JP5252622B2 - Double-stranded RNA capable of expressing high nuclease resistance and excellent RNA interference effect - Google Patents

Description

The present invention relates to a modified RNA that can effectively suppress the expression of a target gene. More particularly, the present invention is 23-29 bases having a sequence complementary to the sense strand RN A及 beauty the sense strand RNA consisting of 21 to 27 bases (sequence complementary to a target sequence in a target gene) It is a double-stranded RNA having an antisense strand RNA consisting of the above-mentioned modified RNA having a high nuclease resistance and an excellent RNA interference effect.

  The development of a medicine that efficiently treats intractable diseases such as cancer and AIDS is a major issue in the life science field. One of the promising methods that can overcome this problem is a gene drug that acts only on a specific gene. Among these gene medicines, the RNA interference (RNAi) method using a short double-stranded RNA (siRNA) of 21 bases has recently attracted attention. This RNAi method was first reported by Fire et al. In 1998 (see Non-Patent Document 1). According to the report of Fire et al., By introducing a double-stranded RNA of about 100 base pairs homologous to a specific region of the gene whose function is to be inhibited into the cell, 2 to about 20-25 base pairs by the action of Dicer in the cytoplasm. It is decomposed into single-stranded RNA, and then forms multiple RNA / protein complexes with this protein (this complex is called RICS: RNA-induced silencing complex) and binds to the homologous site of mRNA produced from the target gene. It strongly suppresses gene expression. However, in mammalian cells, when a long double-stranded RNA of about 30 base pairs or more is introduced, an interferon reaction, which is a viral response reaction, is induced, resulting in cell death. In mammalian cell systems, The RNAi method was considered difficult to apply. Therefore, Tuschl et al. Chemically synthesized a 21-base long double-stranded RNA with a dangling end (overhang) at the 3 'end and introduced it directly into mammalian cells, thereby avoiding the interferon response and sequence-specificity. It was reported that the gene expression suppression ability was high (refer nonpatent literature 2). They also synthesize short double-stranded RNAs with double-stranded regions of 19 base pairs and various dangling end strands at the 3 'end or 5' end to examine the RNA interference effect. As a result, a 21-base siRNA with a 2-base dangling end at the 3 'end of the sense and antisense strands showed a very high RNA interference effect, but all other types of short double strands. No significant RNA interference effect was observed in RNA. According to this report, RNA interferometry using double-stranded RNA that is 21 bases long and has a 2 base dangling end at the 3 'end of both the sense strand and antisense strand is now common. . Here, the method of inhibiting target gene expression using a short double-stranded RNA having a length of 21 bases is referred to as the siRNA method in distinction from the RNAi method.

  Since this siRNA method uses synthetic RNA, sample preparation is relatively easy, the handling operation is simple, and it has a very powerful effect, so it attracts great attention not only in the life science field but also in the biobusiness field. ing.

However, this excellent siRNA method also has a problem that must be solved. The siRNA as described above are composed of RNA molecules are rapidly degraded by the action of nuclease contained in cells and in culture ground. The double-stranded RNA region shows relatively high nuclease resistance compared to single-stranded RNA, but double-stranded RNA consisting of 19 base pairs hardly shows the conventional RNA interference effect. Therefore, synthetic siRNA shows a high gene expression suppression effect for about 2 days to 4 days after introduction into cells with the target gene sequence, but after that, the RNA interference effect declines sharply, and after about 7 days the RNA interference effect Has been reported to almost disappear.

  Recently, various chemically modified siRNAs have been reported in order to obtain a long-term and highly active RNA interference effect in synthetic siRNAs with excellent cell transduction properties. For example, in order to acquire degradation resistance from exonuclease, siRNA in which the end of siRNA is modified with an amino group, a thiol group, or basic is synthesized. However, it has already been reported that the RNA interference effect is significantly reduced in most cases of 21-base siRNA modified at the ends.

  Moreover, in order to give cell introduction property and tissue selectivity, cholesterol, long-chain alkyl, sugar chain, peptide and the like modified at the end of siRNA have been reported. SiRNA modified with cholesterol and long alkyl chain has not only improved cell transferability but also has been observed to accumulate in the liver in vivo. In addition, an improvement in cell introduction property has been observed in siRNA modified with sugar chains and peptides. However, in any case, compared with siRNA not modified, the same or lower RNA interference effect was observed, and nuclease resistance was not acquired.

  In addition, double-stranded RNA that exerts an RNA interference effect generally employs a structure having a dangling end at the end, but does not have a dangling end at the end (that is, a structure having a blunt end). ) Is also being studied for RNA interference effects. However, it is suggested that the RNA interference effect is hardly improved or the RNA interference effect is reduced by simply making the 3 ′ end of the sense strand RNA a blunt end (see Non-Patent Document 3). ).

  Furthermore, by combining functional molecules (protein, peptide, cholesterol, etc.) with double-stranded RNA that exhibits RNA interference effects, in addition to RNA interference effects, useful effects based on the functional molecules are also exhibited. It is expected that However, it has been found that simply binding a functional molecule to a double-stranded RNA that exerts an RNA interference effect leads to a significant attenuation of the RNA interference effect. The present situation is that functional molecule-modified RNA having useful effects based on molecules has not been constructed.

As described above, in the prior art, what base length RNA and what structure RNA should be adopted in order to provide excellent nuclease resistance and RNA interference effect for double-stranded RNA that exhibits RNA interference effect? The current situation has not been elucidated.
Fire et.al, Nature, 391, 806-811 (1998) Tuschl et.al., EMBO Journal, 20, 6877-6888 (2001) JT Marques et.al., Nature Biotech., 24, 559-565 (2005)

  An object of the present invention is to provide a novel double-stranded RNA that has high nuclease resistance and can exhibit an excellent RNA interference effect. Furthermore, an object of the present invention is to provide a functional molecule-modified double-stranded RNA having both an excellent RNA interference effect and a useful effect based on a functional molecule.

The present inventors have, when intensive studies for solving the above problems, the antisense strand RNA containing a base sequence complementary to a target sequence in a target gene, and a nucleotide sequence complementary to the antisense strand RNA the have a free Muse Nsu strand RNA, in a double-stranded RNA capable of suppressing the expression of the target gene, by including all the following features (i) ~ (iii), high nuclease resistance, even better RNA It was found that RNA having an interference effect can be obtained.
(i) The sense strand RNA is composed of 21 to 27 nucleotides, and the antisense strand RNA is composed of 23 to 29 nucleotides.
(ii) The 3 ′ end of the sense strand RNA is a blunt end, and the 3 ′ end of the antisense strand has a dangling end.
(iii) A substituent is bonded only to at least one of the first to sixth nucleotides from the 3 ′ end side of the sense strand RNA.

The present invention has been completed by making further improvements based on such findings. That is, the present invention provides the following modified RNAs.
Item 1. Antisense strand RNA comprising a nucleotide sequence complementary to a target sequence in a target gene, and has a ruse Nsu stranded RNA having a nucleotide sequence complementary to the antisense strand RNA, and the expression of the target gene A double-stranded RNA that can be suppressed, wherein the sense strand RNA consists of 21 to 27 nucleotides, and the antisense strand RNA consists of 23 to 29 nucleotides,
The 3 ′ end side of the sense strand RNA is a blunt end, the 3 ′ end of the antisense strand has a dangling end, and the 1st to 6th nucleotides from the 3 ′ end side of the sense strand RNA A modified RNA, wherein a substituent is bonded to at least one.
Item 2. Item 2. The modified RNA according to Item 1, wherein the sense strand RNA is composed of 21 nucleotides, and the antisense strand RNA is composed of 23 nucleotides.
Item 3. Item 2. The modified RNA according to Item 1, wherein the sense strand RNA is composed of 23 nucleotides, and the antisense strand RNA is composed of 25 nucleotides.
Item 4. Item 2. The modified RNA according to Item 1, wherein the sense strand RNA is composed of 25 nucleotides, and the antisense strand RNA is composed of 27 nucleotides.
Item 5. Item 5. The modified RNA according to any one of Items 1 to 4, wherein the first and second nucleotides from the 3 ′ end side of the sense strand RNA are composed of deoxyribonucleotides.
Item 6. Item 6. The modified RNA according to any one of Items 1 to 5, wherein one substituent is bound only to the first nucleotide from the 3 ′ end of the sense strand RNA.
Item 7. Item 7. The modified RNA according to any one of Items 1 to 6, wherein the substituent is an aminoalkyl group, DNA, a group having a lipid, or a group having cholesterol.
Item 8. Item 7. The modified form according to any one of Items 1 to 6, wherein the substituent is a group having an aminoalkyl group having 1 to 40 carbon atoms, a DNA having a base length of 5 to 50, or a fatty acid having 6 to 50 carbon atoms. RNA.
Item 9. Item 8. The lipid-modified double-stranded RNA according to Item 7, wherein the group having lipid is a group having lauric acid, stearic acid, myristylic acid, or palmitic acid.

The modified RNA of the present invention comprises: (i) the sense strand RNA is composed of 21 to 27 nucleotides, and the antisense strand RNA is composed of 23 to 29 nucleotides. (Ii) 3 ′ of the sense strand RNA The terminal side is a blunt end, the 3 ′ end of the antisense strand has a dangling end, and (iii) at least one of the first to sixth nucleotides from the 3 ′ end side of the sense strand RNA It satisfies all three characteristics that the substituent is bonded only to this. By adopting such a configuration, the modified RNA of the present invention can achieve a dramatic improvement in RNA interference effect and can also have excellent nuclease resistance. Therefore, the modified RNA of the present invention can be applied as a gene drug effective for the treatment of diseases such as cancer and AIDS.

Modified RNA of the present invention is composed of 21 to 27 nucleotides, comprising a sense strand RNA that is homologous to the nucleotide sequence to a target sequence in a target gene.

  Here, the target gene is a gene whose gene expression is to be suppressed by the RNA interference effect. In the modified RNA of the present invention, the target gene is not particularly limited, and can be appropriately selected based on the use of the modified RNA.

  The target sequence in the target gene is not particularly limited as long as it is a sequence capable of suppressing gene expression due to RNA interference effect, and is appropriately determined by a known method, specifically using NCBI BLAST search or the like. Can do. For example, a region consisting of 19 to 30 bases following the base “AA” in the exon portion 50 to 100 bases downstream from the start codon of the target gene coding region (ORF) and having a GC content of around 50% May be the target sequence. Employing such a complementary strand to the target sequence has empirically revealed in the art that an excellent RNA interference effect can be obtained. Further, for example, the target sequence can be set according to the manual (Dicer Substrate RNAi Design) of IDT (Integrated DNA Technologies, INC). Recently, (i) the 5 ′ end of the antisense strand RNA is an A / U pair, (ii) the 5 ′ end of the sense strand RNA is a G / C pair, and (iii) the antisense strand RNA 5 'It has a high RNA interference effect by designing double-stranded RNA that has about 5 A / U pairs on the end side and (vi) no more than 9 G / C pairs in the double strand 2 It has been reported that single-stranded RNA can be designed (Ui-Tei et. Al, Nucleic Acids Res., 32, 936-948 (2004)).

Sense strand RNA in modified RNA of the present invention is a sequence consisting of homologous 21-27 nucleotides in the target sequence in the target gene. Here, the number of nucleotides of the sense strand RNA referred means the number of nucleotides constituting the homologous nucleotide sequence to the target sequence in the target gene, the sense strand RNA nucleic acid such as DNA or PNA as a substituent The number of nucleotides constituting these substituents is not included.

  From the viewpoint of providing an excellent RNA interference effect with the modified RNA of the present invention, the number of nucleotides constituting the sense strand RNA is preferably 21, 23, 27 or 25, more preferably 21, 23 or 25. The

  Further, all nucleotides constituting the sense strand RNA may be constituted by ribonucleotides, but deoxyribonucleotides may be contained. Particularly preferred sense strand RNAs include those in which the first and second nucleotides from the 3 'end of the sense strand RNA are composed of deoxyribonucleotides. By adding deoxyribonucleotides to the sense strand in such a configuration, it is converted to double-stranded RNA (siRNA) having a 2nt dangling end at the 3 'end with high efficiency by undergoing Dicer processing. And an excellent RNA interference effect can be achieved.

  In addition, a substituent is bonded to at least one of the first to sixth nucleotides from the 3 ′ end of the sense strand RNA of the modified RNA of the present invention. On the other hand, in the modified RNA of the present invention, no substituent is bonded to a site other than the 3 ′ end side of the sense strand RNA. That is, the portion other than the 3 'terminal side of the sense strand RNA and the antisense strand RNA portion described later are not substituted with a substituent, and are composed only of nucleotides or deoxyribonucleotides. As described above, since the substituent is bonded only to the 3 ′ end side of the sense strand RNA, it is possible to express a significantly superior RNA interference effect.

  Here, the substituent is not particularly limited, and examples thereof include an amino group; a mercapto group; a nitro group; an alkyl group having 1 to 40 carbon atoms (preferably 2 to 20, more preferably 4 to 12); carbon An aminoalkyl group having 1 to 40 (preferably 2 to 20, more preferably 4 to 12); a thioalkyl group having 1 to 40 (preferably 2 to 20, more preferably 4 to 12) carbon atoms; 40 (preferably 2-20, more preferably 4-12) alkoxyl group; 1-40 carbon atoms (preferably 2-20, more preferably 4-12) aminoalkoxyl groups; 1-40 carbon atoms (preferably Is a thioalkoxyl group having 2 to 20 and more preferably 4 to 12); a mono- or dialkylamino group having 1 to 40 carbon atoms (preferably 2 to 20, more preferably 4 to 12); An alkylthio group having 1 to 40 (preferably 2 to 20 and more preferably 4 to 12); a polyethylene oxide group having 2 to 40 (preferably 2 to 20 and more preferably 4 to 12) carbon atoms; 39 (preferably 3 to 21, more preferably 3 to 12) polypropylene oxide groups. By binding these substituents, the RNA interference effect can be remarkably enhanced.

  Further, the substituent may be a group having a functional molecule other than those described above. Thus, when the substituent is a group having a functional molecule, it is possible to combine an excellent RNA interference effect and a useful effect based on the functional molecule.

  Here, examples of functional molecules include sugars, proteins, peptides, amino acids, DNA, RNA (including tRNA), aptamers, modified nucleotides, low molecular organic / inorganic materials, cholesterol, dendrimers, lipids, polymer materials, etc. Is done.

  Examples of the sugar include monosaccharides such as glucose, galactose, glucosamine, and galactosamine, and oligosaccharides or polysaccharides in which these are arbitrarily combined.

As said protein, the protein which exists in the living body, the protein which has a pharmacological action, the protein which has a molecule recognition effect etc. can be used, Importin beta protein, avidin, an antibody etc. can be mentioned as an example of this protein.

  Specific examples of the DNA include DNA having a base length of 5 to 50, preferably DNA having a base length of 5 to 25.

  Examples of the peptide include R8, nuclear localization signal peptide sequences (such as HIV-1 Tat and SV40T antigen), nuclear export signal peptides (such as HIV-1 Rev and MAPKK), and cell membrane fusion peptides. .

  Examples of the modified nucleotide include those obtained by modifying a phosphate skeleton such as phosphorothioate-type or boranophosphate-type DNA / RNA; 2′-modified nucleotides such as 2′-0Me-modified RNA and 2′-F-modified RNA; LNA (Locked Nucleic Acid) and ENA (2'-O, 4'-C-Ethylene-bridged nucleic acids), etc., modified nucleotides that are cross-linked sugar molecules; PNA (peptide nucleic acid), morpholino nucleotides, etc. Modified nucleotides and the like are different.

  Examples of the low-molecular organic / inorganic material include fluorescent substances such as Cy3 and Cy5; biotin; quantum dots; gold fine particles.

  As said dendrimer, a polyamide amine dendrimer etc. are mentioned, for example.

  Examples of the lipid include fatty acids having 6 to 50 carbon atoms, DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), etc., in order to dramatically improve the RNA interference effect and nuclease resistance. Preferably, lauric acid, stearic acid, myristylic acid and palmitic acid are used.

  Examples of the polymer material include polyethylene glycol and polyethyleneimine.

  Here, the group having a functional molecule may be a residue of the functional molecule itself, or a group represented by the following general formula (I), that is, a functional molecule residue of a bifunctional linker. It may be a group to which one functional group is bonded. That is, in the former case, the functional molecule is directly bonded to a predetermined site of the sense strand RNA, and in the latter case, the functional molecule is attached to a predetermined site of the sense strand RNA via a bifunctional linker. Are connected. The group having a functional molecule is preferably the latter group represented by the following general formula (I).

  Here, the bifunctional linker is not particularly limited as long as it includes two functional groups. For example, N-succinimidyl = 3- (2-pyridyldithio) propinate, N-4-maleimidobutyric acid, S- (2-pyridyldithio) cysteamine, iodoacetoxysuccinimide, N- (4-maleimidobutyryloxy) succinimide, N- [5- (3′-maleimidopropylamide) -1-carboxypentyl] iminodiacetic acid, N -(5-aminopentyl) -iminodiacetic acid etc. can be used.

  In addition to the above, those having the following structure can also be used as the bifunctional linker.

  Here, in the general formulas (L-4) to (L-21), n1 represents an integer of 1 to 40, preferably an integer of 2 to 20, and more preferably an integer of 2 to 12.

  In the general formulas (L-22) and (L-23), n2 represents an integer of 1 to 20, preferably an integer of 1 to 10, and more preferably an integer of 1 to 6.

  In the linkers represented by the general formulas (L-4) to (L-23), a functional molecule may be bonded to either the right side or the left side. Preferably, a functional molecule is bound on the left side, and a predetermined site of the sense strand RNA is bound on the right side.

  The bifunctional linker may be appropriately selected and used according to the type of functional molecule to be bound. For example, when cholesterol is used as the functional molecule, a linker of general formula (L-22) or (L-23), particularly a linker of general formula (L-22) is preferably used.

  When the functional molecule is a lipid, the aminoalkyl group is bonded to the 3 ′ end of the sense strand RNA, and the amino group of the aminoalkyl group and the carboxyl group of the lipid are bonded by an amide bond. Thus, it is desirable to link a lipid-containing group to the 3 ′ end side of the sense strand RNA. Further, when the functional molecule is DNA, it is desirable to bind the DNA directly to the 3 ′ end side of the sense strand RNA without using a linker.

  Among the groups having functional molecules, DNA, cholesterol-containing groups, and lipid-containing groups are preferable. When DNA is employed as the substituent, it becomes possible to form a complex by further hybridizing DNA or RNA having a base sequence complementary to the DNA, and to provide a further useful function based on the DNA of the present invention. The modified RNA can be prepared. In addition, when a group having cholesterol or a group having lipid is employed as the substituent, an advantage that the modified RNA of the present invention can be introduced into cells without using a gene introduction agent can be obtained.

  Preferable examples of the substituent include an aminoalkyl group, a thioalkyl group, a group having cholesterol, a group having lipid, and a particularly preferable example is an aminoalkyl group. For example, when an aminoalkyl group and a thioalkyl group are employed as the substituent, the RNA interference effect can be dramatically improved, and further, a membrane-permeable peptide, sugar, protein, or not via a linker. The functional molecule such as can be covalently bound, and the advantage that various functions can be imparted to the RNA molecule can be obtained. Furthermore, for example, when a group containing cholesterol is employed as the substituent, the modified RNA of the present invention can be introduced into cells without using a gene introduction agent in addition to exhibiting a desired RNA interference effect. The advantage of becoming

  In the sense strand RNA, the nucleotide to which the substituent is bound is not particularly limited as long as it is the 1st to 6th nucleotide from the 3 ′ end side of the sense strand RNA, but preferably 1 to 3 from the 3 ′ end side. The 4th nucleotide, more preferably the 1st and / or 2nd nucleotide from the 3 ′ end side, particularly preferably the 3 ′ end (1st from the 3 ′ end side) nucleotide.

  Further, the binding site of the substituent is not particularly limited, but the substituent is bonded to the hydrogen atom constituting the hydroxyl group of the phosphate portion of the predetermined nucleotide of the sense strand RNA. It is preferable.

  The number of the substituents bound to the modified RNA of the present invention is not particularly limited, and examples thereof include 1 to 3, preferably 1 or 2, and more preferably 1.

  The binding of the substituent to the nucleotide on the 3 'end side of the sense strand RNA is performed according to a known chemical synthesis method depending on the type of the substituent used.

  In addition, the modified RNA of the present invention can be obtained by hybridizing antisense strand RNA to the above sense strand RNA so that the 3 ′ end of the sense strand RNA becomes a blunt end (brand end). This chain is formed. Here, the structure in which the 3 ′ end of the sense strand RNA is a blunt end is not only the 3 ′ end of the nucleotide constituting the sense strand RNA but also the dangling end of the 5 ′ end of the nucleotide constituting the antisense strand RNA. Means a structure that does not have

  The antisense strand RNA is composed of 23 to 29 nucleotides, and the sense strand RNA is in a state where the 3 ′ end side of the sense strand RNA (that is, the 5 ′ end side of the antisense strand RNA) is a blunt end. RNA that can hybridize with and form a double strand. That is, the antisense strand RNA has a structure in which 1 to 8 nucleotides constituting the dangling end are bound to the 3 ′ end side of RNA consisting of a nucleotide sequence complementary to the sense strand RNA. Is. In the modified RNA of the present invention, the dangling end formed on the 3 'end side of the antisense strand is preferably composed of 2 to 4, more preferably 2 nucleotides.

  In addition, the nucleotide constituting the antisense strand RNA may contain deoxyribonucleotides as in the case of the sense strand RNA.

  As a preferred embodiment of the modified RNA of the present invention, (1) composed of a sense strand RNA consisting of 21 nucleotides and the antisense strand RNA consisting of 23 nucleotides having the above-mentioned substituent on the 3 ′ end side (Ie, the 3 ′ end of the sense strand RNA is a blunt end, and the 3 ′ end of the antisense strand has a dangling end composed of 2 nucleotides); (2) 3 'Consisting of a sense strand RNA consisting of 23 nucleotides and the antisense strand RNA consisting of 25 nucleotides having the above-mentioned substituent on the terminal side (that is, the 3' terminal side of the sense strand RNA is a blunt end Wherein the antisense strand has a dangling end composed of 2 nucleotides at the 3 ′ end); and (3) 25 nucleotides having the above substituent on the 3 ′ end side. A sense strand RNA and an antisense strand RNA consisting of 27 nucleotides (ie, the 3 ′ end of the sense strand RNA is a blunt end and the 3 ′ end of the antisense strand is 2 And those having a dangling end composed of nucleotides). By employing a modified RNA having such a structure, it becomes possible to express the RNA interference effect more effectively.

  As described above, the modified RNA of the above aspects (1) to (3) may contain deoxyribonucleotides as nucleotides constituting the sense strand RNA and / or the antisense strand RNA. In the embodiments of (1) to (3), the first and second nucleotides from the 3 ′ end side of the sense strand RNA may be constituted by deoxyribonucleotides. In particular, in the above embodiment (3), it is preferable that the 1st and 2nd nucleotides from the 3 ′ end of the sense strand RNA are constituted by deoxyribonucleotides, since they can exhibit a particularly excellent RNA interference effect. is there. The modified RNAs of the above aspects (1) to (3) may be phosphorylated at the 5 ′ end of the sense strand. For example, in the above aspects (1) to (3), the sense RNA 3 In the case where the first and second nucleotides from the terminal side are constituted by deoxyribonucleotides, it is preferable that the 5 ′ end of the sense strand is phosphorylated. Among them, the above embodiment (3) is particularly preferable.

  The modified RNA of the present invention synthesizes a sense strand RNA having a substituent having the above structure and an antisense strand RNA having the above structure, and hybridizes these sense strand RNA and antisense strand RNA according to a known method. It is prepared by.

The modified RNA of the present invention is used by being introduced into cells. The modified RNA of the present invention is introduced into a target cell and used in the same manner as RNA conventionally used as siRNA.

  Examples are given below to illustrate the present invention in more detail. These examples are illustrative only and do not limit the invention. In the following, double-stranded RNA is a complete double-stranded RNA having no dangling end (single-stranded region) (double-stranded RNA in which both the 5 ′ end side and the 3 ′ end side of the sense strand RNA are blunt ends. ) DS (double strand) RNA; double-stranded RNA with dangling ends (overhangs) at both ends of the double-stranded RNA; Si RNA; the 3 'end of the sense strand is a blunt end and the antisense strand Double-stranded RNA having a dangling end at the 3 ′ end is referred to as LO (Left Overhang) RNA.

Example 1 Terminal amino-modified double-stranded RNA-1
Has a Renilla luciferase and a phase complementary sequences, can suppress gene expression of Renilla luciferase, and 27-base-long antisense strand RNA oligonucleotides, 2 bases of DNA have 3 'end of the antisense strand and the phase complementary sequences The following experiment was performed using a double-stranded RNA (hereinafter referred to as 25D / 27 dsRNA) consisting of a 25-base long RNA-DNA chimeric oligonucleotide. 25D / 27 dsRNA is characterized in that it has a two-base dangling end (single-stranded region) at the 3 ′ end of the antisense strand, and the 3 ′ end of the sense strand is a blunt end. The sequence of 25D / 27 dsRNA used is as follows. In addition, dN (N = A, G, C, T) in the following sequence indicates DNA. P means phosphoric acid (PO ₃ H).
27nt dsRNA sense strand: 5'-pGGCCUUUCACUACUCCUACGAGCdAdC-3 '
Antisense strand: 3'-GACCGGAAAGUGAUGAGGAUGCUCGUG-5 '
For comparison, a similar experiment was performed using 21 siRNA consisting of 21 bases of RNA containing a 2 nt dangling end at the 3 ′ end, which is commonly used in RNA interference reactions. The sequence of 21 siRNA is shown below.
21 siRNA sense strand: 5'-GGCCUUUCACUACUCCUACGA-3 '
Antisense strand: 3'-GACCGGAAAGUGAUGAGGAUG-5 '

1. Structure of terminal amino-modified 25D / 27 dsRNA
A terminal amino-modified 25D / 27 dsRNA in which the 5 ′ end or 3 ′ end of the sense strand and antisense strand of 25D / 27 dsRNA was aminated was synthesized. The structure of the terminal amino-modified 25D / 27 dsRNA synthesized is shown in FIG. A specific synthesis method is shown below. First, the terminal amino-modified RNA is a single-stranded RNA (purchased from Hayashi Kasei Co., Ltd .; HPLC purified and MALDI-TOF MS analyzed), and 5′-terminal amination is 5′-Amino-Modifier C6 ( Glen Research), 3 'terminal amination was synthesized using 3'-Amino-Modifer C7 GPG (Glen Research). The synthesized terminal amino-modified 25D / 27 dsRNA has a — (CH ₂ ) ₆ at the end (the hydroxyl portion of the phosphate portion of the first nucleotide from the 5 ′ end side) when the 5 ′ end is aminated. When -NH ₂ is bonded and the 3 'end is aminated,-(CH ₂ ) ₆ -NH is added to the end (the hydroxyl portion of the phosphate portion of the first nucleotide from the 3' end). ₂ are combined. The concentration of the synthesized single-stranded RNA was calculated by measuring the absorbance at 260 nm using a UV spectrum detector. In addition, in the universal buffer (Hayashi Kasei Co., Ltd.), the same strand of sense and antisense strands were mixed, heated at 92 ° C for 2 minutes, and then gradually lowered to 4 ° C. . 25D / 27 dsRNA with unmodified ends 25D / 27 RNA A; 25D / 27 dsRNA with 25D / 27 RNA B aminated only at the 3 'end of the sense strand; only 3' end of the antisense strand aminated 25D / 27 dsRNA with 25D / 27 RNA C; 25D / 27 dsRNA with amino group at both 3 'ends of sense and antisense strands 25D / 27 RNA D; only 5' end of antisense strand 25D / 27 dsRNA aminated 25D / 27 RNA E; 3D end of sense strand and 5D end of antisense strand aminated 25D / 27 dsRNA 25D / 27 RNA F; 5 'end of sense strand only 25D / 27 dsRNA amidating 25D / 27 RNA G; 25D / 27 dsRNA amination of 5 'end of sense strand and 3' end of antisense strand 25D / 27 RNA H; 5 'end of sense strand 25D / 27 dsRNA in which the 5 ′ end of the antisense strand was aminated was designated as 25D / 27 RNA I. Of these end-modified 25D / 27 dsRNAs, 25D / 27 RNA B corresponds to the modified RNA of the present invention.

2. Degradation enzyme resistance of terminal amino-modified 25D / 27 dsRNA The nuclease resistance of terminal amino-modified 25D / 27 dsRNA (27B to 27I) was examined. The experiment was performed at 37 ° C in RPMI-1640 medium (Invitrogen) containing 10% FBS (Sanko Junyaku Co., Ltd.) with terminal amino-modified 25D / 27 dsRNA adjusted to a final concentration of 2 µM (final volume 110 µl). After incubation, 10 μl was taken after 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h, and added to a sample tube containing 2 μl of loading die. In order to stop the decomposition reaction, the sample was frozen in liquid nitrogen immediately after sampling and stored at -20 ° C. The obtained product was subjected to electrophoresis using a 20% polyacrylamide gel at 250 V for 70 minutes. Thereafter, the product was stained with a silver staining kit (GE Healthcare Bioscience) (see the product manual for staining conditions), and gel analysis was performed with ChemiImager 4000 (Alpha Innotech Corporation). For comparison, 21 siRNAs having a length of 21 bases were similarly examined.

  The results are shown in FIG. As a result, unmodified 25D / 27 dsRNA A possesses a higher resistance to degrading enzymes than a 21-base siRNA having a 2 dangling end at the 3 'end, which is often used in RNA interference reactions. I found out. In addition, modified 25D / 27 dsRNA in which the end of 25D / 27 dsRNA was modified with an amino group also showed higher degrading enzyme resistance than 21 siRNA. In particular, 25D / 27 dsRNA 3D end of 25D / 27 dsRNA modified with an amino group 25D / 27 dsRNA B or 25D / 27 dsRNA 3D end of both sense strand and antisense strand 25D / 27 dsRNA D is more resistant to degradative enzymes than other modified 25D / 27 dsRNA and unmodified 25D / 27 dsRNA, and these two modified 25D / 27 dsRNAs have excellent stability even in medium containing serum. It was confirmed to show.

3. Processing of terminal amino-modified 25D / 27 dsRNA by Dicer Next, processing of each amino-modified 25D / 27 dsRNA by Dicer was examined. Dicer cleavage experiments were performed using 20 U Tris-HCl (pH 8.0), 15 mM NaCl, 2.5 mM Mg ₂ Cl solution with 0.5 U recombinant Dicer (Gene Therapy Systems) adjusted to a final concentration of 2 μM. 10 μl of / 27 dsRNA was prepared in a sample tube and incubated in an incubator set at 37 ° C. for 12 hours. Thereafter, in order to stop the cleavage reaction by Dicer, 2 μl of Dicer Stop Solution (Gene Therapy Systems) was added to the reaction solution, and further 2 μl of loading dye was added. The obtained product was subjected to electrophoresis using a 20% polyacrylamide gel at 250 V for 70 minutes. Thereafter, the product was stained with a silver staining kit (GE Healthcare Bioscience) (see the product manual for staining conditions), and gel analysis was performed with ChemiImager 4000 (Alpha Innotech Corporation). For comparison, a 21-base siRNA having a 2-base dangling end at the 3 ′ end was also electrophoresed. The processing results of 25D / 27 dsRNA by recombinant Dicer are shown in FIG.

  As a result, the unmodified 25D / 27 dsRNA A band was confirmed at the same position as 21siRNA in the presence of the recombinant Dicer, and a 21-base-long siRNA containing a 2-base dangling end was generated by Dicer cleavage. It was strongly suggested that In addition, 25D / 27 dsRNA B in which the 3 ′ end of the sense strand of 25D / 27 dsRNA was modified with an amino group was confirmed to have a band at the same position as 21siRNA in the presence of recombinant Dicer, as in 25D / 27 dsRNA A. It was strongly suggested that a 21-base siRNA containing a 2-base dangling end was generated. On the other hand, 25D / 27 dsRNA C, which has an amino group at the 3 'end of the antisense strand of 25D / 27 dsRNA, and 25D / 27 dsRNA D, which has an amino group at the 3' end of both the sense strand and antisense strand, are 21 siRNA A gel electrophoresis band is obtained that suggests that no processing has been performed to the 21 siRNA by Dicer, and no modification has been obtained. It became clear this time to have a big influence. In the same way as above, other amination-modified 25D / 27 dsRNAs are also processed by Dicer into siRNAs with a length of 21 bases, only partially processed, or siRNAs other than 21 bases are generated. It has been clarified that the position of amino modification, etc. greatly affects the processing efficiency.

4). Next, the RNA interference effect of each terminal amino-modified 25D / 27 dsRNA was evaluated using Renilla luciferase as a target. Before the experiment, HeLa cells (human cervical cancer cells, Tohoku University Institute for Aging Medicine) adjusted to 1 × 10 ⁵ cells / ml were plated on a 96-well plate, and incubated at 37 ° C. overnight. The next day, remove the old medium in the wells, add 80 μl each of fresh medium without antibiotics to the wells, and add firefly and Renilla luciferase-expressing vectors (psiCHECK ^TM -2 Vector: Promega) and Lipofectamine ^TM 2000 (trade name) , Invitrogen) was added to each well containing 10 μl of HeLa cells. Here, the expression vector was set to 0.02 μg per well and Lipofectamine ^™ 2000 was set to 0.2 μl per well, and the required amount was adjusted with OptiMem (Invitrogen). In order to form a complex, the expression vector and Lipofectamine ^™ 2000 were mixed using OptiMem and then incubated at room temperature for 30 minutes. After adding the complex solution, the cells were incubated for 4 hours at 37 ° C. in the presence of 5% CO ₂ . Thereafter, renilla 21 siRNA and 25D / 27 dsRNA comprising genetic sequence and a phase complementary antisense sequences of luciferase, a terminal amino-modified 25D / 27 dsRNA final concentration 0 nM, 0.2 nM, 0.5 nM, 1 nM, 2 nM, 5 nM A complex was formed with Lipofectamine ^™ 2000 (Invitrogen) to 10 nM, and 10 μl of the complex solution was added to HeLa cells into which the expression vector was introduced. Here, the final volume per well is 100 μl. A complex solution of RNA and Lipofectamine ^™ 2000 was prepared by mixing 5 µl of RNA aqueous solution and 5 µl of Lipofectamine ^™ 2000 (0.2 µl) OptiMem solution per well and incubating at room temperature for 30 minutes. After introducing the RNA, incubate for 48 hours, measure the expression level of firefly and Renilla luciferase using Dula-Glo ^™ Luciferase Assay System (Promega) with a luminometer (MicroLumat LB96p: BERTHOLD), and express the level of firefly luciferase As a control, the expression suppression effect of Renilla luciferase was calculated.

  FIG. 4 shows the results of the RNA interference effect of the terminal amino-modified 25D / 27 dsRNA at a concentration of 0.2 nM. As a result, 25D / 27 dsRNA A was observed to have a slightly higher RNA interference effect than 21 siRNA, and was found to effectively suppress the expression of the target gene. In addition, 25D / 27 dsRNA B, which is aminated only at the 3 'end of the sense strand of 25D / 27 dsRNA, has a significantly higher RNA interference effect compared to 21 siRNA and 25D / 27 dsRNA A, and extremely suppressed gene expression. It became clear that it had the ability. On the other hand, terminal amino-modified 25D / 27 dsRNA other than 25D / 27 dsRNA B showed only an RNA interference effect comparable to or inferior to 25D / 27 dsRNA A.

  From the results obtained this time, the efficiency of cleavage by Dicer in the cell, the structure of the subsequent 21 siRNA, the efficiency of complex formation with RISC, the efficiency of cleaving mRNA, and the resistance to degrading enzymes have a major influence on the RNA interference effect. It was confirmed that the RNA interference effect was dramatically improved by modifying only the 3 ′ end of the sense strand of 25D / 27 dsRNA.

5. Persistence of RNA interference effect of terminal amino-modified 25D / 27 dsRNA Next, the persistence of RNA interference effect of terminal amino-modified 25D / 27 dsRNA was examined. In order to evaluate the persistence of RNA interference effect, terminal amino-modified 25D / 27 dsRNA adjusted to 50 nM for 7 days (168 hours), HeLa cells (human cervical cancer cells, Institute of Aging Medicine, Tohoku University) And the subsequent RNA interference effect was followed. The target used in the gene expression suppression experiment was Renilla luciferase, and a vector (psiCHECK ^TM -2 Vector: Promega) containing the firefly and Renilla luciferase genes was introduced into the cells 48 hours before measurement using Lipofectamine ^TM 2000 . Terminal amino-modified 25D / 27 dsRNA was also introduced into cells using Lipofectamine ^™ 2000, and the medium was changed every two days. For gene expression suppression analysis, firefly and Renilla luciferase expression levels were measured with a luminometer using the Dula-Glo ^™ Luciferase Assay System (Promega), and the Renilla luciferase expression suppression effect was calculated using the firefly luciferase expression level as a control did. The expression vector and RNA used here were complexed with Lipofectamine ^™ 2000 in the same manner as described above, and 10 μl of each sample was added to the cells. The final volume of the cell solution was adjusted to 100 μl. For comparison, 21siRNA was also examined in the same manner.

  The obtained results are shown in FIG. As a result, 21 siRNA showed a high RNA interference effect on the second day after addition to the cells and suppressed the expression of Renilla luciferase by 80% or more, but the RNA interference effect on the fourth day after the addition of the cells. However, only about 40% of the gene expression could be suppressed on the 7th day. In contrast, 25D / 27 dsRNA B modified with an amino group at the 3 'end of the sense strand shows a high persistence of RNA interference effect, and most modified and unmodified 25D / 27 dsRNAs on day 7 In addition, about 80% of the target gene was suppressed. On the other hand, no long-lasting RNA interference effect was observed with 25D / 27 dsRNA E or 25D / 27 dsRNA F.

Example 2 Terminal amino-modified double-stranded RNA-2
As a double-stranded RNA that has a homologous sequence to Renilla luciferase and can suppress the gene expression of Renilla luciferase, the 3 'end of the sense strand has a blunt end and a 2-base dangling end only at the 3' end of the antisense strand. The double stranded RNA possessed was designated as LO RNA. This LO RNA has a 21-base RNA in the sense strand and a 23-base RNA in the antisense strand, and the 3 'end of the sense strand is a blunt end and a 2-base dangling end only at the 3' end of the antisense strand. LO 21AN5 / 23B RNA with LO 21A / 23B RNA and LO 21A5 / 23B RNA aminated at the 5 'end of the sense strand of LO 21A / 23B RNA, LO 21AN3 / 23B RNA aminated at the 3' end of the sense strand of LO 21A / 23B RNA Designed. Similarly, the sense strand has 23 base RNA, the antisense strand has 25 base RNA, and the 3 'end of the sense strand is a blunt end and a 2 base dangling end only at the 3' end of the antisense strand. LO 23AN5 / 25B RNA with LO 23A / 25B RNA and LO 23A5 / 25B RNA aminated at the 5 ′ end of LO 23A / 25B RNA, LO 23AN3 / 25B RNA with aminated 3 ′ end of the sense strand of LO 23A / 25B RNA Designed. In addition, a 25-base RNA in the sense strand and a 27-base RNA in the antisense strand, and the 3 'end of the sense strand is a blunt end and a 2-base dangling end only at the 3' end of the antisense strand LO 25AN5 / 27B RNA aminated at the 5 'end of the sense strand of 25A / 27B RNA and LO 25A / 27B RNA, LO 25AN3 / 27B RNA aminated at the 3' end of the sense strand of LO 25A / 27B RNA were designed . In addition, LO 21DA / 23B and LO 23DA / 25B were prepared by using two deoxyribonucleotides linked to the 3 ′ ends of 19- and 21-base RNAs as LO RNA sense strands. LO 21DAN3 / 23B and LO 23DAN3 / 25B were designed by amination of the 3 ′ end of the RNA sense strand (deoxyribonucleotide at the 3 ′ end). In addition, double-stranded RNA having a 2-base dangling end at the 3 ′ end of both the sense strand and antisense strand was compared with LO RNA as siRNA. With this siRNA, si21A5 / 21B RNA consisting of 21-base RNA and si21AN5 / 21B RNA aminated at the 5 'end of si21A / 21B RNA, and si21AN3 / 21B RNA aminated at the 3' end of si21A / 21B RNA are designed. did. Similarly, si23AN5 / with 23 base RNA si23A / 23B RNA and si23A / 23B RNA 5 'end with 2 base dangling ends at the 3' end of both sense strand and antisense strand SiBAN3 / 23B RNA in which the 3 ′ end of 23B RNA, si23A / 23B RNA was aminated was designed. In addition, si25AN5 / 25B, which has a dangling end of 2 bases at the 3 'end of both the sense strand and antisense strand and amination of the 5' end of si25A / 25B RNA and si25A / 25B RNA consisting of 25 base RNA Si25AN3 / 25B RNA in which the 3 ′ end of RNA, si25A / 25B RNA was aminated was designed. The RNA sequences used are as follows. In addition, dN (N = A, G, C, T) in the following sequences indicates deoxyribonucleotides. P means phosphoric acid (PO ₃ H).
<Sense strand>
21nt 21A: 5'-GGCCUUUCACUACUCCUACGA-3 '
21DA: 5'-pGGCCUUUCACUACUCCUAC dGdA -3 '
21AN5: 5'NH _2- (CH ₂ ) ₆ -PO ₃ -GGCCUUUCACUACUCCUACGA-3 '
21AN3: 5'-GGCCUUUCACUACUCCUACGA-PO _3- (CH ₂ ) ₆ -NH ₂ 3 '
21DAN3: 5'-pGGCCUUUCACUACUCCUAC dGdA -PO _3- (CH ₂ ) ₆ -NH ₂ 3 '
23nt 23A: 5'-GGCCUUUCACUACUCCUACGAGCGC-3 '
23DA: 5'-pGGCCUUUCACUACUCCUACGA dGdC- 3 '
23AN5: 5'-NH _2- (CH ₂ ) ₆ -PO ₃ -GGCCUUUCACUACUCCUACGAGC-3 '
23AN3: 5'-GGCCUUUCACUACUCCUACGAGCGC-PO _3- (CH ₂ ) ₆ -NH ₂ 3 '
23DAN3: 5'-pGGCCUUUCACUACUCCUACGA dGdC -PO _3- (CH ₂ ) ₆ -NH ₂ 3 '
25nt 25A: 5'-GGCCUUUCACUACUCCUACGAGCAC-3 '
25AN5: 5'NH _2- (CH ₂ ) ₆ -PO ₃ -GGCCUUUCACUACUCCUACGAGCAC-3 '
25AN3: 5'GGCCUUUCACUACUCCUACGAGCAC-PO _3- (CH ₂ ) ₆ -NH ₂ 3 '
<Antisense strand>
23nt 23B: 5'-UCGUAGGAGUAGUGAAAGGCCAG-3 '
25nt 25B: 5'-GCUCGUAGGAGUAGUGAAAGGCCAG -3 '
27nt 27B: 5'-GUGCUCGUAGGAGUAGUGAAAGGCCAG-3 '

1. Structure of terminal amino-modified double-stranded RNA
A terminal amino-modified double-stranded RNA was synthesized by amination of the 5 ′ end and 3 ′ end of the sense strand of the double-stranded RNA. A specific synthesis method is shown below. First, the terminal amino-modified RNA is a single-stranded RNA (purchased from Hayashi Kasei Co., Ltd .; HPLC purified and MALDI-TOF MS analyzed), and 5′-terminal amination is 5′-Amino-Modifier C6 ( Glen Research), 3 'terminal amination was synthesized using 3'-Amino-Modifer C7 GPG (Glen Research). In the synthesized terminal amino-modified double-stranded RNA, the 5 ′ end is aminated and — (CH ₂ ) ₆ —NH ₂ is bound to the end (the first nucleotide from the 5 ′ end side). In addition,-(CH ₂ ) ₆ -NH ₂ is bonded to the end (the first nucleotide from the 3 ′ end side) of the aminated 3 ′ end. The concentration of the synthesized single-stranded RNA was calculated by measuring the absorbance at 260 nm using a UV spectrum detector. In addition, in the universal buffer (Hayashi Kasei Co., Ltd.), the same strand of sense and antisense strands were mixed, heated at 92 ° C for 2 minutes, and then gradually lowered to 4 ° C. . Each synthesized double-stranded RNA is electrophoresed for 60 minutes under a 250V condition using a 20% polyacrylamide gel, and then stained with a silver staining kit (GE Healthcare Bioscience). si RNA (sense strand: 21 nt to 25 nt) and LO RNA (sense strand: 21 nt to 25 nt) were confirmed. The structure of each siRNA and LO RNA is shown in FIG. Among the LO RNAs shown in FIG. 6, LO 21AN3 / 23B RNA, LO 23AN3 / 25B RNA, LO 25AN3 / 27B RNA, LO 21AN3 / 23B RNA, and LO 23AN3 / 25B RNA correspond to the modified RNA of the present invention.

2. Degradation enzyme resistance unmodified LO RNA of terminal amino-modified double-stranded RNA and nuclease resistance of 5′-terminal amino-modified and 3′-amino-modified LO RNA were examined. In addition, 21A / 21B siRNA consisting of 21-base RNA commonly used in RNA interference reactions and having a 2nt dangling end at the 3 'end of the sense and antisense strands was also examined for comparison. . The experiment was performed in the same manner as in Example 1 above.

  The results are shown in FIG. In FIG. 7, “A” is an unmodified LO RNA, “B” is an LO RNA in which the 5 ′ end of the sense strand is modified with an amino group, and “C” is an LO RNA in which the 3 ′ end of the sense strand is modified with an amino group. Is the result of As a result, each of the unmodified and modified LO RNAs (sense strand: 21 nt to 25 nt) showed higher degrading enzyme resistance than 21A / 21B siRNA. In addition, a comparison of LO RNAs with different RNA lengths revealed that the longer the RNA chain length, the higher the resistance to degrading enzymes. In addition, examination of differences in resistance to degrading enzymes due to differences in unmodified and modified RNA, as well as modification positions, revealed that modified LO RNA is more resistant to degrading enzymes than unmodified LO RNA. Showed higher resistance to the degradation of LO RNA modified with an amino group at the 3 ′ end of the sense strand than LO RNA modified with an amino group at the 5 ′ end of the sense strand. From these results, it was clarified that LO RNA exhibits higher decomposing enzyme resistance than siRNA, and that high degrading enzyme resistance can be obtained by applying a modifying group to the 3 'end of the sense strand.

3. Processing of terminal amino-modified double-stranded RNA with Dicer Next, we will examine the processing of unmodified and modified siRNA (sense strand: 21 nt to 25 nt) and LO RNA (sense strand: 21 nt to 25 nt) with recombinant Dicer did. The experiment was performed in the same manner as in Example 1 above. In addition, 21 siRNA (si21A / 21B RNA) composed of double-stranded RNA of 21 base length not treated with Dicer was used as a control.

  The results are shown in FIG. In FIG. 8, “A” is si RNA (sense strand: 21 nt to 25 nt), “B” is LO RNA (sense strand: 21 nt to 25 nt), and “C” includes two depoxyribonucleotides in the sense strand. It is a result of LO RNA (sense strand: 21 nt to 23 nt). Note that 21 siRNA (si21A / 21B RNA) is not shown in the figure because no particular change was observed in the sensing with the recombinant Dicer.

  In siRNA, unmodified siRNA si23A / 23B RNA and si25A / 25B RNA were observed as unprocessed RNA in si23A / 23B RNA, but 21-base siRNA by Dicer in almost all RNA Processing was confirmed.

  In si23AN5 / 23B RNA and si25AN5 / 25B RNA in which the 5 ′ end of the sense strand was modified with an amino group, si25AN5 / 25B RNA was almost completely processed into a 21-base siRNA, whereas si23AN5 / 25B In RNA, about half of the RNA processing was confirmed. It was also confirmed that si23AN3 / 23B RNA and si25AN3 / 25B RNA in which the 3 'end of the sense strand was modified with an amino group were not processed by Dicer.

  In LO RNA as well, differences in processing by Dicer were observed depending on the RNA chain length, the presence or absence of a modifying group, and the position of the modifying group. In unmodified LO RNA (sense strand: 21 nt to 25 nt), LO 21A / 23B RNA with a short RNA strand was processed by Dicer into a 21-base siRNA, and remained unprocessed. A double-stranded RNA such as the one was confirmed. On the other hand, it was confirmed that LO 23A / 25 RNA and LO 25A / 27B RNA having a long RNA chain were processed by dicer into 21-base siRNA in almost all RNA molecules. For LO RNA containing two deoxyribonucleotides at the 3 ′ end of the sense strand, LO 21DA / 23B was hardly processed by Dicer and LO 23DA / 25B was processed by Dicer into a 21nt siRNA. Similar results were obtained when the 3 'end of the sense strand was aminated.

  This result clearly shows that LO RNA (sense strand: 21 nt to 25 nt) modified with an amino group at the 5 'end of the sense strand is less susceptible to processing by Dicer than unmodified LO RNA with the same RNA strand length became. Furthermore, LO RNA in which the 3 ′ end of the sense strand is modified with an amino group (sense strand: 21 nt to 25 nt) is similarly converted into unmodified LO RNA having an RNA strand length or LO RNA in which the 5 ′ end of the sense strand is modified. Compared to Dicer's processing, this result confirmed that it is less susceptible to processing by Dicer.

4). Next, the RNA interference effect of each terminal amino-modified double-stranded RNA was evaluated using Renilla luciferase as a target. The experiment was performed in the same manner as in Example 1 above.

FIG. 9 and FIG. 10 show that the unmodified si RNA or unmodified LO RNA is 100%, the 5′-amino modified or 3′-amino modified siRNA and LO RNA corresponding to the respective RNA strand lengths, and the 3 ′ end of the sense strand. Shows the relative value of the RNA interference effect of LO RNA containing two deoxyribonucleotides. FIG. 9 shows the results of siRNA and FIG. 10 shows the results of LO RNA.
As a result, siRNA (sense strand: 21 nt to 25 nt) is a large RNA between unmodified siRNA with similar RNA strand length and modified siRNA in which the 5 'or 3' end of the sense strand is modified with an amino group No difference in the interference effect was observed, and it was found that the RNA interference effect was not improved even when a modifying group was added to siRNA.

  On the other hand, in LO RNA (sense strand: 21 nt to 25 nt), a large difference in RNA interference effect was observed between unmodified and modified double-stranded RNA having the same RNA chain length. In particular, LO RNA (sense strands: 21AN3, 23AN3, 25AN3) with an amino group added only to the 3 'end of the sense strand is unmodified LO RNA (sense strands: 21A, 23A, 25A) and only the 5' end of the sense strand An RNA interference effect much higher than that of LO RNA (sense strands: 21AN5, 23AN5, 25AN5) to which an amino group was added was observed, confirming that expression of the target gene was effectively suppressed. Further, LO RNA having two deoxyribonucleotides at the 3 ′ end of the sense strand and having an amino group introduced also improved the RNA interference effect.

  From these results, double-stranded RNA having a blunt end at the 3 ′ end of the sense strand and a 2 nt dangling end at the 3 ′ end of the antisense strand has a modification group only at the 3 ′ end of the sense strand. As a result, it has been clarified that the effect of suppressing the expression of the target gene can be dramatically improved. It was also revealed that the effect of suppressing gene expression is improved even when deoxyribonucleotide is bound to the 3 ′ end of the sense strand of the modified RNA.

5. Persistence of RNA interference effect of terminal amino-modified double-stranded RNA Next, the persistence of RNA interference effect of terminal amino-modified double-stranded RNA (LO RNA) was examined. The experiment was performed in the same manner as in Example 1 above.

  The obtained results are shown in FIG. As a result, 21 siRNA showed a high RNA interference effect on the second day after addition to the cells and suppressed the expression of Renilla luciferase by 80% or more, but the RNA interference effect on the fourth day after the addition of the cells. However, only about 40% of the gene expression could be suppressed on the 7th day. In contrast, LO RNA showed a high persistence of RNA interference effect. In particular, the modified LO RNA showed a high RNA interference effect and persistence, and among them, the 21A / 23B type LO RNA with a short RNA chain length showed excellent persistence of the RNA interference effect.

Example 3 Terminal DNA-modified double-stranded RNA
A DNA modified sense that has a homologous sequence with Renilla luciferase and is a modified double-stranded RNA that can suppress Renilla luciferase gene expression. A double-stranded RNA (LO 25A-9D / 27B RNA) consisting of a strand RNA (sense strand RNA-DNA) and an antisense strand RNA of 27 bases in length, the sense strand is 25 bases long and the antisense strand is 27 bases Comparison was made with unmodified double stranded RNA consisting of long RNA (LO 25A / 27B RNA). The sequences of RNA and DNA-RNA chimera used are as follows.
<Sense strand>
25A: 5'-GGCCUUUCACUACUCCUACGAGCAC-3 '
25A-9D: 5'- CUGGCCUUUCACUACUCCUACGAGCAC-ttc gca cca -3 '
<Antisense strand>
27B: 5'-GUGCUCGUAGGAGUAGUGAAAGGCCAG-3 '

1. Structure of 5′-end DNA modified dsRNA 25-base long sense strand RNA of a predetermined sequence, DNA-modified sense strand RNA in which 9-base long DNA is bound to the 3 ′ end of 25-base long sense strand RNA, and A 27-base long antisense strand RNA was synthesized (purchased from Hayashi Kasei Co., Ltd .; HPLC purified and MALDI-TOF MS analyzed). The concentration of the synthesized sense strand RNA, DNA modified sense strand RNA, and antisense strand was calculated by measuring the absorbance at 260 nm using a UV spectrum detector. In the universal buffer (Hayashi Kasei Co., Ltd.), the same mole of sense strand or DNA-modified sense strand RNA and antisense strand RNA are mixed, heated at 92 ° C for 2 minutes, and then gradually lowered to 4 ° C. It was created by that. Each synthesized double-stranded RNA is electrophoresed for 60 minutes under a 250V condition using a 20% polyacrylamide gel, and then stained with a silver staining kit (GE Healthcare Bioscience). confirmed. FIG. 12 shows double-stranded RNA in which the 5 ′ end is modified with DNA.

2. Processing of terminal DNA-modified dsRNA by Dicer Next, processing of each DNA-modified double-stranded RNA by Dicer was examined. The experiment was performed in the same manner as in Example 1 above. As a control, 21 siRNA composed of 21-base long double-stranded RNA not treated with Dicer was also measured.

  The results are shown in FIG. As a result, in the presence of recombinant Dicer, LO 25A-9D / 27B RNA was confirmed to have some bands in the same position as 21 siRNA in some RNAs, and several other bands were confirmed. It was revealed that RNA other than 21siRNA was also produced.

3. Next, the RNA interference effect of LO 25A-9D / 27B RNA was evaluated using Renilla luciferase as a target. As a comparison, the same experiment was performed with LO 25A / 27B RNA. The experiment was performed in the same manner as in Example 1 above.

  FIG. 14 shows the results of the RNA interference effect of the terminal DNA-modified double-stranded RNA at a concentration of 0.2 nM. FIG. 14 shows the relative value of the RNA interference effect of LO 25A-9D / 27B RNA when LO 25A / 27B RNA is taken as 100%. As a result, it is clear that LO 25A-9D / 27B RNA in which the end of 25nt double-stranded RNA is modified with DNA has higher gene expression suppression ability and higher RNA interference effect than unmodified LO 25A / 27B RNA. became. From this result, it was clarified that even when DNA was modified at the 3 'end of the sense strand of LO 25A / 27B RNA, the RNA interference effect was improved, and the same effect was obtained except for the amino group.

Example 4 Luciferase gene expression inhibitory effect of 3′-end lipid or cholesterol-modified LO type double-stranded RNA
1. Synthesis of 3'-terminal lipid or cholesterol-modified LO double-stranded RNA targeting the luciferase gene
1-1. Synthesis of terminal lipid-modified double-stranded RNA targeting the luciferase gene Lipid-modified sense-strand RNA was synthesized by binding lipid to the 3 'end of the sense strand of LO-type double-stranded RNA that can suppress the expression of the luciferase gene. In the lipid-modified sense strand RNA, the lipid is covalently bound via an aminoalkyl group (3′-Amino-Modifer C7 GPG; Glen Research) linked to the 3 ′ end of the sense strand RNA. The lipid-modified sense strand RNA was synthesized by reacting a lipid compound having an active ester group (hereinafter referred to as an active esterified lipid compound) and a sense strand RNA aminated at the 3 ′ end in a liquid phase ( Reaction formula 1).

A specific synthesis method is shown below. In order to aminate the 3 'end of RNA, the 3' terminal aminoalkyl group was synthesized by the usual method (phosphoramidite synthesis method) using 3'-Amino-Modifer C7 GPG (Glen Research) on RNA solid phase synthesis. Modified sense strand RNA was synthesized. The 3 ′ terminal aminoalkyl group sense strand RNA can be purchased from Hayashi Kasei Co., Ltd. after HPLC purification and MALDI-TOF MS analysis. In the synthesized 3 ′ terminal aminoalkyl group sense strand RNA, — (CH ₂ ) ₆ —NH ₂ is bonded to the terminal (the hydroxyl part of the phosphate part of the first nucleotide from the 3 ′ terminal side). The concentration of the synthesized single-stranded RNA was calculated by measuring the absorbance at 260 nm using a UV spectrum detector. This aminoalkyl group-modified single-stranded RNA and an active esterified lipid compound [Palmitic acid N-hydroxy succinimide easter (Sigma-Aldrich)] dissolved in DMF (N, N-dimethylformamide) are subjected to a condensation reaction condition. To synthesize lipid-modified sense strand RNA. After the reaction, the reaction solution was purified by HPLC in order to remove unnecessary reagents in the reaction solution containing the lipid-modified sense strand RNA. For HPLC purification, A: 100% 20 mM TEAA (pH 7.0) and B: 70% CH ₃ CN / 20 mM TEAA (pH 7.0) were used as buffers, and 10% B buffer to 100% B buffer were used for 50 minutes. Purification was carried out with a linear gradient. Further, CAP CELL (4.6 × 150 mm, 5 μm; SHISEIDO) was used as a purification column. Lipid-modified sense strand RNA purified by HPLC was lyophilized and dissolved in purified water, and then the concentration and synthesis yield were calculated by UV spectrum analysis.

  The structure model and yield of lipid-modified sense strand RNA are shown below.

The synthesized lipid-modified sense strand RNA was allowed to form a double strand with the same antisense RNA as in Example 2 to obtain a lipid-modified LO type double-stranded RNA. Formation of lipid-modified LO type double-stranded RNA was performed in the same manner as in Example 1, and was confirmed by 20% polyacrylamide gel electrophoresis. The structure of lipid-modified double-stranded RNA is shown in FIG. In FIG. 15, dN indicates DNA (A, G, C, T). P means phosphoric acid (PO ₃ H).

1-2. Synthesis of terminal cholesterol-modified double-stranded RNA targeting the luciferase gene Using 25D / 27 RNA A used in Example 1, a group having cholesterol was bound to the 3 ′ end of the sense strand of 25D / 27 RNA A. Cholesterol-modified 25D / 27 RNA (LO 25DA-chol / 27B RNA) was synthesized. The “group having cholesterol” used here is a group represented by the following formula. The synthesized sense strand RNA in which a group having cholesterol at the 3 ′ end is bonded to the 3 ′ end of the sense strand RNA (the hydroxyl moiety of the phosphate residue of the first nucleotide from the 3 ′ end side) A group represented by the formula is bonded.

The structure of the terminal cholesterol-modified 25D / 27 RNA (LO 25DA-chol / 27B RNA) synthesized is shown in FIG. A specific synthesis method is shown below. First, the terminal cholesterol-modified RNA is a single-stranded RNA (purchased from Hayashi Kasei Co., Ltd .; HPLC purified, MALDI-TOF MS analyzed) and 3'-Cholesteryl-TEG-CPG (Glen Research). And synthesized with a DNA / RNA synthesizer. The concentration of the synthesized single-stranded RNA was calculated by measuring the absorbance at 260 nm using a UV spectrum detector. Formation of cholesterol-modified LO type double-stranded RNA was carried out in the same manner as in Example 1, and was confirmed by 20% polyacrylamide gel electrophoresis. The structure of cholesterol-modified LO type double-stranded RNA is shown in FIG. In FIG. 15, dN indicates DNA (A, G, C, T). P means phosphoric acid (PO ₃ H). The synthesized terminal cholesterol-modified 25D / 27 RNA was LO 25DA-chol / 27B RNA.

2. The nuclease resistance of lipid-cholesterol-modified LO double-stranded RNA and degradation enzyme-resistant lipid and cholesterol-modified LO RNA (LO 25A-C16 / 27, LO 25DA-C16 / RNA, LO25DA-Chol / 27) were examined. The experiment was performed in the same manner as in Example 1 above. The comparison with the 21-base long siRNA used in Example 1 was also performed. The results of gel electrophoresis are shown in FIG.

  As a result, the lipid and cholesterol-modified LO-type RNA showed much higher nuclease resistance than 21 siRNA, and the double-stranded RNA was alive even after 48 hours. From this result, a new finding was obtained that lipid-modified double-stranded RNA possesses significantly higher in vivo stability than 21 siRNA, which is widely used in general.

3. Processing of Lipid or Cholesterol-Modified LO Double-Strand RNA by Dicer Targeting Luciferase Gene Processing of the synthesized lipid and cholesterol-modified LO-type double-stranded RNA by recombinant Dicer was examined. The experiment was performed in the same manner as in Example 1 above. The results are shown in FIG.

  As a result, in the lipid / cholesterol-modified LO double-stranded RNA, processing differences by Dicer were observed depending on the RNA chain length, the presence or absence of the modifying group, and the position of the modifying group. In LO 21A-C16 / 23 and LO 21DA-C16 / 23B, processing to 21nt siRNA was not confirmed even in the presence of Dicer, and the intact structure remained after the reaction. LO 23DA-C16 / 25 was partly processed to 21nt siRNA in the presence of Dicer, but it was confirmed that it remained intact without processing. LO 23A-C16 / 25, LO 25A-C16 / 27, LO 25DA-C16 / 27, and LO 25DA-Chol / 27 are almost all RNA molecules processed by Dicer into 21-base siRNA. confirmed.

4). Inhibition of luciferase gene expression of lipid-modified LO-type double-stranded RNA Next, the RNA interference effect of each terminal lipid-modified LO-type double-stranded RNA was evaluated using Renilla luciferase as a target. The experiment was performed in the same manner as in Example 1 above.

  Figure 18 shows the relative value of the RNA interference effect of the 3'-end lipid-modified LO-type double-stranded RNA corresponding to each RNA chain length, with unmodified LO RNA (LO 25A / 27B and LO 25DA / 27B) as 100%. Indicates.

  As a result, it became clear that LO RNA with a lipid at the 3 'end (sense strand: 21nt to 25nt) has a higher RNA interference effect than unmodified LO RNA with similar RNA strand length. . The effect of the lipid-modified LO-type RNA having two deoxyribonucleotides at the 3 'end of the sense strand was also dramatically improved in the RNA interference effect compared to the unmodified type.

  From these results, the sense strand is a double-stranded RNA having a blunt end at the 3 ′ end and a 2 nt dangling end at the 3 ′ end of the antisense strand, and a lipid only at the 3 ′ end of the sense strand. It has been clarified that the effect of suppressing the target gene expression can be drastically improved by the application. It has also been clarified that the gene expression suppressing effect is improved even when the 3 ′ end of the sense strand of the modified RNA is composed of deoxyribonucleotides.

5. RNA interference effect of lipid or cholesterol-modified LO double-stranded RNA targeting the luciferase gene (no gene transfer agent)
Next, it was examined whether lipid-cholesterol-modified LO double-stranded RNA alone was introduced into cells without using a gene introduction agent such as Lipofectamine ^™ 2000, and also exhibited RNA interference effects.

Before the experiment, HeLa cells (human cervical cancer cells, Tohoku University Institute for Aging Medicine) adjusted to 1 × 10 ⁵ cells / ml were plated on a 96-well plate, and incubated at 37 ° C. overnight. The next day, remove the old medium on the wells, each 80μl added a new medium without antibiotics to the wells, vectors expressing firefly and Renilla luciferase (psiCHECK ^TM -2 Vector: Promega) and Lipofectamine ^TM 2000 (trade name, 10 μl of Invitrogen) complex solution was added to each well containing HeLa cells. Here, the expression vector was set to 0.02 μg per well and Lipofectamine ^™ 2000 was set to 0.2 μl per well, and the required amount was adjusted with OptiMem (Invitrogen). In order to form a complex, the expression vector and Lipofectamine ^™ 2000 were mixed using OptiMem and then incubated at room temperature for 30 minutes. After adding the complex solution, the cells were incubated for 4 hours at 37 ° C. in the presence of 5% CO ₂ . Thereafter, in order to remove Lipofectamine ^™ 2000 from the medium, each well was washed three times with 100 μl of the medium. Then added medium containing antibiotics 90 [mu] l to the cells, the unmodified double-stranded RNA and lipid and cholesterol-modified LO type double-stranded RNA type comprising genetic sequence and a phase complementary antisense sequences of the Renilla luciferase Samples adjusted with OptiMem to final concentrations of 0 nM, 25 nM, 50 nM, 100 nM, 200 nM, 400 nM, 600 n, and 800 nM were added to 10 μl cells and incubated at 37 ° C. for 48 hours. The expression level of firefly and Renilla luciferase was measured with a luminometer (MicroLumat LB96p: BERTHOLD) using Dula-Glo ^™ Luciferase Assay System (Promega). For comparison, unmodified LO 25DA / 27B (25D / 27 RNA A) was also examined for RNA interference effect under the same conditions as described above. For the RNA interference effect, the expression level of Renilla luciferase was calculated using the expression level of firefly luciferase as a control. The results of RNA interference effects of LO 25DA / 27B and LO 25DA-C16 / 27B when the final concentration is 50 nM to 400 nM are shown in FIG. 19, and LO 25DA / 27B and LO 25DA-Chol / when the final concentration is 50 nM to 800 nM. The result of the RNA interference effect of 27B is shown in FIG. As a result, LO 25DA-C16 / 27B in which palmitic acid was modified at the 3 ′ end of the sense strand suppressed Renilla luciferase expression in a double-stranded RNA concentration-dependent manner. However, it was revealed that it was introduced into cells and caused an RNA interference reaction. On the other hand, unmodified LO 25DA / 27B (25D / 27 RNA A) was not confirmed to have a sufficient gene expression suppressing effect even in a high concentration region. This also confirms that palmitic acid-modified LO-type double-stranded RNA has an excellent ability to introduce into cells, and exhibits excellent ability to suppress gene expression without the use of a gene introduction agent. It was done.
In addition, LO 25DA-Chol / 27B, in which cholesterol is modified at the 3 'end of the sense strand, also suppresses Renilla luciferase expression depending on the concentration of double-stranded RNA. It was revealed that an RNA interference reaction occurred.

6). Examination of lipid / cholesterol-modified double-stranded RNA into cells: HeLa cells (human cervical cancer cells, Institute of Aging Medicine, Tohoku University) and A549 cells (human lung cancer cells) adjusted to 1x10 ⁵ cell / ml before the experiment , Tohoku University Institute of Aging Medicine), 1 ml each in a 24-well plate, in a medium containing 10% fetal bovine serum (FBS: Sanko Junyaku Co., Ltd.) and antibiotics in the presence of 5% CO ₂ at 37 ° C In culture. The antibiotics and medium used here were MEM medium (Invitrogen) for HeLa cells and RPMI-1640 (Invitrogen) for A549 cells. Prior to introduction of the fluorescently labeled oligonucleotide, the medium was changed to a medium (450 μl) containing no antibiotics. Fluorescently-labeled oligonucleotide uses 27 nt antisense strand RNA with 6-FAM labeling at the 5 ′ end, 25 nt with unmodified 25 nt sense strand RNA and 3 ′ end modified with aminoalkyl, lipid, cholesterol A double strand was formed with the sense strand RNA. In order to form a complex with fluorescently labeled oligonucleotide and Lipofectamine ^TM 2000 (manufactured by Invitrogen), the cell introduction experiment was carried out by using 25 μl of a mixed solution of 10 μl of 10 μM fluorescent labeled oligonucleotide aqueous solution and 15 μl of OptiMem solution, and Lipofectamine ^TM 2000. 50 μl of the mixed solution (2 μl of Invitrogen) and 25 μl of the mixed solution of 23 μl of OptiMem solution were incubated at room temperature for 30 minutes. When Lipofectamine ^™ 2000 (manufactured by Invitrogen) is not used (C in Fig. 21; -LF2000), replace the 2 µl Lipofectamine ^™ 2000 solution in the above complex formation conditions with the OptiMem solution, and perform the same procedure. Adjusted. 50 μl of the prepared fluorescently labeled oligonucleotide complex was added to 450 μl of the cells prepared above (final double-stranded RNA concentration: 200 nM) and incubated at 37 ° C. for 4 hours in the presence of 5% CO ₂ . . Thereafter, the cells were washed three times with PBS (−) or a medium, and cell introduction was evaluated with a confocal fluorescence laser microscope and flow cytometry.

The confocal fluorescence laser microscope used a Radiance 2000 system (Bio Rad), and observed fluorescence using an argon laser. For flow cytometry, a coulter EPICS XL cytometer (Beckman coulter) was used to measure the cell transferability per 10,000 counts of cells. For flow cytometry analysis, XL EXPO32 ^™ software (Beckman coulter) was used.

The results are shown in FIG. C (-LF2000) in FIG. 21 is the result when Lipofectamine ^TM 2000 is not used, and A and B (+ LF2000) in FIG. 21 are the results when Lipofectamine ^TM 2000 is used as the introduction agent. . In FIG. 21, A is the result of cell transfection of various double-stranded RNAs when Lipofectamine ^™ 2000 is used as an introduction agent for HeLa cells and B is A549 cells. FIG. 21C shows the results of cell introduction properties of various double-stranded RNAs when no commercially available introduction agent was used for HeLa cells.

As a result, unmodified LO-type double-stranded RNA and LO-type double-stranded RNA modified with aminoalkyl groups, lipids, or cholesterol in the presence of Lipofectamine ^TM 2000 are introduced into all cells (HeLa cells, A549 cells). Was confirmed. In particular, LO 25DA-C16 / 27B and LO 25DA-Chol / 27B modified with palmitic acid or cholesterol at the 3 'end of the sense strand are unmodified LO type double stranded RNA (LO 25DA / 27B) and aminoalkyl modified LO type Compared with double-stranded RNA (LO 25DAN3 / 27B), a very high cell transduction property was observed in confocal fluorescence microscopy and flow cytometry. Moreover, confocal fluorescence microscopy suggested that the LO-type double-stranded RNA modified with palmitic acid and cholesterol was positively localized in the cytoplasm in the cell. Further, in the absence of Lipofectamine ^™ 2000, LO-type double-stranded RNA modified with palmitic acid and cholesterol was confirmed by flow cytometry analysis to have a higher cell introduction property than unmodified double-stranded RNA. In particular, high cell transduction was confirmed with LO 25DA-Chol / 27B modified with cholesterol. From this result, it is possible to dramatically improve cell transduction by covalently binding lipids such as palmitic acid and cholesterol to the 3 ′ end of the sense strand of LO-type double-stranded RNA, and localize in the cytoplasm within the cell. The knowledge that it is possible to make it.

Example 5 Inhibition of VEGF gene expression by 5 'lipid-modified double-stranded RNA
1. Synthesis of aminoalkyl or lipid-modified LO double-stranded RNA targeting the VEGF gene
1-1. Sequence of sense strand RNA and antisense strand RNA
We designed a double-stranded RNA of 25-base long sense strand RNA and 27-base antisense strand RNA that has homologous sequence to VEGF (vascular endothelial growth factor) and can suppress VEGF gene expression. The following experiment was conducted using these double-stranded RNAs. 25D / 27 RNA (VEGF) has a 2 base dangling end (single-stranded region) at the 3 'end of the antisense strand, the 3' end of the sense strand is a blunt end, and the 3 'end of the sense strand. It is LO type double stranded RNA whose terminal two bases are DNA. The sequence of RNA used is as follows. dN indicates DNA (A, G, C, T). The synthesis of LO type double-stranded RNA was carried out in the same manner as in Example 1.
25D / 27 VEGF
Sense strand 25D: 5'-UCCUACAGCACAACAAAUGUGAA dTdG- 3 '
Antisense strand 27: 3'-GAAGGAUGUCGUGUUGUUUACACUUAC-5 '
1-2. Synthesis of lipid-modified double-stranded RNA targeting the VEGF gene
A modified double-stranded RNA in which an aminoalkyl group or a lipid was bound to the 3 ′ end of the sense strand of the double-stranded RNA capable of suppressing the expression of the VEGF gene was synthesized. In the lipid-modified double-stranded RNA, the lipid is covalently bonded through the aminoalkyl group modified at the 5 ′ end of the sense strand RNA. Aminoalkyl-modified single-stranded RNA (sense strand) was synthesized by the same method as in Example 1, and lipid-modified single-stranded RNA (sense strand) was synthesized by the same method as in Example 4.

  The structure and yield of synthesized aminoalkyl or lipid-modified sense strand RNA are shown below.

  The synthesized aminoalkyl or lipid-modified sense strand RNA was formed into a double strand with the antisense strand RNA, thereby obtaining an aminoalkyl or lipid-modified LO double-stranded RNA. Double strand formation was performed in the same manner as in Example 1 and was confirmed by 20% polyacrylamide gel electrophoresis. The structure of unmodified, aminoalkyl or lipid modified double stranded RNA is shown in FIG. 22A. The lipid-modified RNA targeting the VEGF gene also had the same elution time as in Example 4.

2. Processing of aminoalkyl or lipid double-stranded RNA targeting VEGF gene by Dicer We studied the synthesis of unmodified LO double-stranded RNA, aminoalkyl or lipid-modified LO double-stranded RNA synthesized by Dicer, and recombinant Dicer. Dicer cutting experiments were performed in the same manner as in Example 1. The result is shown in FIG. 22B.

  As a result, bands of 25D / 27 VEGF, 25DN3 / 27 VEGF, and 25DC16 / 27 VEGF were confirmed at the same position as unmodified 21 siRNA by the action of recombinant Dicer, and contained a 2-base dangling end by Dicer cleavage. It was strongly suggested that a 21-base long siRNA was generated. In addition, 25DC16 / 27 VEGF was confirmed to be processed into a 21-base long siRNA containing no byproducts. From this result, it is considered that the effect of Dicer processing is small by modifying the 3 ′ end of the sense strand even for LO type double-stranded RNA targeting VEGF.

3. 25D / 27 VEGF without modified VEGF gene expression suppression end of lipid-modified double-stranded RNA, 25DN3 / 27 VEGF with aminoalkyl-modified 3 'end of sense strand RNA, and lipid modification of 3' end of sense strand RNA 25DC16 / 27 The inhibitory effect of VEGF on VEGF gene expression was evaluated using HeLa cells (human cervical cancer cells, Tohoku University Institute of Aging Medicine), A549 cells (human lung cancer cells, Tohoku University Institute of Aging Medicine) .

The experiment was performed as follows. Before the experiment, 500 μl of HeLa cells and A549 cells adjusted to 1 × 10 ⁵ cells / ml were spread on a 24-well plate and incubated overnight at 37 ° C. The next day, the old medium in the wells was removed and 450 μl each of fresh medium without antibiotics was added to the wells. Here, MEM medium was used for HeLa cells, and PRMI-1640 medium was used for A549 cells. Unmodified or aminoalkyl modifying comprising genetic sequence and a phase complementary antisense sequences of VEGF, to form a complex of the lipid-modified double-stranded RNA (25 [mu] l) and Lipofectamine ^TM 2000 (Invitrogen) solution (25 [mu] l), 50 [mu] l Of double stranded RNA was added to 450 μl of the cells. Here, the final volume per well is 500 μl. A complex solution of RNA and Lipofectamine ^™ 2000 was prepared by mixing 25 µl of RNA solution per well and 25 µl of Lipofectamine ^™ 2000 (2 µl) OptiMem solution and incubating at room temperature for 30 minutes. After introducing the RNA, it was incubated at 37 ° C. for 48 hours in the presence of 5% CO ₂ . After incubation, the cells were washed three times with PBS (−), and total RNA in the cells was extracted with RNeasy Plus Mini Kit (Qiagen). Then, RT-PCR reaction was performed in order to measure the amount of mRNA of VEGF. Qiagen OneStep RT-PCR Kit (Qiagen) was used for RT-PCR reaction, and 5'-CCC TGA TGA GAT CGA GTA CAT CTT-3 'and 5'-ACC GCC TCG GCT TGT CAC- 3 'was used. As a control, the GADPH gene was measured by the same method. 5′-GGAAAGCTGTGGCGTGATG-3 ′ and 5′-CTGTTGCTGTAGCCGTATTC-3 ′ were used as primers for GAPDH. RT-PCR reaction is RT (Reverse Transcripratase) reaction at 50 ° C for 30 minutes, PCR reaction is 92 ° C for 30 seconds double strand dissociation, 55 ° C for 30 seconds annealing reaction, 68 ° C for 45 seconds extension reaction This was repeated 25 to 28 times (depending on the cells used), and finally incubated at 68 ° C. for 10 minutes, and the temperature was lowered to 4 ° C. to complete the reaction. Reagents, total-RNA, primers and the like used for RT-PCR were prepared according to the reaction conditions of Qiagen OneStep RT-PCR Kit (Qiagen). After RT-PCR reaction, 2 μl of loading dye was added, and RT-PCR products from VEGF and GADPH mRNA were confirmed on a 2% agarose gel. For the evaluation of the gene expression suppression effect, double-stranded RNA (including unmodified and modified) was introduced when the expression level of VEGF gene in control cells (cells into which double-stranded RNA was not introduced) was 100%. The measurement was performed by measuring the expression level of VEGF in the cells. Moreover, the error in the expression level between cells was corrected by the gene expression level of the control gene (GADPH).

  FIG. 23 shows the results of RNA interference effects of unmodified double-stranded RNA, aminoalkyl-modified, and lipid-modified double-stranded RNA when VEGF is the target and the double-stranded RNA concentration is 200 nM. In FIG. 23, A is a graph showing the VEGF gene expression inhibitory effect of unmodified double-stranded RNA, aminoalkyl-modified, and lipid-modified double-stranded RNA on HeLa cells and B is A549 cells.

  From this result, 25DN3 / 27 and 25DC16 / 27, which were modified with aminoalkyl group and lipid at the 3 ′ end of the sense strand of 25D / 27 VEGF, were much better than unmodified double-stranded RNA (25D / 27 VEGF). It was revealed that it possesses a high VEGF gene expression inhibitory effect. In particular, 25DC16 / 27 VEGF modified with palmitic acid has a much higher ability to suppress gene expression than 25D / 27 VEGF, and by modifying lipids such as palmitic acid to double-stranded RNA, the RNA interference effect is dramatically improved. It was confirmed that it could be improved. From this result, it is clear that the double-stranded RNA targeting VEGF used this time has high sequence specificity and suppresses the expression of the target gene, and side effects on cells by binding lipid to the double-stranded RNA It was suggested that can also be reduced.

FIG. 3 is a diagram showing the structure of a terminal amino-modified 25D / 27 dsRNA synthesized in Example 1. In Example 1, it is a figure which shows the nuclease resistance result of 25D / 27 dsRNA which amino-modified the terminal. In Example 1, it is a figure which shows the result of having examined the processing by 25D / 27 dsRNA which carried out amino modification of the terminal by Dicer. In Example 1, it is a figure which shows the result of the RNA interference effect of 25D / 27 dsRNA which carried out the amino modification of the terminal. In Example 1, it is a figure which shows the result of having evaluated the persistence of the RNA interference effect of 25D / 27 dsRNA which carried out amino modification of the terminal. FIG. 3 is a diagram showing the structure of a terminal amino-modified double-stranded RNA synthesized in Example 2. In Example 2, it is a figure which shows the nuclease resistance result of terminal amino modification double stranded RNA. In Example 2, it is a figure which shows the result of having investigated the processing by Dicer of terminal amino modification double stranded RNA. In Example 2, it is a figure which shows the result of having evaluated the RNA interference effect of terminal amino modification double stranded RNA (siRNA). In Example 2, it is a figure which shows the result of having evaluated the RNA interference effect of terminal amino modification double stranded RNA (LO RNA). In Example 2, it is a figure which shows the result of having evaluated the persistence of the RNA interference effect of terminal amino modification double stranded RNA. FIG. 3 is a view showing the structure of a terminal DNA-modified double-stranded RNA synthesized in Example 3. In Example 3, it is a figure which shows the result of having examined the processing by Dicer of terminal DNA modification double stranded RNA. In Example 3, it is a figure which shows the result of having evaluated the RNA interference effect of terminal DNA modification double stranded RNA. FIG. 4 is a diagram showing the structures of terminal lipids and cholesterol-modified double-stranded RNA synthesized in Example 4. In Example 4, it is a figure which shows the nuclease resistance result of a terminal lipid and cholesterol modification double stranded RNA. In Example 4, it is a figure which shows the result of having examined the processing by the dicer of a terminal lipid and a cholesterol modification double strand RNA. In Example 4, it is a figure which shows the result of having evaluated the RNA interference effect of terminal lipid and a cholesterol modification double strand RNA (siRNA). In Example 4, it is a figure which shows the result of having evaluated the RNA interference effect of terminal lipid modification double stranded RNA (siRNA) without using a gene introduction agent. In Example 4, it is a figure which shows the result of having evaluated the RNA interference effect of terminal cholesterol modification double stranded RNA (siRNA) without using a gene introduction agent. In Example 4, the presence Lipofectamine ^TM 2000, which is a graph showing the results of evaluating the cellular uptake against HeLa, A549 cells terminus and cholesterol modification double-stranded RNA. In the figure, FL represents an image taken with a fluorescence microscope, trans represents an image obtained by photographing the same field of view as the FL with a phase contrast microscope, and merge represents an image obtained by superimposing the FL and trans. In Example 4, it is a figure which shows the result of having evaluated the intracellular introduction | transduction property with respect to the HeLa cell of a terminal lipid and cholesterol modification double stranded RNA in the absence of Lipofectamine ^™ 2000. In the figure, FL represents an image taken with a fluorescence microscope, trans represents an image obtained by photographing the same field of view as the FL with a phase contrast microscope, and merge represents an image obtained by superimposing the FL and trans. FIG. 6 shows the structures of terminal aminoalkyl and lipid-modified double-stranded RNA synthesized in Example 5, and the results of processing by Dicer. FIG. 6 shows the results of RNA interference effects of terminal aminoalkyl and lipid-modified double-stranded RNA synthesized in Example 5 on the VEGF gene.

Claims (4)

An antisense strand RNA comprising a base sequence complementary to the target sequence in the target gene, and a sense strand RNA having a base sequence complementary to the antisense strand RNA, and capable of suppressing the expression of the target gene Single-stranded RNA,
The sense strand RNA consists of 25 nucleotides, the antisense strand RNA consists of 27 nucleotides,
The 3 ′ end of the sense strand RNA is a blunt end, and the 3 ′ end of the antisense strand has a dangling end;
And the substituent is bonded only to the phosphate group of the nucleotide on the 3 ′ end side of the sense strand RNA,
The substituent is a group and an amino group of A aminoalkyl group having 4 to 12 carbon atoms, a carboxyl group of a fatty acid having 6 to 50 carbon atoms obtained by amide bonds, modified RNA. The modified RNA according to claim 1, wherein the first and second nucleotides from the 3 'end of the sense strand RNA are composed of deoxyribonucleotides. The modified RNA according to claim 1 or 2, wherein the aminoalkyl group has 6 carbon atoms. Wherein the fatty acid having 6 to 50 carbon atoms are lauric acid, stearic acid, Mi Risuchi phosphate, or palmitic acid, lipid-modified double-stranded RNA according to claim 1 or 2.