCAAGGUCU UGCAUUUC 1463
104 ACUCUGAU G CUUGAAUU 497 AAUUCAAG GCCGAAAGGCGAGUCAAGGUCU AUCAGAGU 1464
114 UUGAAUUU G UUCUCCCU 557 AGGGAGAA GCCGAAAGGCGAGUCAAGGUCU AAAUUCAA 1465
127 CCCUUCUU G CCAGAAAG 499 CUUUCUGG GCCGAAAGGCGAGUCAAGGUCU AAGAAGGG 1466
151 . AUAACUCG G UGUCAAAG 558 CUUUGACA GCCGAAAGGCGAGUCAAGGUCU CGAGUUAU 1467
153 AACUCGGU G UCAAAGCC 559 GGCUUUGA GCCGAAAGGCGAGUCAAGGUCU ACCGAGUU 1468
159 GUGUCAAA G CCAAGACA 5S0 UGUCUUGG GCCGAAAGGCGAGUCAAGGUCU UUUGACAC 1469
194 UUCCAAAA G CUUCACGU 561 ACGUGAAG GCCGAAAGGCGAGUCAAGGUCU UUUUGGAA 1470
201 AGCUUCAC G UUACAGCA 562 UGCUGUAA GCCGAAAGGCGAGUCAAGGUCU GUGAAGCU 1471
207 ACGUUACA G CAUGGAAG 563 CUUCCAUG GCCGAAAGGCGAGUCAAGGUCU UGUAACGU 1472
215 GCAUGGAA G CUGUUGCC 564 GGCAACAG GCCGAAAGGCGAGUCAAGGUCU UUCCAUGC 1473
218 UGGAAGCU G UUGCCAAG 565 CUUGGCAA GCCGAAAGGCGAGUCAAGGUCU AGCUUCCA 1474
221 AAGCUGUU G CCAAGUUU 500 AAACUUGG GCCGAAAGGCGAGUCAAGGUCU AACAGCUU 1475
226 GUUGCCAA G UUUGAUUU 566 AAAUCAAA GCCGAAAGGCGAGUCAAGGUCU UUGGCAAC 1476
239 AUUUCACU G CUUCAGGU 502 ACCUGAAG GCCGAAAGGCGAGUCAAGGUCU AGUGAAAU 1477
246 UGCUUCAG G UGAGGAUG 567 CAUCCUCA GCCGAAAGGCGAGUCAAGGUCU CUGAAGCA 1478
261 UGAACUGA G CUUUCACA 568 UGUGAAAG GCCGAAAGGCGAGUCAAGGUCU UCAGUUCA 1479
278 CUGGAGAU G UUUUGAAG 569 CUUCAAAA GCCGAAAGGCGAGUCAAGGUCU AUCUCCAG 1480
294 GAUUUUAA G UAACCAAG 570 CUUGGUUA GCCGAAAGGCGAGUCAAGGUCU UUAAAAUC 1481
307 CAAGAGGA G UGGUUUAA 571 UUAAACCA GCCGAAAGGCGAGUCAAGGUCU UCCUCUUG 1482
310 GAGGAGUG G UUUAAGGC 572 GCCUUAAA GCCGAAAGGCGAGUCAAGGUCU CACUCCUC 1483
317 GGUUUAAG G CGGAGCUU 573 AAGCUCCG GCCGAAAGGCGAGUCAAGGUCU CUUAAACC 1484
322 AAGGCGGA G CUUGGGAG 574 CUCCCAAG GCCGAAAGGCGAGUCAAGGUCU UCCGCCUU 1485
330 GCUUGGGA G CCAGGAAG 575 CUUCCUGG GCCGAAAGGCGAGUCAAGGUCU UCCCAAGC 1486
344 AAGGAUAU G UGCCCAAG 576 CUUGGGCA GCCGAAAGGCGAGUCAAGGUCU AUAUCCUU 1487
346 GGAUAUGU G CCCAAGAA 507 UUCUUGGG GCCGAAAGGCGAGUCAAGGUCU ACAUAUCC 1488
370 GACAUCCA G UUUCCCAA 577 UUGGGAAA GCCGAAAGGCGAGUCAAGGUCU UGGAUGUC 1489
382 CCCAAAUG G UUUCACGA 578 UCGUGAAA GCCGAAAGGCGAGUCAAGGUCU CAUUUGGG 1490
393 UCACGAAG G CCUCUCUC 579 GAGAGAGG GCCGAAAGGCGAGUCAAGGUCU CUUCGUGA 1491
410 GACACCAG G CAGAGAAC 580 GUUCUCUG GCCGAAAGGCGAGUCAAGGUCU CUGGUGUC 1492
429 ACUCAUGG G CAAGGAGG 581 CCUCCUUG GCCGAAAGGCGAGUCAAGGUCU CCAUGAGU 1493
437 GCAAGGAG G UUGGCUUC 582 GAAGCCAA GCCGAAAGGCGAGUCAAGGUCU CUCCUUGC 1494
441 GGAGGUUG G CUUCUUCA 583 UGAAGAAG GCCGAAAGGCGAGUCAAGGUCU CAACCUCC 1495
458 UCAUCCGG G CCAGCCAG 584 CUGGCUGG GCCGAAAGGCGAGUCAAGGUCU CCGGAUGA 1496
462 CCGGGCCA G CCAGAGCU 585 AGCUCUGG GCCGAAAGGCGAGUCAAGGUCU UGGCCCGG 1497
468 CAGCCAGA G CUCCCCAG 586 CUGGGGAG GCCGAAAGGCGAGUCAAGGUCU UCUGGCUG 1498
494 CCAUCUCU G UCAGGCAU 587 AUGCCUGA GCCGAAAGGCGAGUCAAGGUCU AGAGAUGG 1499
499 UCUGUCAG G CAUGAGGA 588 UCCUCAUG GCCGAAAGGCGAGUCAAGGUCU CUGACAGA 1500
512 AGGAUGAC G UUCAACAC 589 GUGUUGAA GCCGAAAGGCGAGUCAAGGUCU GUCAUCCU 1501
527 ACUUCAAG G UCAUGCGA 590 UCGCAUGA GCCGAAAGGCGAGUCAAGGUCU CUUGAAGU 1502
532 AAGGUCAU G CGAGACAA 512 UUGUCUCG GCCGAAAGGCGAGUCAAGGUCU AUGACCUU 1503
546 CAACAAGG G UAAUUACU 591 AGUAAUUA GCCGAAAGGCGAGUCAAGGUCU CCUUGUUG 1504
559 UACUUUCU G UGGACUGA 592 UCAGUCCA GCCGAAAGGCGAGUCAAGGUCU AGAAAGUA 1505 571 ACUGAGAA G UUUCCAUC 593 GAUGGAAA GCCGAAAGGCGAGUCAAGGUCU UUCUCAGU 1506
589 CUAAAUAA G CUGGUAGA 594 UCUACCAG GCCGAAAGGCGAGUCAAGGUCU UUAUUUAG 1507
593 AUAAGCUG G UAGACUAC 595 GUAGUCUA GCCGAAAGGCGAGUCAAGGUCU CAGCUUAU 1508
631 AGACAGAA G CAGAUCUU 596 AAGAUCUG GCCGAAAGGCGAGUCAAGGUCU UUCUGUCU 1509
669 AGACCAGG G UCACCGGG 597 CCCGGUGA GCCGAAAGGCGAGUCAAGGUCU CCUGGUCU 1510
678 UCACCGGG G CAACAGCC 598 GGCUGUUG GCCGAAAGGCGAGUCAAGGUCU CCCGGUGA 1511
684 GGGCAACA G CCUGGACC 599 GGUCCAGG GCCGAAAGGCGAGUCAAGGUCU UGUUGCCC 1512
697 GACCGGAG G UCCCAGGG 600 CCCUGGGA GCCGAAAGGCGAGUCAAGGUCU CUCCGGUC 1513
708 CCAGGGAG G CCCACACC 601 GGUGUGGG GCCGAAAGGCGAGUCAAGGUCU CUCCCUGG 1514
720 ACACCUCA G UGGGGCUG 602 CAGCCCCA GCCGAAAGGCGAGUCAAGGUCU UGAGGUGU 1515
725 UCAGUGGG G CUGUGGGA 603 UCCCACAG GCCGAAAGGCGAGUCAAGGUCU CCCACUGA 1516
728 GUGGGGCU G UGGGAGAA 604 UUCUCCCA GCCGAAAGGCGAGUCAAGGUCU AGCCCCAC 1517
763 AACCGGAA G CUGUCGGA 605 UCCGACAG GCCGAAAGGCGAGUCAAGGUCU UUCCGGUU 1518
766 CGGAAGCU G UCGGAUCA 606 UGAUCCGA GCCGAAAGGCGAGUCAAGGUCU AGCUUCCG 1519
793 CUUCCCCU G CAGCAGCA 520 UGCUGCUG GCCGAAAGGCGAGUCAAGGUCU AGGGGAAG 1520
796 CCCCUGCA G CAGCACCA 607 UGGUGCUG GCCGAAAGGCGAGUCAAGGUCU UGCAGGGG 1521
799 CUGCAGCA G CACCAGCA 608 UGCUGGUG GCCGAAAGGCGAGUCAAGGUCU UGCUGCAG 1522
805 CAGCACCA G CACCAGCC 609 GGCUGGUG GCCGAAAGGCGAGUCAAGGUCU UGGUGCUG 1523
811 CAGCACCA G CCACAGCC 610 GGCUGUGG GCCGAAAGGCGAGUCAAGGUCU UGGUGCUG 1524
817 CAGCCACA G CCUCCGCA 611 UGCGGAGG GCCGAAAGGCGAGUCAAGGUCU UGUGGCUG 1525
823 CAGCCUCC G CAAUAUGC 521 GCAUAUUG GCCGAAAGGCGAGUCAAGGUCU GGAGGCUG 1526
830 CGCAAUAU G CCCCAGCG 522 CGCUGGGG GCCGAAAGGCGAGUCAAGGUCU AUAUUGCG 1527
836 AUGCCCCA G CGCCCCAG 612 CUGGGGCG GCCGAAAGGCGAGUCAAGGUCU UGGGGCAU 1528
838 GCCCCAGC G CCCCAGCA 523 UGCUGGGG GCCGAAAGGCGAGUCAAGGUCU GCUGGGGC 1529
844 GCGCCCCA G CAGCUGCA 613 UGCAGCUG GCCGAAAGGCGAGUCAAGGUCU UGGGGCGC 1530
847 CCCCAGCA G CUGCAGCA 614 UGCUGCAG GCCGAAAGGCGAGUCAAGGUCU UGCUGGGG 1531
850 CAGCAGCU G CAGCAGCC 524 GGCUGCUG GCCGAAAGGCGAGUCAAGGUCU AGCUGCUG 1532
853 CAGCUGCA G CAGCGCCC 615 GGGGGCUG GCCGAAAGGCGAGUCAAGGUCU UGCAGCUG 1533
856 CUGCAGCA G CCCCCACA 616 UGUGGGGG GCCGAAAGGCGAGUCAAGGUCU UGCUGCAG 1534
865 CCCCCACA G CAGCGAUA 617 UAUCGCUG GCCGAAAGGCGAGUCAAGGUCU UGUGGGGG 1535
868 CCACAGCA G CGAUAUCU 618 AGAUAUCG GCCGAAAGGCGAGUCAAGGUCU UGCUGUGG 1536
877 CGAUAUCU G CAGCACCA 526 UGGUGCUG GCCGAAAGGCGAGUCAAGGUCU AGAUAUCG 1537
880 UAUCUGCA G CACCACCA 619 UGGUGGUG GCCGAAAGGCGAGUCAAGGUCU UGCAGAUA 1538
903 CCAGGAAC G CCGAGGAG 527 CUCCUCGG GCCGAAAGGCGAGUCAAGGUCU GUUCCUGG 1539
912 CCGAGGAG G CAGCCUUG 620 CAAGGCUG GCCGAAAGGCGAGUCAAGGUCU CUCCUCGG 1540
915 AGGAGGCA G CCUUGACA 621 UGUCAAGG GCCGAAAGGCGAGUCAAGGUCU UGCCUCCU 1541
934 AAUGAUGG G CAUUGUGG 622 CCACAAUG GCCGAAAGGCGAGUCAAGGUCU CCAUCAUU 1542
939 UGGGCAUU G UGGCACCG 623 CGGUGCCA GCCGAAAGGCGAGUCAAGGUCU AAUGCCCA 1543
942 GCAUUGUG G CACCGGCU 624 AGCCGGUG GCCGAAAGGCGAGUCAAGGUCU CACAAUGC 1544
948 UGGCACCG G CUUGGGCA 625 UGCCCAAG GCCGAAAGGCGAGUCAAGGUCU CGGUGCCA 1545
954 CGGCUUGG G CAGUGAAA 626 UUUCACUG GCCGAAAGGCGAGUCAAGGUCU CCAAGCCG 1546
957 CUUGGGCA G UGAAAUGA 627 UCAUUUCA GCCGAAAGGCGAGUCAAGGUCU UGCCCAAG 1547
968 AAAUGAAU G CGGCCCUC 533 GAGGGCCG GCCGAAAGGCGAGUCAAGGUCU AUUCAUUU 1548
971 UGAAUGCG G CCCUCAUG 628 CAUGAGGG GCCGAAAGGCGAGUCAAGGUCU CGCAUUCA 1549
979 GCCCUCAU G CAUCGGAG 534 CUCCGAUG GCCGAAAGGCGAGUCAAGGUCU AUGAGGGC 1550
1001 CAGACCCA G UGCAGCUC 629 GAGCUGCA GCCGAAAGGCGAGUCAAGGUCU UGGGUCUG 1551
1003 GACCCAGU G CAGCUCCA 535 UGGAGCUG GCCGAAAGGCGAGUCAAGGUCU ACUGGGUC 1552
1006 CCAGUGCA G CUCCAGGC 630 GCCUGGAG GCCGAAAGGCGAGUCAAGGUCU UGCACUGG 1553
1013 AGCUCCAG G CGGCAGGG 631 CCCUGCCG GCCGAAAGGCGAGUCAAGGUCU CUGGAGCU 1554
1016 UCCAGGCG G CAGGGCGA 632 UCGCCCUG GCCGAAAGGCGAGUCAAGGUCU CGCCUGGA 1555
1021 GCGGCAGG G CGAGUGCG 633 CGCACUCG GCCGAAAGGCGAGUCAAGGUCU CCUGCCGC 1556
1025 CAGGGCGA G UGCGGUGG 634 CCACCGCA GCCGAAAGGCGAGUCAAGGUCU UCGCCCUG 1557
1027 GGGCGAGU G CGGUGGGC 537 GCCCACCG GCCGAAAGGCGAGUCAAGGUCU ACUCGCCC 1558
1030 CGAGUGCG G UGGGCCCG 635 CGGGCCCA GCCGAAAGGCGAGUCAAGGUCU CGCACUCG 1559 1034 UGCGGUGG G CCCGGGCG 636 CGCCCGGG GCCGAAAGGCGAGUCAAGGUCU CCACCGCA 1560
1040 GGGCCCGG G CGCUGUAU 637 AUACAGCG GCCGAAAGGCGAGUCAAGGUCU CCGGGCCC 1561
1042 GCCCGGGC G CUGUAUGA 538 UCAUACAG GCCGAAAGGCGAGUCAAGGUCU GCCCGGGC 1562
1045 CGGGCGCU G UAUGACUU 638 AAGUCAUA GCCGAAAGGCGAGUCAAGGUCU AGCGCCCG 1563
1058 ACUUUGAG G CCCUGGAG 639 CUCCAGGG GCCGAAAGGCGAGUCAAGGUCU CUCAAAGU 1564
1075 GAUGACGA G CUGGGGUU 640 AACCCCAG GCCGAAAGGCGAGUCAAGGUCU UCGUCAUC 1565
1081 GAGCUGGG G UUCCACAG 641 CUGUGGAA GCCGAAAGGCGAGUCAAGGUCU CCCAGCUC 1566
1089 GUUCC CA G CGGGGAGG 642 CCUCCCCG GCCGAAAGGCGAGUCAAGGUCU UGUGGAAC 1567
1097 GCGGGGAG G UGGUGGAG 643 CUCCACCA GCCGAAAGGCGAGUCAAGGUCU CUCCCCGC 1568
1100 GGGAGGUG G UGGAGGUC 644 GACCUCCA GCCGAAAGGCGAGUCAAGGUCU CACCUCCC 1569
1106 UGGUGGAG G UCCUGGAU 645 AUCCAGGA GCCGAAAGGCGAGUCAAGGUCU CUCCACCA 1570
1116 CCUGGAUA G CUCCAACC 646 GGUUGGAG GCCGAAAGGCGAGUCAAGGUCU UAUCCAGG 1571
1132 CCAUCCUG G UGGACCGG 647 CCGGUCCA GCCGAAAGGCGAGUCAAGGUCU CAGGAUGG 1572
1140 GUGGACCG G CCGCCUGC 648 GCAGGCGG GCCGAAAGGCGAGUCAAGGUCU CGGUCCAC 1573
1143 GACCGGCC G CCUGCACA 543 UGUGCAGG GCCGAAAGGCGAGUCAAGGUCU GGCCGGUC 1574
1147 GGCCGCCU G CACAACAA 544 UUGUUGUG GCCGAAAGGCGAGUCAAGGUCU AGGCGGCC 1575
1156 CACAACAA G CUGGGCCU 649 AGGCCCAG GCCGAAAGGCGAGUCAAGGUCU UUGUUGUG 1576
1161 CAAGCUGG G CCUCUUCC 650 GGAAGAGG GCCGAAAGGCGAGUCAAGGUCU CCAGCUUG 1577
1172 UCUUCCCU G CCAACUAC 545 GUAGUUGG GCCGAAAGGCGAGUCAAGGUCU AGGGAAGA 1578
1181 CCAACUAC G UGGCACCC 651 GGGUGCCA GCCGAAAGGCGAGUCAAGGUCU GUAGUUGG 1579
1184 ACUACGUG G CACCCAUG 652 CAUGGGUG GCCGAAAGGCGAGUCAAGGUCU CACGUAGU 1580
1220 GGACAGAA G CUUUUUGU 653 ACAAAAAG GCCGAAAGGCGAGUCAAGGUCU UUCUGUCC 1581
1227 AGCUUUUU G UCUGGAGC 654 GCUCCAGA GCCGAAAGGCGAGUCAAGGUCU AAAAAGCU 1582
1234 UGUCUGGA G CUGCCCAC 655 GUGGGCAG GCCGAAAGGCGAGUCAAGGUCU UCCAGACA 1583
1237 CUGGAGCU G CCCACAAG 548 CUUGUGGG GCCGAAAGGCGAGUCAAGGUCU AGCUCCAG 1584
1253 GAAAGAGG G CAAGGAAA 656 UUUCCUUG GCCGAAAGGCGAGUCAAGGUCU CCUCUUUC 1585
1266 GAAAAAAG G CUGGACUC 657 GAGUCCAG GCCGAAAGGCGAGUCAAGGUCU CUUUUUUC 1586
Input Sequence = HSA011736. Cut Site = G/Y Stem Length = 8 . Core Sequence = GCcgaaagGCGaGuCaaGGuCu
HSA011736 (Homo sapiens mRNA for growth factor receptor binding protein (GRBLG) ; 1303 bp)
Table VII: Human GRID DNAzyme and Substrate Sequence
Pos Substrate SeqID DNAzyme SeqID
11 GAGGCACA G UUAAUGGA 550 TCCATTAA GGCTAGCTACAACGA TGTGCCTC 1587
15 CACAGUUA A UGGAUCUG 658 CAGATCCA GGCTAGCTACAACGA TAACTGTG 1588
19 GUUAAUGG A UCUGUAAA 659 TTTACAGA GGCTAGCTACAACGA CCATTAAC 1589
23 AUGGAUCU G UAAACUUG 551 CAAGTTTA GGCTAGCTACAACGA AGATCCAT 1590
27 AUCUGUAA A CUUGCACC 660 GGTGCAAG GGCTAGCTACAACGA TTACAGAT 1591
31 GUAAACUU G CACCCUCU 493 AGAGGGTG GGCTAGCTACAACGA AAGTTTAC 1592
33 AAACUUGC A CCCUCUUU 183 AAAGAGGG GGCTAGCTACAACGA GCAAGTTT 1593
46 CUUUCAGA G UGGUACAU 552 ATGTACCA GGCTAGCTACAACGA TCTGAAAG 1594
49 UCAGAGUG G UACAUGGA 553 TCCATGTA GGCTAGCTACAACGA CACTCTGA 1595
51 AGAGUGGU A CAUGGAAG 10 CTTCCATG GGCTAGCTACAACGA ACCACTCT 1596
53 AGUGGUAC A UGGAAGAC 189 GTCTTCCA GGCTAGCTACAACGA GTACCACT 1597
60 CAUGGAAG A CAGCACAA 661 TTGTGCTG GGCTAGCTACAACGA CTTCCATG 1598
63 GGAAGACA G CACAAAGU 554 ACTTTGTG GGCTAGCTACAACGA TGTCTTCC 1599
65 AAGACAGC A CAAAGUGG 191 CCACTTTG GGCTAGCTACAACGA GCTGTCTT 1600
70 AGCACAAA G UGGAUCCA 555 TGGATCCA GGCTAGCTACAACGA TTTGTGCT 1601
74 CAAAGUGG A UCCAUACU 662 AGTATGGA GGCTAGCTACAACGA CCACTTTG 1602
78 GUGGAUCC A UACUCUGA 194 TCAGAGTA GGCTAGCTACAACGA GGATCCAC 1603
80 GGAUCCAU A CUCUGAAA 12 TTTCAGAG GGCTAGCTACAACGA ATGGATCC 1604
88 ACUCUGAA A UGCAGUAA 663 TTACTGCA GGCTAGCTACAACGA TTCAGAGT 1605
90 UCUGAAAU G CAGUAACU 495 AGTTACTG GGCTAGCTACAACGA ATTTCAGA 1606
93 GAAAUGCA G UAACUCUG 556 CAGAGTTA GGCTAGCTACAACGA TGCATTTC 1607
96 AUGCAGUA A CUCUGAUG 664 CATCAGAG GGCTAGCTACAACGA TACTGCAT 1608
102 UAACUCUG A UGCUUGAA 665 TTCAAGCA GGCTAGCTACAACGA CAGAGTTA 1609
104 ACUCUGAU G CUUGAAUU 497 AATTCAAG GGCTAGCTACAACGA ATCAGAGT 1610
110 AUGCUUGA A UUUGUUCU 666 AGAACAAA GGCTAGCTACAACGA TCAAGCAT 1611
114 UUGAAUUU G UUCUCCCU 557 AGGGAGAA GGCTAGCTACAACGA AAATTCAA 1612
127 CCCUUCUU G CCAGAAAG 499 CTTTCTGG GGCTAGCTACAACGA AAGAAGGG 1613
137 CAGAAAGG A UUCUAAUA 667 TATTAGAA GGCTAGCTACAACGA CCTTTCTG 1614
143 GGAUUCUA A UAACUCGG 668 CCGAGTTA GGCTAGCTACAACGA TAGAATCC 1615
146 UUCUAAUA A CUCGGUGU 669 ACACCGAG GGCTAGCTACAACGA TATTAGAA 1616
151 AUAACUCG G UGUCAAAG 558 CTTTGACA GGCTAGCTACAACGA CGAGTTAT 1617
153 AACUCGGU G UCAAAGCC 559 GGCTTTGA GGCTAGCTACAACGA ACCGAGTT 1618
159 GUGUCAAA G CCAAGACA 560 TGTCTTGG GGCTAGCTACAACGA TTTGACAC 1619
165 AAGCCAAG A CAUAAACU 670 AGTTTATG GGCTAGCTACAACGA CTTGGCTT 1620
167 GCCAAGAC A UAAACUCA 213 TGAGTTTA GGCTAGCTACAACGA GTCTTGGC 1621
171 AGACAUAA A CUCAAUCU 671 AGATTGAG GGCTAGCTACAACGA TTATGTCT 1622
176 UAAACUCA A UCUCUUCU 672 AGAAGAGA GGCTAGCTACAACGA TGAGTTTA 1623
194 UUCCAAAA G CUUCACGU 561 ACGTGAAG GGCTAGCTACAACGA TTTTGGAA 1624
199 AAAGCUUC A CGUUACAG 223 CTGTAACG GGCTAGCTACAACGA GAAGCTTT 1625
201 AGCUUCAC G UUACAGCA 562 TGCTGTAA GGCTAGCTACAACGA GTGAAGCT 1626
204 UUCACGUU A CAGCAUGG 43 CCATGCTG GGCTAGCTACAACGA AACGTGAA 1627
207 ACGUUACA G CAUGGAAG 563 CTTCCATG GGCTAGCTACAACGA TGTAACGT 1628
209 GUUACAGC A UGGAAGCU 225 AGCTTCCA GGCTAGCTACAACGA GCTGTAAC 1629
215 GCAUGGAA G CUGUUGCC 564 GGCAACAG GGCTAGCTACAACGA TTCCATGC 1630
218 UGGAAGCU G UUGCCAAG 565 CTTGGCAA GGCTAGCTACAACGA AGCTTCCA 1631
221 AAGCUGUU G CCAAGUUU 500 AAACTTGG GGCTAGCTACAACGA AACAGCTT 1632
226 GUUGCCAA G UUUGAUUU 566 AAATCAAA GGCTAGCTACAACGA TTGGCAAC 1633
231 CAAGUUUG A UUUCACUG 673 CAGTGAAA GGCTAGCTACAACGA CAAACTTG 1634
236 UUGAUUUC A CUGCUUCA 229 TGAAGCAG GGCTAGCTACAACGA GAAATCAA 1635
239 AUUUCACU G CUUCAGGU 502 ACCTGAAG GGCTAGCTACAACGA AGTGAAAT 1636 246 UGCUUCAG G UGAGGAUG 567 CATCCTCA GGCTAGCTACAACGA CTGAAGCA 1637
252 AGGUGAGG A UGAACUGA 674 TCAGTTCA GGCTAGCTACAACGA CCTCACCT 1638
256 GAGGAUGA A CUGAGCUU 675 AAGCTCAG GGCTAGCTACAACGA TCATCCTC 1639
261 UGAACUGA G CUUUCACA 568 TGTGAAAG GGCTAGCTACAACGA TCAGTTCA 1640
267 GAGCUUUC A CACUGGAG 235 CTCCAGTG GGCTAGCTACAACGA GAAAGCTC 1641
269 GCUUUCAC A CUGGAGAU 236 ATCTCCAG GGCTAGCTACAACGA GTGAAAGC 1642
276 CACUGGAG A UGUUUUGA 676 TCAAAACA GGCTAGCTACAACGA CTCCAGTG 1643
278 CUGGAGAU G UUUUGAAG 569 CTTCAAAA GGCTAGCTACAACGA ATCTCCAG 1644
287 UUUUGAAG A UUUUAAGU 677 ACTTAAAA GGCTAGCTACAACGA CTTCAAAA 1645
294 GAUUUUAA G UAACCAAG 570 CTTGGTTA GGCTAGCTACAACGA TTAAAATC 1646
297 UUUAAGUA A CCAAGAGG 678 CCTCTTGG GGCTAGCTACAACGA TACTTAAA 1647
307 CAAGAGGA G UGGUUUAA 571 TTAAACCA GGCTAGCTACAACGA TCCTCTTG 1648
310 GAGGAGUG G UUUAAGGC 572 GCCTTAAA GGCTAGCTACAACGA CACTCCTC 1649
317 GGUUUAAG G CGGAGCUU 573 AAGCTCCG GGCTAGCTACAACGA CTTAAACC 1650
322 AAGGCGGA G CUUGGGAG 574 CTCCCAAG GGCTAGCTACAACGA TCCGCCTT 1651
330 GCUUGGGA G CCAGGAAG 575 CTTCCTGG GGCTAGCTACAACGA TCCCAAGC 1652
340 CAGGAAGG A UAUGUGCC 679 GGCACATA GGCTAGCTACAACGA CCTTCCTG 1653
342 GGAAGGAU A UGUGCCCA 67 TGGGCACA GGCTAGCTACAACGA ATCCTTCC 1654
344 AAGGAUAU G UGCCCAAG 576 CTTGGGCA GGCTAGCTACAACGA ATATCCTT 1655
346 GGAUAUGU G CCCAAGAA 507 TTCTTGGG GGCTAGCTACAACGA ACATATCC 1656
354 GCCCAAGA A UUUCAUAG 680 CTATGAAA GGCTAGCTACAACGA TCTTGGGC 1657
359 AGAAUUUC A UAGACAUC 246 GATGTCTA GGCTAGCTACAACGA GAAATTCT 1658
363 UUUCAUAG A CAUCCAGU 681 ACTGGATG GGCTAGCTACAACGA CTATGAAA 1659
365 UCAUAGAC A UCCAGUUU 247 AAACTGGA GGCTAGCTACAACGA GTCTATGA 1660
370 GACAUCCA G UUUCCCAA 577 TTGGGAAA GGCTAGCTACAACGA TGGATGTC 1661
379 UUUCCCAA A UGGUUUCA 682 TGAAACCA GGCTAGCTACAACGA TTGGGAAA 1662
382 CCCAAAUG G UUUCACGA 578 TCGTGAAA GGCTAGCTACAACGA CATTTGGG 1663
387 AUGGUUUC A CGAAGGCC 253 GGCCTTCG GGCTAGCTACAACGA GAAACCAT 1664
393 UCACGAAG G CCUCUCUC 579 GAGAGAGG GGCTAGCTACAACGA CTTCGTGA 1665
403 CUCUCUCG A CACCAGGC 683 GCCTGGTG GGCTAGCTACAACGA CGAGAGAG 1666
405 CUCUCGAC A CCAGGCAG 258 CTGCCTGG GGCTAGCTACAACGA GTCGAGAG 1667
410 GACACCAG G CAGAGAAC 580 GTTCTCTG GGCTAGCTACAACGA CTGGTGTC 1668
417 GGCAGAGA A CUUACUCA 684 TGAGTAAG GGCTAGCTACAACGA TCTCTGCC 1669
421 GAGAACUU A CUCAUGGG 83 CCCATGAG GGCTAGCTACAACGA AAGTTCTC 1670
425 ACUUACUC A UGGGCAAG 264 CTTGCCCA GGCTAGCTACAACGA GAGTAAGT 1671
429 ACUCAUGG G CAAGGAGG 581 CCTCCTTG GGCTAGCTACAACGA CCATGAGT 1672
437 GCAAGGAG G UUGGCUUC 582 GAAGCCAA GGCTAGCTACAACGA CTCCTTGC 1673
441 GGAGGUUG G CUUCUUCA 583 TGAAGAAG GGCTAGCTACAACGA CAACCTCC 1674
449 GCUUCUUC A UCAUCCGG 268 CCGGATGA GGCTAGCTACAACGA GAAGAAGC 1675
452 UCUUCAUC A UCCGGGCC 269 GGCCCGGA GGCTAGCTACAACGA GATGAAGA 1676
458 UCAUCCGG G CCAGCCAG 584 CTGGCTGG GGCTAGCTACAACGA CCGGATGA 1677
462 CCGGGCCA G CCAGAGCU 585 AGCTCTGG GGCTAGCTACAACGA TGGCCCGG 1678
468 CAGCCAGA G CUCCCCAG 586 CTGGGGAG GGCTAGCTACAACGA TCTGGCTG 1679
480 CCCAGGGG A CUUCUCCA 685 TGGAGAAG GGCTAGCTACAACGA CCCCTGGG 1680
488 ACUUCUCC A UCUCUGUC 283 GACAGAGA GGCTAGCTACAACGA GGAGAAGT 1681
494 CCAUCUCU G UCAGGCAU 587 ATGCCTGA GGCTAGCTACAACGA AGAGATGG 1682
499 UCUGUCAG G CAUGAGGA 588 TCCTCATG GGCTAGCTACAACGA CTGACAGA 1683
501 UGUCAGGC A UGAGGAUG 287 CATCCTCA GGCTAGCTACAACGA GCCTGACA 1684
507 GCAUGAGG A UGACGUUC 686 GAACGTCA GGCTAGCTACAACGA CCTCATGC 1685
510 UGAGGAUG A CGUUCAAC 687 GTTGAACG GGCTAGCTACAACGA CATCCTCA 1686
512 AGGAUGAC G UUCAACAC 589 GTGTTGAA GGCTAGCTACAACGA GTCATCCT 1687
517 GACGUUCA A CACUUCAA 688 TTGAAGTG GGCTAGCTACAACGA TGAACGTC 1688
519 CGUUCAAC A CUUCAAGG 289 CCTTGAAG GGCTAGCTACAACGA GTTGAACG 1689
527 ACUUCAAG G UCAUGCGA 590 TCGCATGA GGCTAGCTACAACGA CTTGAAGT 1690 530 UCAAGGUC A UGCGAGAC 292 GTCTCGCA GGCTAGCTACAACGA GACCTTGA 1691
532 AAGGUCAU G CGAGACAA 512 TTGTCTCG GGCTAGCTACAACGA ATGACCTT 1692
537 CAUGCGAG A CAACAAGG 689 CCTTGTTG GGCTAGCTACAACGA CTCGCATG 1693
540 GCGAGACA A CAAGGGUA 690 TACCCTTG GGCTAGCTACAACGA TGTCTCGC 1694
546 CAACAAGG G UAAUUACU 591 AGTAATTA GGCTAGCTACAACGA CCTTGTTG 1695
549 CAAGGGUA A UUACUUUC 691 GAAAGTAA GGCTAGCTACAACGA TACCCTTG 1696
552 GGGUAAUU A CUUUCUGU 106 ACAGAAAG GGCTAGCTACAACGA AATTACCC 1697
559 UACUUUCU G UGGACUGA 592 TCAGTCCA GGCTAGCTACAACGA AGAAAGTA 1698
563 UUCUGUGG A CUGAGAAG 692 CTTCTCAG GGCTAGCTACAACGA CCACAGAA 1699
571 ACUGAGAA G UUUCCAUC 593 GATGGAAA GGCTAGCTACAACGA TTCTCAGT 1700
577 AAGUUUCC A UCCCUAAA 299 TTTAGGGA GGCTAGCTACAACGA GGAAACTT 1701
585 AUCCCUAA A UAAGCUGG 693 CCAGCTTA GGCTAGCTACAACGA TTAGGGAT 1702
589 CUAAAUAA G CUGGUAGA 594 TCTACCAG GGCTAGCTACAACGA TTATTTAG 1703
593 AUAAGCUG G UAGACUAC 595 GTAGTCTA GGCTAGCTACAACGA CAGCTTAT 1704
597 GCUGGUAG A CUACUACA 694 TGTAGTAG GGCTAGCTACAACGA CTACCAGC 1705
600 GGUAGACU A CUACAGGA 117 TCCTGTAG GGCTAGCTACAACGA AGTCTACC 1706
603 AGACUACU A CAGGACAA 118 TTGTCCTG GGCTAGCTACAACGA AGTAGTCT 1707
608 ACUACAGG A CAAAUUCC 695 GGAATTTG GGCTAGCTACAACGA CCTGTAGT 1708
612 CAGGACAA A UUCCAUCU 696 AGATGGAA GGCTAGCTACAACGA TTGTCCTG 1709
617 CAAAUUCC A UCUCCAGA 309 TCTGGAGA GGCTAGCTACAACGA GGAATTTG 1710
625 AUCUCCAG A CAGAAGCA 697 TGCTTCTG GGCTAGCTACAACGA CTGGAGAT 1711
631 AGACAGAA G CAGAUCUU 596 AAGATCTG GGCTAGCTACAACGA TTCTGTCT 1712
635 AGAAGCAG A UCUUCCUU 698 AAGGAAGA GGCTAGCTACAACGA CTGCTTCT 1713
648 CCUUAGAG A CAGAACCC 699 GGGTTCTG GGCTAGCTACAACGA CTCTAAGG 1714
653 GAGACAGA A CCCGAGAA 700 TTCTCGGG GGCTAGCTACAACGA TCTGTCTC 1715
663 CCGAGAAG A CCAGGGUC 701 GACCCTGG GGCTAGCTACAACGA CTTCTCGG 1716
669 AGACCAGG G UCACCGGG 597 CCCGGTGA GGCTAGCTACAACGA CCTGGTCT 1717
672 CCAGGGUC A CCGGGGCA 323 TGCCCCGG GGCTAGCTACAACGA GACCCTGG 1718
678 UCACCGGG G CAACAGCC 598 GGCTGTTG GGCTAGCTACAACGA CCCGGTGA 1719
681 CCGGGGCA A CAGCCUGG 702 CCAGGCTG GGCTAGCTACAACGA TGCCCCGG 1720
684 GGGCAACA G CCUGGACC 599 GGTCCAGG GGCTAGCTACAACGA TGTTGCCC 1721
690 CAGCCUGG A CCGGAGGU 703 ACCTCCGG GGCTAGCTACAACGA CCAGGCTG 1722
697 GACCGGAG G UCCCAGGG 600 CCCTGGGA GGCTAGCTACAACGA CTCCGGTC 1723
708 CCAGGGAG G CCCACACC 601 GGTGTGGG GGCTAGCTACAACGA CTCCCTGG 1724
712 GGAGGCCC A CACCUCAG 335 CTGAGGTG GGCTAGCTACAACGA GGGCCTCC 1725
714 AGGCCCAC A CCUCAGUG 336 CACTGAGG GGCTAGCTACAACGA GTGGGCCT 1726
720 ACACCUCA G UGGGGCUG 602 CAGCCCCA GGCTAGCTACAACGA TGAGGTGT 1727
725 UCAGUGGG G CUGUGGGA 603 TCCCACAG GGCTAGCTACAACGA CCCACTGA 1728
728 GUGGGGCU G UGGGAGAA 604 TTCTCCCA GGCTAGCTACAACGA AGCCCCAC 1729
740 GAGAAGAA A UCCGACCU 704 AGGTCGGA GGCTAGCTACAACGA TTCTTCTC 1730
745 GAAAUCCG A CCUUCGAU 705 ATCGAAGG GGCTAGCTACAACGA CGGATTTC 1731
752 GACCUUCG A UGAACCGG 706 CCGGTTCA GGCTAGCTACAACGA CGAAGGTC 1732
756 UUCGAUGA A CCGGAAGC 707 GCTTCCGG GGCTAGCTACAACGA TCATCGAA 1733
763 AACCGGAA G CUGUCGGA 605 TCCGACAG GGCTAGCTACAACGA TTCCGGTT 1734
766 CGGAAGCU G UCGGAUCA 606 TGATCCGA GGCTAGCTACAACGA AGCTTCCG 1735
771 GCUGUCGG A UCACCCCC 708 GGGGGTGA GGCTAGCTACAACGA CCGACAGC 1736
774 GUCGGAUC A CCCCCCGA 346 TCGGGGGG GGCTAGCTACAACGA GATCCGAC 1737
782 ACCCCCCG A CCCUUCCC 709 GGGAAGGG GGCTAGCTACAACGA CGGGGGGT 1738
793 CUUCCCCU G CAGCAGCA 520 TGCTGCTG GGCTAGCTACAACGA AGGGGAAG 1739
796 CCCCUGCA G CAGCACCA 607 TGGTGCTG GGCTAGCTACAACGA TGCAGGGG 1740
799 CUGCAGCA G CACCAGCA 608 TGCTGGTG GGCTAGCTACAACGA TGCTGCAG 1741
801 GCAGCAGC A CCAGCACC 361 GGTGCTGG GGCTAGCTACAACGA GCTGCTGC 1742
805 CAGCACCA G CACCAGCC 609 GGCTGGTG GGCTAGCTACAACGA TGGTGCTG 1743
807 GCACCAGC A CCAGCCAC 364 GTGGCTGG GGCTAGCTACAACGA GCTGGTGC 1744 811 CAGCACCA G CCACAGCC 610 GGCTGTGG GGCTAGCTACAACGA TGGTGCTG 1745
814 CACCAGCC A CAGCCUCC 368 GGAGGCTG GGCTAGCTACAACGA GGCTGGTG 1746
817 CAGCCAGA G CCUCCGCA 611 TGCGGAGG GGCTAGCTACAACGA TGTGGCTG 1747
823 CAGCCUCC G CAAUAUGC 521 GCATATTG GGCTAGCTACAACGA GGAGGCTG 1748
826 CCUCCGCA A UAUGCCCC 710 GGGGCATA GGCTAGCTACAACGA TGCGGAGG 1749
828 UCCGCAAU A UGCCCCAG 139 CTGGGGCA GGCTAGCTACAACGA ATTGCGGA 1750
830 CGCAAUAU G CCCCAGCG 522 CGCTGGGG GGCTAGCTACAACGA ATATTGCG 1751
836 AUGCCCCA G CGCCCCAG 612 CTGGGGCG GGCTAGCTACAACGA TGGGGCAT 1752
838 GCCCCAGC G CCCCAGCA 523 TGCTGGGG GGCTAGCTACAACGA GCTGGGGC 1753
844 GCGCCCCA G CAGCUGCA 613 TGCAGCTG GGCTAGCTACAACGA TGGGGCGC 1754
847 CCCCAGCA G CUGCAGCA 614 TGCTGCAG GGCTAGCTACAACGA TGCTGGGG 1755
850 CAGCAGCU G CAGCAGCC 524 GGCTGCTG GGCTAGCTACAACGA AGCTGCTG 1756
853 CAGCUGCA G CAGCCCCC 615 GGGGGCTG GGCTAGCTACAACGA TGCAGCTG 1757
856 CUGCAGCA G CCCCCACA 616 TGTGGGGG GGCTAGCTACAACGA TGCTGCAG 1758
862 CAGCCCCC A CAGCAGCG 390 CGCTGCTG GGCTAGCTACAACGA GGGGGCTG 1759
865 CCCCCACA G CAGCGAUA 617 TATCGCTG GGCTAGCTACAACGA TGTGGGGG 1760
868 CCACAGCA G CGAUAUCU 618 AGATATCG GGCTAGCTACAACGA TGCTGTGG 1761
871 CAGCAGCG A UAUCUGCA 711 TGCAGATA GGCTAGCTACAACGA CGCTGCTG 1762
873 GCAGCGAU A UCUGCAGC 140 GCTGCAGA GGCTAGCTACAACGA ATCGCTGC 1763
877 CGAUAUCU G CAGCACCA 526 TGGTGCTG GGCTAGCTACAACGA AGATATCG 1764
880 UAUCUGCA G CACCACCA 619 TGGTGGTG GGCTAGCTACAACGA TGCAGATA 1765
882 UCUGCAGC A CCACCAUU 395 AATGGTGG GGCTAGCTACAACGA GCTGCAGA 1766
885 GCAGCACC A CCAUUUCC 397 GGAAATGG GGCTAGCTACAACGA GGTGCTGC 1767
888 GCACCACC A UUUCCACC 399 GGTGGAAA GGCTAGCTACAACGA GGTGGTGC 1768
894 CCAUUUCC A CCAGGAAC 401 GTTCCTGG GGCTAGCTACAACGA GGAAATGG 1769
901 CACCAGGA A CGCCGAGG 712 CCTCGGCG GGCTAGCTACAACGA TCCTGGTG 1770
903 CCAGGAAC G CCGAGGAG 527 CTCCTCGG GGCTAGCTACAACGA GTTCCTGG 1771
912 CCGAGGAG G CAGCCUUG 620 CAAGGCTG GGCTAGCTACAACGA CTCCTCGG 1772
915 AGGAGGCA G CCUUGACA 621 TGTCAAGG GGCTAGCTACAACGA TGCCTCCT 1773
921 CAGCCUUG A CAUAAAUG 713 CATTTATG GGCTAGCTACAACGA CAAGGCTG 1774
923 GCCUUGAC A UAAAUGAU 408 ATCATTTA GGCTAGCTACAACGA GTCAAGGC 1775
927 UGACAUAA A UGAUGGGC 714 GCCCATCA GGCTAGCTACAACGA TTATGTCA 1776
930 CAUAAAUG A UGGGCAUU 715 AATGCCCA GGCTAGCTACAACGA CATTTATG 1777
934 AAUGAUGG G CAUUGUGG 622 CCACAATG GGCTAGCTACAACGA CCATCATT 1778
936 UGAUGGGC A UUGUGGCA 409 TGCCACAA GGCTAGCTACAACGA GCCCATCA 1779
939 UGGGCAUU G UGGCACCG 623 CGGTGCCA GGCTAGCTACAACGA AATGCCCA 1780
942 GCAUUGUG G CACCGGCU 624 AGCCGGTG GGCTAGCTACAACGA CACAATGC 1781
944 AUUGUGGC A CCGGCUUG 410 CAAGCCGG GGCTAGCTACAACGA GCCACAAT 1782
948 UGGCACCG G CUUGGGCA 625 TGCCCAAG GGCTAGCTACAACGA CGGTGCCA 1783
954 CGGCUUGG G CAGUGAAA 626 TTTCACTG GGCTAGCTACAACGA CCAAGCCG 1784
957 CUUGGGCA G UGAAAUGA 627 TCATTTCA GGCTAGCTACAACGA TGCCCAAG 1785
962 GCAGUGAA A UGAAUGCG 716 CGCATTCA GGCTAGCTACAACGA TTCACTGC 1786
966 UGAAAUGA A UGCGGCCC 717 GGGCCGCA GGCTAGCTACAACGA TCATTTCA 1787
968 AAAUGAAU G CGGCCCUC 533 GAGGGCCG GGCTAGCTACAACGA ATTCATTT 1788
971 UGAAUGCG G CCCUCAUG 628 CATGAGGG GGCTAGCTACAACGA CGCATTCA 1789
977 CGGCCCUC A UGCAUCGG 417 CCGATGCA GGCTAGCTACAACGA GAGGGCCG 1790
979 GCCCUCAU G CAUCGGAG 534 CTCCGATG GGCTAGCTACAACGA ATGAGGGC 1791
981 CCUCAUGC A UCGGAGAC 418 GTCTCCGA GGCTAGCTACAACGA GCATGAGG 1792
988 CAUCGGAG A CACACAGA 718 TCTGTGTG GGCTAGCTACAACGA CTCCGATG 1793
990 UCGGAGAC A CACAGACC 419 GGTCTGTG GGCTAGCTACAACGA GTCTCCGA 1794
992 GGAGACAC A CAGACCCA 420 TGGGTCTG GGCTAGCTACAACGA GTGTCTCC 1795
996 ACACACAG A CCCAGUGC 719 GCACTGGG GGCTAGCTACAACGA CTGTGTGT 1796
1001 CAGACCCA G UGCAGCUC 629 GAGCTGCA GGCTAGCTACAACGA TGGGTCTG 1797
1003 GACCCAGU G CAGCUCCA 535 TGGAGCTG GGCTAGCTACAACGA ACTGGGTC 1798 1006 CCAGUGCA G CUCCAGGC 630 GCCTGGAG GGCTAGCTACAACGA TGCACTGG 1799
1013 AGCUCCAG G CGGCAGGG 631 CCCTGCCG GGCTAGCTACAACGA CTGGAGCT 1800
1016 UCCAGGCG G CAGGGCGA 632 TCGCCCTG GGCTAGCTACAACGA CGCCTGGA 1801
1021 GCGGCAGG G CGAGUGCG 633 CGCACTCG GGCTAGCTACAACGA CCTGCCGC 1802
1025 CAGGGCGA G UGCGGUGG 634 CCACCGCA GGCTAGCTACAACGA TCGCCCTG 1803
1027 GGGCGAGU G CGGUGGGC 537 GCCCACCG GGCTAGCTACAACGA ACTCGCCC 1804
1030 CGAGUGCG G UGGGCCCG 635 CGGGCCCA GGCTAGCTACAACGA CGCACTCG 1805
1034 UGCGGUGG G CCCGGGCG 636 CGCCCGGG GGCTAGCTACAACGA CCACCGCA 1806
1040 GGGCCCGG G CGCUGUAU 637 ATACAGCG GGCTAGCTACAACGA CCGGGCCC 1807
1042 GCCCGGGC G CUGUAUGA 538 TCATACAG GGCTAGCTACAACGA GCCCGGGC 1808
1045 CGGGCGCU G UAUGACUU 638 AAGTCATA GGCTAGCTACAACGA AGCGCCCG 1809
1047 GGCGCUGU A UGACUUUG 152 CAAAGTCA GGCTAGCTACAACGA ACAGCGCC 1810
1050 GCUGUAUG A CUUUGAGG 720 CCTCAAAG GGCTAGCTACAACGA CATACAGC 1811
1058 ACUUUGAG G CCCUGGAG 639 CTCCAGGG GGCTAGCTACAACGA CTCAAAGT 1812
1068 CCUGGAGG A UGACGAGC 721 GCTCGTCA GGCTAGCTACAACGA CCTCCAGG 1813
1071 GGAGGAUG A CGAGCUGG 722 CCAGCTCG GGCTAGCTACAACGA CATCCTCC 1814
1075 GAUGACGA G CUGGGGUU 640 AACCCCAG GGCTAGCTACAACGA TCGTCATC 1815
1081 GAGCUGGG G UUCCACAG 641 CTGTGGAA GGCTAGCTACAACGA CCCAGCTC 1816
1086 GGGGUUCC A CAGCGGGG 439 CCCCGCTG GGCTAGCTACAACGA GGAACCCC 1817
1089 GUUCCACA G CGGGGAGG 642 CCTCCCCG GGCTAGCTACAACGA TGTGGAAC 1818
1097 GCGGGGAG G UGGUGGAG 643 CTCCACCA GGCTAGCTACAACGA CTCCCCGC 1819
1100 GGGAGGUG G UGGAGGUC 644 GACCTCCA GGCTAGCTACAACGA CACCTCCC 1820
1106 UGGUGGAG G UCCUGGAU 645 ATCCAGGA GGCTAGCTACAACGA CTCCACCA 1821
1113 GGUCCUGG A UAGCUCCA 723 TGGAGCTA GGCTAGCTACAACGA CCAGGACC 1822
1116 CCUGGAUA G CUCCAACC 646 GGTTGGAG GGCTAGCTACAACGA TATCCAGG 1823
1122 UAGCUCCA A CCCAUCCU 724 AGGATGGG GGCTAGCTACAACGA TGGAGCTA 1824
1126 UCCAACCC A UCCUGGUG 448 CACCAGGA GGCTAGCTACAACGA GGGTTGGA 1825
1132 CCAUCCUG G UGGACCGG 647 CCGGTCCA GGCTAGCTACAACGA CAGGATGG 1826
1136 CCUGGUGG A CCGGCCGC 725 GCGGCCGG GGCTAGCTACAACGA CCACCAGG 1827
1140 GUGGACCG G CCGCCUGC 648 GCAGGCGG GGCTAGCTACAACGA CGGTCCAC 1828
1143 GACCGGCC G CCUGCACA 543 TGTGCAGG GGCTAGCTACAACGA GGCCGGTC 1829
1147 GGCCGCCU G CACAACAA 544 TTGTTGTG GGCTAGCTACAACGA AGGCGGCC 1830
1149 CCGCCUGC A CAACAAGC 455 GCTTGTTG GGCTAGCTACAACGA GCAGGCGG 1831
1152 CCUGCACA A CAAGCUGG 726 CCAGCTTG GGCTAGCTACAACGA TGTGCAGG 1832
1156 CACAACAA G CUGGGCCU 649 AGGCCCAG GGCTAGCTACAACGA TTGTTGTG 1833
1161 CAAGCUGG G CCUCUUCC 650 GGAAGAGG GGCTAGCTACAACGA CCAGCTTG 1834
1172 UCUUCCCU G CCAACUAC 545 GTAGTTGG GGCTAGCTACAACGA AGGGAAGA 1835
1176 CCCUGCCA A CUACGUGG 727 CCACGTAG GGCTAGCTACAACGA TGGCAGGG 1836
1179 UGCCAACU A CGUGGCAC 164 GTGCCACG GGCTAGCTACAACGA AGTTGGCA 1837
1181 CCAACUAC G UGGCACCC 651 GGGTGCCA GGCTAGCTACAACGA GTAGTTGG 1838
1184 ACUACGUG G CACCCAUG 652 CATGGGTG GGCTAGCTACAACGA CACGTAGT 1839
1186 UACGUGGC A CCCAUGAC 468 GTCATGGG GGCTAGCTACAACGA GCCACGTA 1840
1190 UGGCACCC A UGACCCGA 471 TCGGGTCA GGCTAGCTACAACGA GGGTGCCA 1841
1193 CACCCAUG A CCCGAUAA 728 TTATCGGG GGCTAGCTACAACGA CATGGGTG 1842
1198 AUGACCCG A UAAACUCU 729 AGAGTTTA GGCTAGCTACAACGA CGGGTCAT 1843
1202 CCCGAUAA A CUCUUCAG 730 CTGAAGAG GGCTAGCTACAACGA TTATCGGG 1844
1214 UUCAGGGG A CAGAAGCU 731 AGCTTCTG GGCTAGCTACAACGA CCCCTGAA 1845
1220 GGACAGAA G CUUUUUGU 653 ACAAAAAG GGCTAGCTACAACGA TTCTGTCC 1846
1227 AGCUUUUU G UCUGGAGC 654 GCTCCAGA GGCTAGCTACAACGA AAAAAGCT 1847
1234 UGUCUGGA G CUGCCCAC 655 GTGGGCAG GGCTAGCTACAACGA TCCAGACA 1848
1237 CUGGAGCU G CCCACAAG 548 CTTGTGGG GGCTAGCTACAACGA AGCTCCAG 1849
1241 AGCUGCCC A CAAGAAAG 483 CTTTCTTG GGCTAGCTACAACGA GGGCAGCT 1850
1253 GAAAGAGG G CAAGGAAA 656 TTTCCTTG GGCTAGCTACAACGA CCTCTTTC 1851
1266 GAAAAAAG G CUGGACUC 657 GAGTCCAG GGCTAGCTACAACGA CTTTTTTC 1852 1271 AAGGCUGG A CUCCAUGA 732 TCATGGAG GGCTAGCTACAACGA CCAGCCTT 1853
1276 UGGACUCC A UGACUAUA 489 TATAGTCA GGCTAGCTACAACGA GGAGTCCA 1854
1279 ACUCCAUG A CUAUAUAU 733 ATATATAG GGCTAGCTACAACGA CATGGAGT 1855
1282 CCAUGACU A UAUAUACA 175 TGTATATA GGCTAGCTACAACGA AGTCATGG 1856
1284 AUGACUAU A UAUACAUA 176 TATGTATA GGCTAGCTACAACGA ATAGTCAT 1857
1286 GACUAUAU A UACAUACA 177 TGTATGTA GGCTAGCTACAACGA ATATAGTC 1858
1288 CUAUAUAϋ A CAUACAUC 178 GATGTATG GGCTAGCTACAACGA ATATATAG 1859
1290 AUAUAUAC A UACAUCUA 491 TAGATGTA GGCTAGCTACAACGA GTATATAT 1860
1292 AUAUACAU A CAUCUAUC 179 GATAGATG GGCTAGCTACAACGA ATGTATAT 1861
1294 AUACAUAC A UCUAUCUA 492 TAGATAGA GGCTAGCTACAACGA GTATGTAT 1862
Input Sequence = HSA011736. Cut Site = R/Y Stem Length = 8 . Core Sequence = GGCTAGCTACAACGA
HSA011736 (Homo sapiens mRNA for growth factor receptor binding protein (GRBLG) ; 1303 bp)
400.013
Table VIII: Human GRID Amberzyme and Substrate Sequence
Pos Substrate SeqED Amberzyme SeqID
11 GAGGCACA G UUAAUGGA 550 UCCAUUAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUGCCUC 1863
17 CAGUUAAU G GAUCUGUA 734 UACAGAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUAACUG 1864
18 AGUUAAUG G AUCUGUAA 735 UUACAGAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUUAACU 1865
23 AUGGAUCU G UAAACUUG 551 CAAGUUUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAUCCAU 1866
31 GUAAACUU G CACCCUCU 493 AGAGGGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGUUUAC 1867
44 CUCUUUCA G AGUGGUAC 736 GUACCACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAAAGAG 1868
46 CUUUCAGA G UGGUACAU 552 AUGUACCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUGAAAG 1869
48 UUCAGAGU G GUACAUGG 737 CCAUGUAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUCUGAA 1870
49 UCAGAGUG G UACAUGGA 553 UCCAUGUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACUCUGA 1871
55 UGGUACAU G GAAGACAG 738 CUGUCUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGUACCA 1872
56 GGUACAUG G AAGACAGC 739 GCUGUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUGUACC 1873
59 ACAUGGAA G ACAGCACA 740 UGUGCUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCCAUGU 1874
63 GGAAGACA G CACAAAGU 554 ACUUUGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUCUUCC 1875
70 AGCACAAA G UGGAUCCA 555 UGGAUCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUGUGCU 1876
72 CACAAAGU G GAUCCAUA 741 UAUGGAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUUUGUG 1877
73 ACAAAGUG G AUCCAUAC 742 GUAUGGAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACUUUGU 1878
85 CAUACUCU G AAAUGCAG 494 CUGCAUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAGUAUG 1879
90 UCUGAAAU G CAGUAACU 495 AGUUACUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUUCAGA 1880
93 GAAAUGCA G UAACUCUG 556 CAGAGUUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCAUUUC 1881
101 GUAACUCU G AUGCUUGA 496 UCAAGCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAGUUAC 1882
104 ACUCUGAU G CUUGAAUU 497 AAUUCAAG. GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCAGAGU 1883
108 UGAUGCUU G AAUUUGUU 498 AACAAAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGCAUCA 1884
114 UUGAAUUU G UUCUCCCU 557 AGGGAGAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAUUCAA 1885
127 CCCUUCUU G CCAGAAAG 499 CUUUCUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGAAGGG 1886
131 UCUUGCCA G AAAGGAUU 743 AAUCCUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGCAAGA 1887
135 GCCAGAAA G GAUUCUAA 744 UUAGAAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUCUGGC 1888
136 CCAGAAAG G AUUCUAAU 745 AUUAGAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUUCUGG 1889
150 AAUAACUC G GUGUCAAA 746 UUUGACAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAGUUAUU 1890
151 AUAACUCG G UGUCAAAG 558 CUUUGACA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGAGUUAU 1891
153 AACUCGGU G UCAAAGCC 559 GGCUUUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCGAGUU 1892
400.013
159 GUGUCAAA G CCAAGACA 560 UGUCUUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUGACAC 1893
164 AAAGCCAA G ACAUAAAC 747 GUUUAUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGGCUUU 1894
194 UUCCAAAA G CUUCACGU 561 ACGUGAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUUGGAA 1895
201 AGCUUCAC G UUACAGCA 562 UGCUGUAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUGAAGCU 1896
207 ACGUUACA G CAUGGAAG 563 CUUCCAUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUAACGU 1897
211 UACAGCAU G GAAGCUGU 748 ACAGCUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGCUGUA 1898
212 ACAGCAUG G AAGCUGUU 749 AACAGCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUGCUGU 1899
215 GCAUGGAA G CUGUUGCC 564 GGCAACAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCCAUGC 1900
218 UGGAAGCU G UUGCCAAG 565 CUUGGCAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCUUCCA 1901
221 AAGCUGUU G CCAAGUUU 500 AAACUUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AACAGCUU 1902
226 GUUGCCAA G UUUGAUUU 566 AAAUCAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGGCAAC 1903
230 CCAAGUUU G AUUUCACU 501 AGUGAAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAACUUGG 1904
239 AUUUCACU G CUUCAGGU 502 ACCUGAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGUGAAAU 1905
245 CUGCUUCA G GUGAGGAU 750 AUCCUCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAAGCAG 1905
246 UGCUUCAG G UGAGGAUG 567 CAUCCUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGAAGCA 1907
248 CUUCAGGU G AGGAUGAA 503 UUCAUCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCUGAAG 1908
250 UCAGGUGA G GAUGAACU 751 AGUUCAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCACCUGA 1909 ∞
251 CAGGUGAG G AUGAACUG 752 CAGUUCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCACCUG 1910
254 GUGAGGAU G AACUGAGC 504 GCUCAGUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCCUCAC 1911
259 GAUGAACU G AGCUUUCA 505 UGAAAGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGUUCAUC 1912
261 UGAACUGA G CUUUCACA 568 UGUGAAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCAGUUCA 1913
272 UUCACACU G GAGAUGUU 753 AACAUCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGUGUGAA 1914
273 UCACACUG G AGAUGUUU 754 AAACAUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGUGUGA 1915
275 ACACUGGA G AUGUUUUG 755 CAAAACAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCAGUGU 1916
278 CUGGAGAU G UUUUGAAG 569 CUUCAAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCUCCAG 1917
283 GAUGUUUU G AAGAUUUU 506 AAAAUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAACAUC 1918
286 GUUUUGAA G AUUUUAAG 756 CUUAAAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCAAAAC 1919
294 GAUUUUAA G UAACCAAG 570 CUUGGUUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUAAAAUC 1920
302 GUAACCAA G AGGAGUGG 757 CCACUCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGGUUAC 1921
304 AACCAAGA G GAGUGGUU 758 AACCACUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUUGGUU 1922
305 ACCAAGAG G AGUGGUUU 759 AAACCACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCUUGGU 1923
307 CAAGAGGA G UGGUUUAA 571 UUAAACCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUCUUG 1924
309 AGAGGAGU G GUUUAAGG 760 CCUUAAAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUCCUCU 1925
310 GAGGAGUG G UUUAAGGC 572 GCCUUAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACUCCUC 1926
400.013
316 UGGUUUAA G GCGGAGCU 761 AGCUCCGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUAAACCA 1927
317 GGUUUAAG G CGGAGCUU 573 AAGCUCCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUAAACC 1928
319 UUUAAGGC G GAGCUUGG 762 CCAAGCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG- GCCUUAAA 1929
320 UUAAGGCG G AGCUUGGG 763 CCCAAGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGCCUUAA 1930
322 AAGGCGGA G CUUGGGAG 574 CUCCCAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCGCCUU 1931
326 CGGAGCUU G GGAGCCAG 764 CUGGCUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGCUCCG 1932
327 GGAGCUUG G GAGCCAGG 765 CCUGGCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAGCUCC 1933
328 GAGCUUGG G AGCCAGGA 766 UCCUGGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAAGCUC 1934
330 GCUUGGGA G CCAGGAAG 575 CUUCCUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCCAAGC 1935
334 GGGAGCCA G GAAGGAUA 767 UAUCCUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGCUCCC 1936
335 GGAGCCAG G AAGGAUAU 768 AUAUCCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGGCUCC 1937
338 GCCAGGAA G GAUAUGUG 769 CACAUAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCCUGGC 1938
339 CCAGGAAG G AUAUGUGC 770 GCACAUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUCCUGG 1939
344 AAGGAUAU G UGCCCAAG 576 CUUGGGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUAUCCUU 1940 o
346 GGAUAUGU G CCCAAGAA 507 UUCUUGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAUAUCC 1941 r
352 GUGCCCAA G AAUUUCAU 771 AUGAAAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGGGCAC 1942
362 AUUUCAUA G ACAUCCAG 772 CUGGAUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAUGAAAU 1943
370 GACAUCCA G UUUCCCAA 577 UUGGGAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGAUGUC 1944
381 UCCCAAAU G GUUUCACG 773 CGUGAAAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUUGGGA 1945
382 CCCAAAUG G UUUCACGA 578 UCGUGAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUUUGGG 1946
389 GGUUUCAC G AAGGCCUC 508 GAGGCCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUGAAACC 1947
392 UUCACGAA G GCCUCUCU 774 AGAGAGGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCGUGAA 1948
393 UCACGAAG G CCUCUCUC 579 GAGAGAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUCGUGA 1949
402 CCUCUCUC G ACACCAGG 509 CCUGGUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAGAGAGG 1950
409 CGACACCA G GCAGAGAA 775 UUCUCUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGUGUCG 1951
410 GACACCAG G CAGAGAAC 580 GUUCUCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGGUGUC 1952
413 ACCAGGCA G AGAACUUA 776 UAAGUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCCUGGU 1953
415 CAGGCAGA G AACUUACU 777 AGUAAGUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUGCCUG 1954
427 UUACUCAU G GGCAAGGA 778 UCCUUGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGAGUAA 1955
428 UACUCAUG G GCAAGGAG 779 CUCCUUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUGAGUA 1956
429 ACUCAUGG G CAAGGAGG 581 CCUCCUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAUGAGU 1957
433 AUGGGCAA G GAGGUUGG 780 CCAACCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGCCCAU 1958
434 UGGGCAAG G AGGUUGGC 781 GCCAACCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUGCCCA 1959
436 GGCAAGGA G GUUGGCUU 782 AAGCCAAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUUGCC 1960
400.013
437 GCAAGGAG G UUGGCUUC 582 GAAGCCAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCUUGC 1961
440 AGGAGGUU G GCUUCUUC 783 GAAGAAGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AACCUCCU 1962
441 GGAGGUUG G CUUCUUCA 583 UGAAGAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAACCUCC 1963
456 CAUCAUCC G GGCCAGCC 784 GGCUGGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGAUGAUG 1964
457 AUCAUCCG G GCCAGCCA 785 UGGCUGGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGGAUGAU 1965
458 UCAUCCGG G CCAGCCAG 584 CUGGCUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCGGAUGA 1966
462 CCGGGCCA G CCAGAGCU 585 AGCUCUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGCCCGG 1967
466 GCCAGCCA G AGCUCCCC 786 GGGGAGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGCUGGC 1968
468 CAGCCAGA G CUCCCCAG 586 CUGGGGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUGGCUG 1969
476 GCUCCCCA G GGGACUUC 787 GAAGUCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGGGAGC 1970
477 CUCCCCAG G GGACUUCU 788 AGAAGUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGGGGAG 1971
478 UCCCCAGG G GACUUCUC 789 GAGAAGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUGGGGA 1972
479 CCCCAGGG G ACUUCUCC 790 GGAGAAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCUGGGG 1973
494 CCAUCUCU G UCAGGCAU 587 AUGCCUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAGAUGG 1974
498 CUCUGUCA G GCAUGAGG 791 CCUCAUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGACAGAG 1975 oo
499 UCUGUCAG G CAUGAGGA 588 UCCUCAUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGACAGA 1976 ω
503 UCAGGCAU G AGGAUGAC 510 GUCAUCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGCCUGA 1977
505 AGGCAUGA G GAUGACGU 792 ACGUCAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCAUGCCU 1978
506 GGCAUGAG G AUGACGUU 793 AACGUCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCAUGCC 1979
509 AUGAGGAU G ACGUUCAA 511 UUGAACGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCCUCAU 1980
512 AGGAUGAC G UUCAACAC 589 GUGUUGAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUCAUCCU 1981
526 CACUUCAA G GUCAUGCG 794 CGCAUGAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGAAGUG 1982
527 ACUUCAAG G UCAUGCGA 590 UCGCAUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUGAAGU 1983
532 AAGGUCAU G CGAGACAA 512 UUGUCUCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGACCUU 1984
534 GGUCAUGC G AGACAACA 513 UGUUGUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCAUGACC 1985
536 UCAUGCGA G ACAACAAG 795 CUUGUUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCGCAUGA 1986
544 GACAACAA G GGUAAUUA 796 UAAUUACC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGUUGUC 1987
545 ACAACAAG G GUAAUUAC 797 GUAAUUAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUGUUGU 1988
546 CAACAAGG G UAAUUACU 591 AGUAAUUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUUGUUG 1989
559 UACUUUCU G UGGACUGA 592 UCAGUCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAAAGUA 1990
561 CUUUCUGU G GACUGAGA 798 UCUCAGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAGAAAG 1991
562 UUUCUGUG G ACUGAGAA 799 UUCUCAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACAGAAA 1992
566 UGUGGACU G AGAAGUUU 514 AAACUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGUCCACA 1993
568 UGGACUGA G AAGUUUCC 800 GGAAACUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCAGUCCA 1994
400.013
571 ACUGAGAA G UUUCCAUC 593 GAUGGAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUCAGU 1995
589 CUAAAUAA G CUGGUAGA 594 UCUACCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUAUUUAG 1996
592 AAUAAGCU G GUAGACUA 801 UAGUCUAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCUUAUU 1997
593 AUAAGCUG G UAGACUAC 595 GUAGUCUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGCUUAU 1998
596 AGCUGGUA G ACUACUAC 802 GUAGUAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UACCAGCU 1999
606 CUACUACA G GACAAAUU 803 AAUUUGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUAGUAG 2000
607 UACUACAG G ACAAAUUC 804 GAAUUUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGUAGUA 2001
624 CAUCUCCA G ACAGAAGC 805 GCUUCUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGAGAUG 2002
628 UCCAGACA G AAGCAGAU 806 AUCUGCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUCUGGA 2003
631 AGACAGAA G CAGAUCUU 596 AAGAUCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUGUCU 2004
634 CAGAAGCA G AUCUUCCU 807 AGGAAGAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCUUCUG 2005
645 CUUCCUUA G AGACAGAA 808 UUCUGUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAAGGAAG 2006
647 UCCUUAGA G ACAGAACC 809 GGUUCUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUAAGGA 2007
651 UAGAGACA G AACCCGAG 810 CUCGGGUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUCUCUA 2008
657 CAGAACCC G AGAAGACC 515 GGUCUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGUUCUG 2009
659 GAACCCGA G AAGACCAG 811 CUGGUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCGGGUUC 2010 oo
662 CCCGAGAA G ACCAGGGU 812 ACCCUGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUCGGG 2011
667 GAAGACCA G GGUCACCG 813 CGGUGACC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGUCUUC 2012
668 AAGACCAG G GUCACCGG 814 CCGGUGAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGGUCUU 2013
669 AGACCAGG G UCACCGGG 597 CCCGGUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUGGUCU 2014
675 GGGUCACC G GGGCAACA 815 UGUUGCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGUGACCC 2015
676 GGUCACCG G GGCAACAG 816 CUGUUGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGGUGACC 2016
677 GUCACCGG G GCAACAGC 817 GCUGUUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCGGUGAC 2017
678 UCACCGGG G CAACAGCC 598 GGCUGUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCGGUGA 2018
684 GGGCAACA G CCUGGACC 599 GGUCCAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUUGCCC 2019
688 AACAGCCU G GACCGGAG 818 CUCCGGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGCUGUU 2020
689 ACAGCCUG G ACCGGAGG 819 CCUCCGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGGCUGU 2021
693 CCUGGACC G GAGGUCCC 820 GGGACCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGUCCAGG 2022
694 CUGGACCG G AGGUCCCA 821 UGGGACCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGGUCCAG 2023
696 GGACCGGA G GUCCCAGG 822 CCUGGGAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCGGUCC 2024
697 GACCGGAG G UCCCAGGG 600 CCCUGGGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCGGUC 2025
703 AGGUCCCA G GGAGGCCC 823 GGGCCUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGGACCU 2026
704 GGUCCCAG G GAGGCCCA 824 UGGGCCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGGGACC 2027
705 GUCCCAGG G AGGCCCAC 825 GUGGGCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUGGGAC 2028
400.013
707 CCCAGGGA G GCCCACAC 826 GUGUGGGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCCUGGG 2029
708 CCAGGGAG G CCCACACC 601 GGUGUGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCCUGG 2030
720 ACACCUCA G UGGGGCUG 602 CAGCCCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAGGUGU 2031
722 ACCUCAGU G GGGCUGUG 827 CACAGCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUGAGGU 2032
723 CCUCAGUG G GGCUGUGG 828 CCACAGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACUGAGG 2033
724 CUCAGUGG G GCUGUGGG 829 CCCACAGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCACUGAG 2034
725 UCAGUGGG G CUGUGGGA 603 UCCCACAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCACUGA 2035
728 GUGGGGCU G UGGGAGAA 604 UUCUCCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCCCCAC 2036
730 GGGGCUGU G GGAGAAGA 830 UCUUCUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAGCCCC 2037
731 GGGCUGUG G GAGAAGAA 831 UUCUUCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACAGCCC 2038
732 GGCUGUGG G AGAAGAAA 832 UUUCUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCACAGCC 2039
734 CUGUGGGA G AAGAAAUC 833 GAUUUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCCACAG 2040
737 UGGGAGAA G AAAUCCGA 834 UCGGAUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUCCCA 2041
744 AGAAAUCC G ACCUUCGA 516 UCGAAGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGAUUUCU 2042
751 CGACCUUC G AUGAACCG 517 CGGUUCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAAGGUCG 2043 o
754 CCUUCGAU G AACCGGAA 518 UUCCGGUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCGAAGG 2044
759 GAUGAACC G GAAGCUGU 835 ACAGCUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGUUCAUC 2045
760 AUGAACCG G AAGCUGUC 836 GACAGCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGGUUCAU 2046
763 AACCGGAA G CUGUCGGA 605 UCCGACAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCCGGUU 2047
766 CGGAAGCU G UCGGAUCA 606 UGAUCCGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCUUCCG 2048
769 AAGCUGUC G GAUCACCC 837 GGGUGAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GACAGCUU 2049
770 AGCUGUCG G AUCACCCC 838 GGGGUGAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGACAGCU 2050
781 CACCCCCC G ACCCUUCC 519 GGAAGGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGGGGUG 2051
793 CUUCCCCU G CAGCAGCA 520 UGCUGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGGGAAG 2052
796 CCCCUGCA G CAGCACCA 607 UGGUGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCAGGGG 2053
799 CUGCAGCA G CACCAGCA 608 UGCUGGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCUGCAG 2054
805 CAGCACCA G CACCAGCC 609 GGCUGGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGUGCUG 2055
811 CAGCACCA G CCACAGCC 610 GGCUGUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGUGCUG 2056
817 CAGCCACA G CCUCCGCA 611 UGCGGAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUGGCUG 2057
823 CAGCCUCC G CAAUAUGC 521 GCAUAUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGAGGCUG 2058
830 CGCAAUAU G CCCCAGCG 522 CGCUGGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUAUUGCG 2059
836 AUGCCCCA G CGCCCCAG 612 CUGGGGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGGGCAU 2060
838 GCCCCAGC G CCCCAGCA 523 UGCUGGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCUGGGGC 2061
844 GCGCCCCA G CAGCUGCA 613 UGCAGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGGGCGC 2062
400.013
847 CCCCAGCA G CUGCAGCA 614 UGCUGCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCUGGGG 2063
850 CAGCAGCU G CAGCAGCG 524 GGCUGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCUGCUG 2064
853 CAGCUGCA G CAGCCCCC 615 GGGGGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCAGCUG 2065
856 CUGCAGCA G CCCCCACA 616 UGUGGGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCUGCAG 2066
865 CCCCCACA G CAGCGAUA 617 UAUCGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUGGGGG 2067
868 CCACAGCA G CGAUAUCU 618 AGAUAUCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCUGUGG 2068
870 ACAGCAGC G AUAUCUGC 525 GCAGAUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCUGCUGU 2069
877 CGAUAUCU G CAGCACCA 526 UGGUGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAUAUCG 2070
880 UAUCUGCA G CAGCACCA 619 UGGUGGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCAGAUA 2071
898 UUCCACCA G GAACGCCG 839 CGGCGUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGUGGAA 2072
899 UCCACCAG G AACGCCGA 840 UCGGCGUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGGUGGA 2073
903 CCAGGAAC G CCGAGGAG 527 CUCCUCGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUUCCUGG 2074
906 GGAACGCC G AGGAGGCA 528 UGCCUCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGCGUUCC 2075
908 AACGCCGA G GAGGCAGC 841 GCUGCCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCGGCGUU 2076
909 ACGCCGAG G AGGCAGCC 842 GGCUGCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCGGCGU 2077 o
911 GCCGAGGA G GCAGCCUU 843 AAGGCUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUCGGC 2078
912 CCGAGGAG G CAGCCUUG 620 CAAGGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCUCGG 2079
915 AGGAGGCA G CCUUGACA 621 UGUCAAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCCUCCU 2080
920 GCAGCCUU G ACAUAAAU 529 AUUUAUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGGCUGC 2081
929 ACAUAAAU G AUGGGCAU 530 AUGCCCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUUAUGU 2082
932 UAAAUGAU G GGCAUUGU 844 ACAAUGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCAUUUA 2083
933 AAAUGAUG G GCAUUGUG 845 CACAAUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUCAUUU 2084
934 AAUGAUGG G CAUUGUGG 622 CCACAAUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAUCAUU 2085
939 UGGGCAUU G UGGCACCG 623 CGGUGCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAUGCCCA 2086
941 GGCAUUGU G GCACCGGC 846 GCCGGUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAAUGCC 2087
942 GCAUUGUG G CACCGGCU 624 AGCCGGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACAAUGC 2088
947 GUGGCACC G GCUUGGGC 847 GCCCAAGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGUGCCAC 2089
948 UGGCACCG G CUUGGGCA 625 UGCCCAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGGUGCCA 2090
952 ACCGGCUU G GGCAGUGA 848 UCACUGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGCCGGU 2091
953 CCGGCUUG G GCAGUGAA 849 UUCACUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAGCCGG 2092
954 CGGCUUGG G CAGUGAAA 626 UUUCACUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAAGCCG 2093
957 CUUGGGCA G UGAAAUGA 627 UCAUUUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCCCAAG 2094
959 UGGGCAGU G AAAUGAAU 531 AUUCAUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUGCCCA 2095
964 AGUGAAAU G AAUGCGGC 532 GCCGCAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUUCACU 2096
400.013
968 AAAUGAAU G CGGCCCUC 533 GAGGGCCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUCAUUU 2097
970 AUGAAUGC G GCCCUCAU 850 AUGAGGGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCAUUCAU 2098
971 UGAAUGCG G CCCUCAUG 628 CAUGAGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGCAUUCA 2099
979 GCCCUCAU G CAUCGGAG 534 CUCCGAUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGAGGGC 2100
984 CAUGCAUC G GAGACACA 851 UGUGUCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAUGCAUG 2101
985 AUGCAUCG G AGACACAC 852 GUGUGUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGAUGCAU 2102
987 GCAUCGGA G ACACACAG 853 CUGUGUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCGAUGC 2103
995 GACACACA G ACCCAGUG 854 CACUGGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUGUGUC 2104
1001 CAGACCCA G UGCAGCUC 629 GAGCUGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGGUCUG 2105
1003 GACCCAGU G CAGCUCCA 535 UGGAGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUGGGUC 2106
1006 CCAGUGCA G CUCCAGGC 630 GCCUGGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCACUGG 2107
1012 CAGCUCCA G GCGGCAGG 855 CCUGCCGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGAGCUG 2108
1013 AGCUCCAG G CGGCAGGG 631 CCCUGCCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGGAGCU 2109
1015 CUCCAGGC G GCAGGGCG 856 CGCCCUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCCUGGAG 2110 oo
1016 UCCAGGCG G CAGGGCGA 632 UCGCCCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGCCUGGA 2111
1019 AGGCGGCA G GGCGAGUG 857 CACUCGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCCGCCU 2112
1020 GGCGGCAG G GCGAGUGC 858 GCACUCGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGCCGCC 2113
1021 GCGGCAGG G CGAGUGCG 633 CGCACUCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUGCCGC 2114
1023 GGCAGGGC G AGUGCGGU 536 ACCGCACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCCCUGCC 2115
1025 CAGGGCGA G UGCGGUGG 634 CCACCGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCGCCCUG 2116
1027 GGGCGAGU G CGGUGGGC 537 GCCCACCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUCGCCC 2117
1029 GCGAGUGC G GUGGGCCC 859 GGGCCCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCACUCGC 2118
1030 CGAGUGCG G UGGGCCCG 635 CGGGCCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGCACUCG 2119
1032 AGUGCGGU G GGCCCGGG 860 CCCGGGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCGCACU 2120
1033 GUGCGGUG G GCCCGGGC 861 GCCCGGGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACCGCAC 2121
1034 UGCGGUGG G CCCGGGCG 636 CGCCCGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCACCGCA 2122
1038 GUGGGCCC G GGCGCUGU 862 ACAGCGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGCCCAC 2123
1039 UGGGCCCG G GCGCUGUA 863 UACAGCGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGGGCCCA 2124
1040 GGGCCCGG G CGCUGUAU 637 AUACAGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCGGGCCC 2125
1042 GCCCGGGC G CUGUAUGA 538 UCAUACAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCCCGGGC 2126
1045 CGGGCGCU G UAUGACUU 638 AAGUCAUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCGCCCG 2127
1049 CGCUGUAU G ACUUUGAG 539 CUCAAAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUACAGCG 2128
1055 AUGACUUU G AGGCCCUG 540 CAGGGCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAGUCAU 2129
1057 GACUUUGA G GCCCUGGA 864 UCCAGGGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCAAAGUC 2130
400.013
1058 ACUUUGAG G CCCUGGAG 639 CUCCAGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCAAAGU 2131
1063 GAGGCCCU G GAGGAUGA 865 UCAUCCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGGCCUC 2132
1064 AGGCCCUG G AGGAUGAC 866 GUCAUCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGGGCCU 2133
1066 GCCCUGGA G GAUGACGA 867 UCGUCAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCAGGGC 2134
1067 CCCUGGAG G AUGACGAG 868 CUCGUCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCAGGG 2135
1070 UGGAGGAU G ACGAGCUG 541 CAGCUCGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCCUCCA 2136
1073 AGGAUGAC G AGCUGGGG 542 CCCCAGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUCAUCCU 2137
1075 GAUGACGA G CUGGGGUU 640 AACCCCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCGUCAUC 2138
1078 GACGAGCU G GGGUUCCA 869 UGGAACCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCUCGUC 2139
1079 ACGAGCUG G GGUUCCAC 870 GUGGAACC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGCUCGU 2140
1080 CGAGCUGG G GUUCCACA 871 UGUGGAAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAGCUCG 2141
1081 GAGCUGGG G UUCCACAG 641 CUGUGGAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCAGCUC 2142
1089 GUUCCACA G CGGGGAGG 642 CCUCCCCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUGGAAC 2143
1091 UCCACAGC G GGGAGGUG 872 CACCUCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCUGUGGA 2144
1092 CCACAGCG G GGAGGUGG 873 CCACCUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGCUGUGG 2145 oo oo
1093 CACAGCGG G GAGGUGGU 874 ACCACCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCGCUGUG 2146
1094 ACAGCGGG G AGGUGGUG 875 CACCACCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCGCUGU 2147
1096 AGCGGGGA G GUGGUGGA 876 UCCACCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCCCGCU 2148
1097 GCGGGGAG G UGGUGGAG 643 CUCCACCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCCCGC 2149
1099 GGGGAGGU G GUGGAGGU 877 ACCUCCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCUCCCC 2150
1100 GGGAGGUG G UGGAGGUC 644 GACCUCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACCUCCC 2151
1102 GAGGUGGU G GAGGUCCU 878 AGGACCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCACCUC 2152
1103 AGGUGGUG G AGGUCCUG 879 CAGGACCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACCACCU 2153
1105 GUGGUGGA G GUCCUGGA 880 UCCAGGAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCACCAC 2154
1106 UGGUGGAG G UCCUGGAU 645 AUCCAGGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCACCA 2155
1111 GAGGUCCU G GAUAGCUC 881 GAGCUAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGACCUC 2156
1112 AGGUCCUG G AUAGCUCC 882 GGAGCUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGGACCU 2157
1116 CCUGGAUA G CUCCAACC 646 GGUUGGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAUCCAGG 2158
1131 CCCAUCCU G GUGGACCG 883 CGGUCCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGAUGGG 2159
1132 CCAUCCUG G UGGACCGG 647 CCGGUCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGGAUGG 2160
1134 AUCCUGGU G GACCGGCC 884 GGCCGGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCAGGAU 2161
1135 UCCUGGUG G ACCGGCCG 885 CGGCCGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACCAGGA 2162
1139 GGUGGACC G GCCGCCUG 886 CAGGCGGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGUCCACC 2163
1140 GUGGACCG G CCGCCUGC 648 GCAGGCGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGGUCCAC 2164
400.013
1143 GACCGGCC G CCUGCACA 543 UGUGCAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGCCGGUC 2165
1147 GGCCGCCU G CACAACAA 544 UUGUUGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGCGGCC 2166
1156 CACAACAA G CUGGGCCU 649 AGGCCCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGUUGUG 2167
1159 AACAAGCU G GGCCUCUU 887 AAGAGGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCUUGUU 2168
1160 ACAAGCUG G GCCUCUUC 888 GAAGAGGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGCUUGU 2169
1161 CAAGCUGG G CCUCUUCC 650 GGAAGAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAGCUUG 2170
1172 UCUUCCCU G CCAACUAC 545 GUAGUUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGGAAGA 2171
1181 CCAACUAC G UGGCACCC 651 GGGUGCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUAGUUGG 2172
1183 AACUACGU G GCACCCAU 889 AUGGGUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACGUAGUU 2173
1184 ACUACGUG G CACCCAUG 652 CAUGGGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACGUAGU 2174
1192 GCACCCAU G ACCCGAUA 546 UAUCGGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGGGUGC 2175
1197 CAUGACCC G AUAAACUC 547 GAGUUUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGUCAUG 2176
1210 ACUCUUCA G GGGACAGA 890 UCUGUCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAAGAGU 2177
1211 CUCUUCAG G GGACAGAA 891 UUCUGUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGAAGAG 2178
1212 UCUUCAGG G GACAGAAG 892 CUUCUGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUGAAGA 2179 o
1213 CUUCAGGG G ACAGAAGC 893 GCUUCUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCUGAAG 2180
1217 AGGGGACA G AAGCUUUU 894 AAAAGCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUCCCCU 2181
1220 GGACAGAA G CUUUUUGU 653 ACAAAAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUGUCC 2182
1227 AGCUUUUU G UCUGGAGC 654 GCUCCAGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAAAAGCU 2183
1231 UUUUGUCU G GAGCUGCC 895 GGCAGCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGACAAAA 2184
1232 UUUGUCUG G AGCUGCCC 896 GGGCAGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGACAAA 2185
1234 UGUCUGGA G CUGCCCAC, 655 GUGGGCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCAGACA 2186
1237 CUGGAGCU G CCCACAAG 548 CUUGUGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCUCCAG 2187
1245 GCCCACAA G AAAGAGGG 897 CCCUCUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGUGGGC 2188
1249 ACAAGAAA G AGGGCAAG 898 CUUGCCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUCUUGU 2189
1251 AAGAAAGA G GGCAAGGA 899 UCCUUGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUUUCUU 2190
1252 AGAAAGAG G GCAAGGAA 900 UUCCUUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCUUUCU 2191
1253 GAAAGAGG G CAAGGAAA 656 UUUCCUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ccucuuuc 2192
1257 GAGGGCAA G GAAAAAAG 901 CUUUUUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGCCCUC 2193
1258 AGGGCAAG G AAAAAAGG 902 CCUUUUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUGCCCU 2194
1265 GGAAAAAA G GCUGGACU 903 AGUCCAGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG uuuuuucc 2195
1266 GAAAAAAG G CUGGACUC 657 GAGUCCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUUUUUC 2196
1269 AAAAGGCU G GACUCCAU 904 AUGGAGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCCUUUU 2197
1270 AAAGGCUG G ACUCCAUG 905 CAUGGAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGCCUUU 2198
400.013
1278 GACUCCAU G ACUAUAUA 549 UAUAUAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGGAGUC 2199
Input Sequence = HSA011736. Cut Site = G/ .
Stem Length = 8. Core Sequence = GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG HΞA011736 (Homo sapiens mRNA for growth factor receptor binding protein (GRBLG) ; 1303 bp)
400.013
Table IX: Human GRID GeneBloc and Substrate Sequence
Figure imgf000092_0001
Upper Case = Ribo
400.013
Lower Case = 2 -O-Methyl s = phosphorothioate linkage
B = inverted deoxyabasic ribonucleotide
Input Sequence = HSA011736 GB Length = 23
HSA011736 (Homo sapiens mRNA for growth, factor receptor binding protein (GRBLG) ; 1303 bp)
C r
400.013
Table X: Human Grid Enzymatic nucleic acid and target sequence
Figure imgf000094_0001
Lower case = 2'-0-methyl nucleotide Upper case = ribonucleotide
B = inverted deoxyabasic moiety

Claims

CLAIMS What is claimed is:
1. A nucleic acid molecule which down regulates expression of a Grb2 -related with Insert
Domain (GRID) gene.
2. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is used to treat conditions selected from the group consisting of tissue/graft rejection and leukemia.
3. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is an enzymatic nucleic acid molecule having at least one binding arm.
4. The nucleic acid molecule of claim 3, wherein one or more binding arms of the enzymatic nucleic acid molecule comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOS. 1-905 and 2256-2279.
5. The nucleic acid molecule of claim 3, wherein the enzymatic nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NOS. 906-2199 and 2280-2304.
6. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is an antisense nucleic acid molecule.
7. The nucleic acid molecule of claim 6, wherein said antisense nucleic acid molecule comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOS. 1-905, 2200-2211 and 2256-2279
8. The nucleic acid molecule of claim 6, wherein said antisense nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NOS. 2212-2235.
9. The nucleic acid molecule of claim 3, wherein said enzymatic nucleic acid molecule is in a hammerhead (HH) motif.
10. The nucleic acid molecule of claim 3, wherein said enzymatic nucleic acid molecule is in a hairpm, hepatitis Delta virus, group I intron, VS nucleic acid, amberzyme, zinzyme or
RNAse P nucleic acid motif.
11. The nucleic acid molecule of claim 3, wherein said enzymatic nucleic acid molecule is in an Inozyme motif.
12. The nucleic acid molecule of claim 3, wherein said enzymatic nucleic acid molecule is in a G-cleaver motif.
13. The nucleic acid molecule of claim 3, wherein said enzymatic nucleic acid molecule is a DNAzyme.
14. The nucleic acid molecule of claim 3, wherein said enzymatic nucleic acid molecule comprises between 12 and 100 bases complementary to the RNA of a GRID gene.
15. The nucleic acid molecule of claim 3, wherein said enzymatic nucleic acid molecule comprises between 14 and 24 bases complementary to the RNA of a GRID gene.
16. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is chemically synthesized.
17. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least one 2 '-sugar modification.
18. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least one nucleic acid base modification.
19. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least one phosphate backbone modification.
20. A mammalian cell including the nucleic acid molecule of claim 1.
21. The mammalian cell of claim 20, wherein said mammalian cell is a human cell.
22. A method of reducing GRID activity in a cell comprising the step of contacting said cell with the nucleic acid molecule of claim 1 under conditions suitable for said reduction of GRID activity.
23. A method of treatment of a patient having a condition associated with the level of GRID, comprising contacting cells of said patient with the nucleic acid molecule of claim 1, under conditions suitable for said treatment.
24. The method of claim 23 further comprising the use of one or more therapies under conditions suitable for said treatment.
25. A method of cleaving RNA of a GRID gene comprising the step of contacting the nucleic acid molecule of claim 1 with said RNA under conditions suitable for the cleavage of said RNA.
26. The method of claim 25, wherein said cleavage is carried out in the presence of a divalent cation.
27. The method of claim 26, wherein said divalent cation is Mg2+.
28. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a cap structure at the 5 '-end, the 3 '-end or both the 5 '-end and the 3 '-end.
29. The nucleic acid molecule of claim 9, wherein one or more binding arms of the hammerhead motif comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOS. 1-179 and 2256-2260.
30. The nucleic acid molecule of claim 11, wherein one or more binding arms of the NCH motif comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOS. 180-492 and 2261-2265.
31. The nucleic acid molecule of claim 12, wherein one or more binding arms of the G- cleaver motif comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOS. 493-657.
32. The nucleic acid molecule of claim 13, wherein one or more binding arms of the DNAzyme comprises a sequence complementary to a sequence selected from the group consisting of substrate sequences shown in Table VII.
33. The nucleic acid molecule of claim 10, wherein one or more binding arms of the zinzyme comprises a sequence complementary to a sequence selected from the group consisting of substrate sequences shown in Table VI.
34. The nucleic acid molecule of claim 10, wherein one or more binding arms of the amberzyme comprises a sequence complementary to a sequence selected from the group consisting of substrate sequences shown in Table VIII.
35. An expression vector comprising a nucleic acid sequence encoding at least one nucleic acid molecule of claim 1 in a manner which allows expression of the nucleic acid molecule.
36. A mammalian cell including the expression vector of claim 35.
37. The mammalian cell of claim 36, wherein said mammalian cell is a human cell.
38. The expression vector of claim 35, wherein said nucleic acid molecule is an enzymatic nucleic acid molecule.
39. The expression vector of claim 35, wherein said expression vector further comprises a sequence for an antisense nucleic acid molecule complementary to the RNA of a GRID gene.
40. The expression vector of claim 35, wherein said expression vector comprises a sequence encoding two or more of said nucleic acid molecules, which may be the same or different.
41. The expression vector of claim 40, wherein said expression vector comprises a nucleic acid sequence encoding an antisense nucleic acid molecule complementary to the RNA of a GRID gene.
42. The expression vector of claim 40, wherein said expression vector comprises a nucleic acid sequence encoding an enzymatic nucleic acid molecule complementary to the RNA of a GRID gene.
43. A method for treatment of tissue/graft rejection comprising the step of administering to a patient the nucleic acid molecule of claim 1 under conditions suitable for said treatment.
44. A method for treatment of leukemia comprising the step of administering to a patient the nucleic acid molecule of claim 1 under conditions suitable for said treatment.
45. An enzymatic nucleic acid molecule which cleaves RNA derived from a GRID gene.
46. The enzymatic nucleic acid molecule of claim 45, wherein said enzymatic nucleic acid molecule is selected from the group consisting of Hammerhead, Hairpin, Inozyme, G- cleaver, DNAzyme, Amberzyme and Zinzyme.
47. The method of any of claims 43 or 44, wherein said method further comprises administering to said patient one or more other therapies.
48. The method of claim 47, wherein said other therapies are therapies selected from the group consisting of radiation, chemotherapy, and cyclosporin treatment.
49. The nucleic acid molecule of claim 7, wherein said nucleic acid molecule comprises at least five ribose residues, at least ten 2'-( -methyl modifications, and a 3'- end modification.
50. The nucleic acid molecule of claim 49, wherein said nucleic acid molecule further comprises a phosphorothioate core with a 3' and a 5' -end modification.
51. The nucleic acid molecule of any of claims 49 and 50, wherein said 3' and/or 5'- end modification is 3 '-3' inverted abasic moiety.
52. The nucleic acid molecule of claim 3, wherein said nucleic acid molecule comprises at least five ribose residues, at least ten 2'-<9-methyl modifications, and a 3'- end modification.
53. The nucleic acid molecule of claim 52, wherein said nucleic acid molecule further comprises phosphorothioate linkages on at least three of the 5' terminal nucleotides.
54. The nucleic acid molecule of claim 52, wherein said 3'- end modification is 3 '-3' inverted abasic moiety.
55. The enzymatic nucleic acid molecule of claim 13, wherein said DNAzyme comprises at least ten 2,-< -methyl modifications and a 3 '-end modification.
56. The enzymatic nucleic acid molecule of claim 55, wherein said DNAzyme further comprises phosphorothioate linkages on at least three of the 5' terminal nucleotides.
57. The enzymatic nucleic acid molecule of claim 55, wherein said 3'- end modification is 3 '-3' inverted abasic moiety.
PCT/US2001/005957 2000-02-24 2001-02-23 Antisense and catalytically acting nucleic acid molecules targeted to grb2- related with insert domain (grid) proteins and their uses WO2001062911A2 (en)

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