WO2016090115A1

WO2016090115A1 - Novel multivalent bioassay reagents

Info

Publication number: WO2016090115A1
Application number: PCT/US2015/063690
Authority: WO
Inventors: Bradley T. Messmer; Laura Ruff
Original assignee: The Regents Of The University Of California
Priority date: 2014-12-04
Filing date: 2015-12-03
Publication date: 2016-06-09

Abstract

One embodiment provided herein relates to a non-naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences wherein the nucleic acid selectively binds to a target molecule when the target molecule is bound to a surface relative to the target molecule in solution.

Description

NOVEL MULTIVALENT BIOASSAY REAGENTS

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Prov. App. No. 62/087,703 filed December 4, 2015 entitled "NOVEL MULTIVALENT BIOASSAY REAGENTS", which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

[0002] This invention was made with government support under NIH Grant/Contract Number R21 CA 143362 awarded by the National Institutes of Health of the United States of America. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

[0003] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled UCSD091001 WOSEQLIST.TXT, created December 1 , 2015, which is 2 Kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

BACKGROUND

[0004] The present technology relates to the fields of molecular biology, biochemistry, medicine, diagnostics and therapeutics. In particular, in some embodiments, a non-naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences, wherein said nucleic acid selectively binds to a target molecule when said target molecule is bound to a surface relative to said target molecule in solution is provided.

[0005] The guiding principle for the creation of biomolecular recognition agents has been that affinity is essential for both strength and specificity. Monoclonal antibodies, the dominant workhorse of affinity reagents, have mono-valent affinities in the μΜ-ηΜ range with apparent affinities that can be sub nM with the bi-valency intrinsic in intact immunoglobulin structure. The avidin-biotin interaction used ubiquitously for biomolecular assembly is femto-molar and both highly specific and essentially irreversible. High affinity has been proclaimed the essential goal for the selection of useful specific aptamers, though there has been disagreement about a tight coupling of affinity and specificity.

[0006] However, high affinity reagents are not able to discriminate between soluble targets and surface bound or aggregated targets. This limitation manifests in a "high dose hook effect" in contexts such as sandwich assay formats that don't wash between sample and secondary, such as lateral flow immunoassay (LFA). High dose hooks effects are particularly pernicious for diagnostics since they result in false negative results in the very samples that are the most positive for the analyte. This is a significant problem for rapid diagnostic assays, including those for prostate and ovarian cancer.

SUMMARY OF THE INVENTION

[0007] One embodiment provided herein relates to a non-naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences wherein the nucleic acid selectively binds to a target molecule when the target molecule is bound to a surface relative to the target molecule in solution. In one aspect of the foregoing embodiment, each of the individual concatemeric sequences has low affinity for the target molecule fixed to the surface and wherein the concatemeric sequences, in multimeric form, have high avidity for the target molecule fixed to the surface. In another aspect of any of the foregoing nucleic acids, the nucleic acid comprises a nanoparticle. In a further aspect of any of the foregoing nucleic acids, the nucleic acid comprises DNA. In yet another aspect of any of the foregoing nucleic acids, the DNA is more than 1 kb in length. In yet another aspect of any of the foregoing nucleic acids, the DNA is more than 2 kb in length. In yet another aspect of any of the foregoing nucleic acids, the DNA is more than 5 kb in length. In yet another aspect of any of the foregoing nucleic acids, the DNA is more than 100 kb in length. In yet another aspect of any of the foregoing nucleic acids, the nucleic acid comprises a sequence encoding a sequence selected from a siRNA, reporter gene, therapeutic protein, and CpG sequence. In yet another aspect of any of the foregoing nucleic acids, the nucleic acid is associated with a therapeutic or diagnostic agent. In yet another aspect of any of the foregoing nucleic acids, the therapeutic agent comprises a drug. In yet another aspect of any of the foregoing nucleic acids, the diagnostic agent comprises an in vivo imaging agent. In yet another aspect of any of the foregoing nucleic acids, the target molecule comprises a peptide or protein. In yet another aspect of any of the foregoing nucleic acids, the target molecule comprises a molecule differentially expressed in individuals with a disease. In yet another aspect of any of the foregoing nucleic acids, the target molecule comprises a molecule differentially expressed in tumors. In yet another aspect of any of the foregoing nucleic acids, the target molecule comprises a marker expressed on tumor vasculature. In yet another aspect of any of the foregoing nucleic acids, the target molecule comprises a molecule found in blood clots. In yet another aspect of any of the foregoing nucleic acids, the target molecule is fibrin. In yet another aspect of any of the foregoing nucleic acids, nucleic acid comprises from about 10 to about 300 copies of the concatemeric sequences. In yet another aspect of any of the foregoing nucleic acids, the target molecule is fibrin. In yet another aspect of any of the foregoing nucleic acids, nucleic acid comprises from about 10 to about 1200 copies of the concatemeric sequences. In yet another aspect of any of the foregoing nucleic acids, the nucleic acid selectively binds to the target molecule when the target molecule is bound to a lateral flow immunoassay support. In yet another aspect of any of the foregoing nucleic acids, the nucleic acid selectively binds to the target molecule when the target molecule is bound to a bead.

[0008] In another embodiment, a library of nanoparticles comprising at least two populations of nucleic acids, wherein each of the at least two populations comprise a non- naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences wherein the nucleic acid selectively binds to a target molecule when the target molecule is bound to a surface relative to the target molecule in solution is provided. In one aspect of the foregoing embodiment, each of the individual concatemeric sequences has low affinity for the target molecule bound to the surface and wherein the concatemeric sequences, in multimeric form, have high avidity for the target molecule bound to the surface. In yet another aspect of any of the foregoing libraries, the nucleic acids comprise a nanoparticle. In yet another aspect of any of the foregoing libraries, the nucleic acids comprise DNA. In yet another aspect of any of the foregoing libraries, the nucleic acids are more than 5 kb in length. In yet another aspect of any of the foregoing libraries, the nucleic acids are more than 100 kb in length. In yet another aspect of any of the foregoing libraries, the nucleic acids comprise a sequence encoding a sequence selected from a siRNA, reporter gene, therapeutic protein, and CpG sequence. In yet another aspect of any of the foregoing libraries, the nucleic acids comprise from about 10 to about 300 copies of the concatemeric sequences. In yet another aspect of any of the foregoing libraries, the nucleic acids comprise from about 10 to about 1200 copies of the concatemeric sequences. In yet another aspect of any of the foregoing libraries, the library comprises about 10¹² or fewer populations of nucleic acids.

[0009] In a further embodiment, a liposome comprising any of the foregoing nucleic acids is provided.

[0010] In an additional embodiment, a pharmaceutical composition comprising any of the foregoing nucleic acids is provided.

[0011] In another embodiment, a method of determining whether a sample contains a target molecule comprising binding the target molecule to a surface and contacting the bound target molecule with a non-naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences wherein the nucleic acid selectively binds to the target molecule when the target molecule is bound to a surface relative to the target molecule in solution is provided. In one aspect of the foregoing method, the target molecule is bound to the surface via an antibody. In another aspect of any of the foregoing methods, the target molecule is bound directly or indirectly to the surface. In another aspect of any of the foregoing methods, the surface comprises a lateral flow immunoassay support.

[0012] In another embodiment, a method of making a non-naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences wherein the nucleic acid selectively binds to a target molecule when the target molecule is bound to a surface relative to the target molecule in solution, the method comprising performing rolling circle amplification on a circular template comprising a sequence to be concatemerized is provided. In some embodiments, the circular template comprises a complementary sequence to the sequence to be concatemerized.

[0013] In another embodiment, a method of making a non-naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences wherein the nucleic acid selectively binds to a target molecule when the target molecule is bound to a surface relative to the target molecule in solution, the method comprising performing a ligation reaction to generate the concatemeric sequences. [0014] In a further embodiment, a method of making a non-naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences wherein the nucleic acid selectively binds to a target molecule when the target molecule is bound to a surface relative to the target molecule in solution, the method comprising performing a Click chemistry reaction to generate the concatemeric sequences.

[0015] In another embodiment, provided herein is a method of overcoming a high dose hook effect in a lateral flow assay comprising binding a target molecule to a surface of a lateral flow immunoassay support, and contacting the bound target molecule with a non- naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences, wherein said nucleic acid selectively binds to the bound target molecule relative to said target molecule in solution.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 A is a schematic of a lateral flow immunoassay (LFA) for monoclonal antibody authentication. The test line and anti-kappa control lines as well as the anti-IgG control lines should appear when the test antibody (Herceptin) is present. FIG. IB depicts prototype LFAs showing a strong high dose hook effect (arrow) above 100 μg/ml, giving rise to false negative results.

[0017] FIG. 2 shows a selection method for DNA nanoparticles that bind to target coated beads.

[0018] FIG. 3 shows graphs depicting selection and characterization of streptavidin binding nanoparticles. Panel A depicts staining of streptavidin selection rounds 1 -5, probe-only, library, and positive control (biotinylated library) are shown. Panel B depicts staining of 4 selected streptavidin clones on streptavidin beads and BSA beads, negative control clone (GlOneg), and biotinylated positive control clone (GlObio) are shown. Panel C depicts free streptavidin competition titration of SA-D7 and SA-D8 clones and GlObio positive control. Nanoparticles were pre-incubated with varying concentrations of free streptavidin, then streptavidin beads were added. Panel D depicts results from streptavidin beads pre-incubated with a 100 bases oligo the same sequence as the concatemer that makes up SA-D8, an irrelevant 100 bases oligo, or buffer, then nanoparticles were added. Panel E shows results from a free biotin/desthiobiotin/2-iminobiotin competition titration done by pre-incubating streptavidin beads with one of the biotin/biotin derivatives (or buffer for the baseline), then adding nanoparticles, and graphed as percent inhibition. Panel F depicts results from a Biotin/desthiobiotin/2-iminobiotin competitive release done by staining streptavidin beads with nanoparticles, then adding biotin/biotin derivative (or buffer for baseline), graphed as percent released from beads.

[0019] FIG. 4A is a schematic of ligation process using restriction sites in short hairpins in which a 5 '-digested oligo (Faul) only can be ligated to a 3 '-digested oligo (BtsCI), via a biotinylated, 40 bases linker. The linker includes 20 bases of non-specific sequence on the 5' end, which allows the linker to be unzipped from the ligated product with the complementary oligo following purification via the biotin or gel extraction. This process is then repeated to produce larger oligos. FIG. 4B shows agarose gels of 5'-digested and 3'- digested oligos that are subsequently ligated (left panel), purification of ligated product from unligated/undigested oligos via streptavidin bead pulldown and elution via temperature (middle panel), and 5'- and 3 '-digestion of 2-copy oligo and subsequent ligation to 4-copy oligo. FIG. 4C is a schematic of ligation process using click chemistry. An oligo with 5' amine and 3' thiol is synthesized; the core 100 bases oligo is the same as in panel A. In one reaction, the amine is converted to alkyne; in another reaction, the thiol is converted to azide. The click reaction will only join the azide to the alkyne, forming a 2-copy oligo. The process is repeated to form oligos with more copies.

[0020] FIG. 5A is a graph showing results from protein G beads incubated with varying concentrations of either the humanized monoclonal antibody rituximab, serving as a model analyte, or Avastin (bevacizumab) together with a rituximab binding nanoparticle that had been fluorescently labeled, after incubation and final washing, the fluorescence on the beads was measured. FIG. 5B is a model of how the observed nanoparticle detection range differs from initial prediction as well as traditional sandwich immunoassay. Traditional sandwich immunoassay loses signal at high analyte concentrations whereas nanoparticles are insensitive to excess free analyte. The unexpected result that nanoparticles are linear over a wide concentration range and achieve high sensitivity suggests an alternate model for the interaction with analyte. [0021] FIG. 6A depicts global changes in particle frequency distribution as the selection against leukemia cells proceeds. Most sequences in the initial library were unique, as expected. As the selection proceeds, an increase in the fraction of sequences with greater population frequency is observed. FIG. 6B depicts a technical control replicate. The same sample was independently amplified in duplicate and the population frequencies compared. FIG. 6C depicts a biological control replicate. The 2nd round of selection was performed in duplicate and independently amplified for sequencing.

[0022] FIG. 7 shows that nanoparticles overcome the high dose hook effect in LFA. Traditional LFA sandwich assays, such as those shown in FIG. 2, suffer from loss of signal at high analyte concentrations since the excess analyte saturates both the capture agent immobilized on the test line and the secondary detecting agent on the colloidal gold. In contrast, nanoparticles only bind to the analyte that is available for multivalent interaction once captured by the primary test line capture agent.

[0023] FIG. 8 shows selection of clot binding nanoparticles. Panel A depicts a nanoparticle library screened as in FIG. 2 in which the population of particles from each round was fluorescently labeled and the bound particles measured on fibrinogen-coated beads. Panel B depicts binding of individual clones regenerated as nanoparticles and tested for binding on fibrinogen- or BSA-coated beads (as well as other proteins not shown). The selected particles only bound to fibrinogen. Panel C depicts selected particles not inhibited from binding to fibrinogen-coated beads by 4 mg/ml free fibrinogen. Fib3 is a non-binding clone that was a "hitchhiker" in the original selection i.e. it bound to other bound clones. G10 and VI 1 are random particles. Panel D depicts at least 10 clones sequenced from each of 3 independent selections against fibrinogen-coated beads. A C/T rich motif was observed in the majority of the particle sequences. Panel E depicts selected particles bind to clots made from thrombin treated plasma. An irrelevant particle was included, and the ratio of the selected to unselected control particle, as measured by quantitative PC after washing the clot, was determined.

[0024] FIG. 9 shows the structure and sequence motif of Streptavidin nanoparticles. Panel A shows structures of the 100 bases oligo that make up the 4 selected streptavidin nanoparticles (mFold); motif is highlighted in the box. Panel B depicts motif from the 4 particles. Panel C is an alignment of the motif.

[0025] FIG. 10A depicts SA-D8 and biotinylated SA-D8 stained on streptavidin sepharose beads and fluorescence was compared to streptavidin magnetic beads. Probe only is also shown. FIG. 10B depicts SA-D8 and biotinylated SA-D8 stained on streptavidin PMMA beads and fluorescence was compared to streptavidin magnetic beads. Probe only is also shown.

[0026] FIG. 1 1A depicts binding of streptavidin nanoparticles made by alteration of time and/or dNTP dilution. Nanoparticles were made with 3 nmol dNTPs for 30 min at 30°C (the standard conditions used throughout), or 93.8 pmol dNTPs for 30 min or 7.5 min at 30°C. A control nanoparticle from a different library was also made for each of these conditions and used as an internal control in the subsequent PCR. This control was mixed with the SA-D7, SA-D8, GlObio, or GlOneg and included in the staining. The stained streptavidin beads and nanoparticle mixes were then analyzed by PCR and quantitated with a standard for the appropriate library (a plasmid containing the 100 bases template used to make nanoparticles). The ratio of the bound particles (streptavidin nanoparticle ontrol nanoparticle) to total particles (streptavidin nanoparticlexontrol nanoparticle) is graphed. FIG. 1 1 B depicts dissociation of streptavidin nanoparticle over time. Nanoparticles were made with standard conditions and used to stain streptavidin magnetic beads. Extensive washing was done to remove unbound nanoparticles. The stained beads were then incubated in 10 ml PBS 1% BSA 10 mM MgCFi for 35 days. Aliquots were taken every week of the total sample (supernatant plus beads) and supernatant only (beads were removed by magnet). PCR was done on all samples/timepoints and % unbound is graphed (nanoparticles in supernatant/ nanoparticles in total * 100%). At day 21 , a biotin knockoff was also done (filled symbols), in which excess biotin was added to an aliquot of total sample, incubated for 30 min, then beads were removed via magnet to obtain supernatant only.

[0027] FIG. 12 shows Rituximab-specific and Herceptin-specific nanoparticle competitive titration and competitive release. Panel A depicts a free Rituximab competition titration of 3Ritl nanoparticles. Nanoparticles were pre-incubated with varying concentrations of free Rituximab or free whole human IgG, then Rituximab beads were added. Panel B depicts a free peptide competition titration done by pre-incubating Rituximab beads with free Rituximab-specific, BSA-conjugated peptide or BSA-conjugated irrelevant peptide, then adding nanoparticles. Panel C depicts a free peptide competitive release done by staining Rituximab beads with nanoparticles, then adding Rituximab-specific, BSA- conjugated peptide or BSA-conjugated irrelevant peptide. Percent release was determined by PCR of supernatant after incubation with free peptide and centrifugation of beads. Beads stained with nanoparticles prior to addition of peptide were amplified by PCR to determine maximum signal. Panels D, E, and F). Experiments with Herceptin and nanoparticles specific for Herceptin were performed the same as Rituximab (Panels A, B, and C).

[0028] FIG. 13 shows a sandwich assay with protein G, Rituximab, and 3Ritl nanoparticles. Protein G was incubated with different concentrations of Rituximab, polyclonal human IgG, or a mix of Rituximab and polyclonal human IgG for lhr. The Rituximab+IgG samples are graphed as amount of Rituximab in the sample (all samples had the same total amount of antibody). Fluorescently-labeled 3Ritl nanoparticles were then added to the sample for 2hr, then samples were washed and fluorescence measured.

[0029] FIG. 14 shows a sandwich assay done in the presence of excess amounts of free antibody. Protein G was incubated with different concentrations of Rituximab or Avastin, and incubated for 1 hr. Alexa Fluor647-labeled 3Ritl nanoparticles, Aval nanoparticles, or MJneg nanoparticles, or Alexa Fluor488-labeled anti-kappa human light chain antibody were then added and incubated for an additional 2 hr, then washed and measured for fluorescence. For anti-kappa light chain samples only: Rituximab and Avastin samples <l-fold free antibody were diluted with mouse IgG2b κ to equal 1-fold total antibody. This was done to bind all free protein G sites before addition of anti-kappa antibody so it would not bind non-specifically.

[0030] FIG. 15A -15D show graphs depicting SA-D7 and SA-D8 binding to streptavidin-coated beads in a variety of buffer conditions. Fluorescently-labeled SA-D7, SA- D8, negative control G10 and positive control GlObio were incubated with streptavidin- coated beads in a variety of different buffer conditions and binding was assessed. FIG. 15A relates to MgCFi concentration. FIG. 15B relates to NaCl concentration. FIG. 15C relates to physiologic buffers. FIG. 15D relates to biologic buffers. [0031] FIG. 16A shows an atomic force micrograph (AFM) of dried DeNAno SA- D8 on poly-L-lysine-coated mica (scale=400 nm). FIG. 16B depicts SA-D8 DeNAno roughly 75 nm in diameter as observed by transmission electron microscopy (TEM) using negative staining (scale=100 nm). FIG. 16C shows a TEM of small SA-D8 DeNAno roughly 58 nm in diameter (scale=100 nm).

DETAILED DESCRIPTION

[0032] In some embodiments, a non-naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences wherein the nucleic acid selectively binds to a target molecule when the target molecule is bound to a surface relative to the target molecule in solution is provided. As used herein, "selectively binds" refers to a binding by a non- naturally occurring nucleic acid that is only or predominantly for the target molecule. In certain embodiments, "selectively binds" refers to a non-naturally occurring nucleic acid binding a target molecule at least 10, 50, 100, 200, 500, 1000, or more times greater than binding to a non-target molecule. In some embodiments the nucleic acid is a nanoparticle. As used herein, "concatemeric sequences" refers to continuous nucleic acid sequences that contain multiple copies of the same nucleic acid sequence linked in series.

[0033] The disclosures of U.S. Patent App. No. 14/528980, entitled "SINGLE MOLECULE NUCLEIC ACID NANOP ARTICLES," filed Oct. 30, 2014; U.S. Patent No. 8,895,242, entitled "SINGLE MOLECULE NUCLEIC ACID NANOPARTICLES, issued Nov. 25, 2014; International App. No. PCT/US2010/053270 entitled "SINGLE MOLECULE NUCLEIC ACID NANOPARTICLES", filed October 19, 2010 and published in English on April 28, 201 1 as WO 201 1/050000; U.S. Prov. App. No. 61/279,408 filed on October 20, 2009; and U.S. Prov. App. No. 61/095,575 filed on September 9, 2008, are incorporated herein by reference in their entireties.

[0034] Although, monoclonal antibodies bind to captured and free targets with huge affinity, in some embodiments, the nucleic acids described herein bind to the captured molecules through high avidity but low affinity interactions. These types of interactions are generally stronger in sum than the net hybridization energy of the unit nanoparticle. In some embodiments, the nucleic acids described herein have increased binding to target molecules when the target molecules are captured or arranged on a surface, compared to binding to target molecules when the target molecules are in solution. This has not been demonstrated with other affinity ligands (i.e. mAb, peptide, aptamer). Thus, in some embodiments, the nucleic acids and nanoparticles described herein provide a biomolecular affinity reagent that replaces single or bi-valent affinity with hyperavidity. These particles need to bind multiple targets to produce a high afilnity/high avidity interaction. Binding of only one or a few targets produces low individual affinity/low overall avidity interaction and the DNA particle does not maintain interaction. The low individual affinity interactions are additive, thus when more target in aggregate is bound by DNA particle, the overall avidity increases to a high level. Thus, the particles preferentially bind aggregated target molecules even in the presence of high concentrations of free target molecule.

[0035] The guiding principle for the creation of biomolecular recognition agents has been that affinity is important for both strength and specificity. Monoclonal antibodies, the dominant workhorse of affinity reagents, have mono-valent affinities in the μΜ-ηΜ range with apparent affinities that can be sub nM with the bi-valency intrinsic in intact immunoglobulin structure. The avidin-biotin interaction used ubiquitously for biomolecular assembly is femto-molar and both highly specific and essentially irreversible. High affinity has been proclaimed an important goal for the selection of useful specific aptamers, though there has been disagreement about a tight coupling of affinity and specificity. In some embodiments, the nucleic acids and nanoparticles described herein achieve specific recognition through an orthogonal approach which substitutes massive avidity for monovalent affinity. As a consequence to their reliance on avidity rather than affinity, the nucleic acids and nanoparticles described herein can perform molecular target recognition in settings where traditional affinity reagents fail. For example, high affinity reagents are not able to discriminate between soluble targets and surface bound or aggregated targets. This limitation manifests in a "high dose hook effect" in sandwich assay formats that don't wash between sample and secondary, such as lateral flow immunoassay (LFA). High dose hook effects are particularly pernicious for diagnostics since they result in false negative results in the very samples that are the most positive for the analyte. This is a significant problem for rapid diagnostic assays, including those for prostate and ovarian cancer. [0036] The nucleic acids and nanoparticles described herein have many applications, including applications to produce novel affinity reagent for immunoassays. Other applications of the present nucleic acids and nanoparticles include in vivo imaging or therapeutic applications using nucleic acids or nanoparticles which are biocompatible and which have a sufficient half-life.

[0037] Targeting cells with nanoparticles without knowing the protein targets is described in Steiner JM, et al , DeNAno: Selectable deoxyribonucleic acid nanoparticle libraries. J Biotechnol. 2010 Feb 15; 145(4):330-3.

[0038] As described herein, nucleic acids or nanoparticles against defined proteins may be selected on protein coated beads whereby the particles bind to the active sites of the protein targets and bind to arrayed proteins but not the same protein in solution. In addition, in some embodiments, the nucleic acids and nanoparticles described herein are far more sensitive as a detection agent than would have been predicted. In some embodiments, the nucleic acids and nanoparticles described herein may be used in immunoassays. In some embodiments, the nucleic acids and nanoparticles described herein have increased binding to target molecules when the target molecules are captured or arranged on a surface, compared to binding to target molecules when the target molecules are not captured or arranged on a surface, for example, when the target molecules are in solution.

[0039] Monoclonal antibodies bind to captured and free targets with high affinity. In some embodiments, the nucleic acids and nanoparticles described herein bind to the captured molecules through high avidity but low affinity interactions. This has not been shown with other affinity ligands (such as mAb, peptide, aptamer).

[0040] In some embodiments, the nucleic acids and nanoparticles described herein comprise about 10 to about 300 copies of a template sequence and can bind to multiple target molecules if they are physically constrained such that the particle can interact with many targets. In some embodiments, the nucleic acids and nanoparticles described herein comprise about 10 to about 1200 copies of a template sequence and can bind to multiple target molecules if they are physically constrained such that the particle can interact with many targets. In some embodiments, the nucleic acids and nanoparticles are DNA. Surprisingly, the nucleic acids and nanoparticles seem to be able to aggregate their targets on the capture surface even when the target is at a very low concentration (see below).

[0041] In some embodiments, the nucleic acids and nanoparticles described herein may be used as affinity reagent for immunoassays. Other applications of the nucleic acids and nanoparticles described herein include in vivo imaging or therapeutic applications.

[0042] The guiding principle for the creation of biomolecular recognition agents has been that affinity is important for both strength and specificity. Monoclonal antibodies, the dominant workhorse of affinity reagents, have mono-valent affinities in the μΜ-ηΜ range with apparent affinities that can be sub nM with the bi-valency intrinsic in intact immunoglobulin structure. The avidin-biotin interaction used ubiquitously for biomolecular assembly is fempto-molar and both highly specific and essentially irreversible. High affinity has been proclaimed an important goal for the selection of useful specific aptamers (Eaton BE, et al., Chem Biol. (1995) 10:633-8), though there has been disagreement about a tight coupling of affinity and specificity (Carothers JM, et al., J Am Chem Soc. 2006 128:7929- 37).

[0043] In some embodiments, the nucleic acids and nanoparticles described herein achieve specific recognition through an orthogonal approach which substitutes massive avidity for monovalent affinity. As a consequence to their reliance on avidity rather than affinity, in some embodiments, the nucleic acids and nanoparticles described herein can perform molecular target recognition in settings where traditional affinity reagents fail. For example, high affinity reagents are not able to discriminate between soluble targets and surface bound or aggregated targets. This limitation manifests in a "high dose hook effect" in sandwich assay formats that don't wash between sample and secondary, such as lateral flow immunoassay (LFA). High dose hook effects are particularly pernicious for diagnostics since they result in false negative results in the very samples that are the most positive for the analyte. This is a significant problem for rapid diagnostic assays, including those for prostate and ovarian cancer (Gillet P, et ah, Malar J. 2009; 8:271 ; Fleseriu M, et al., J Neurooncol. 2006 79:41-3; McCudden CR, et al, Clin Biochem. 2009 42: 121-4; Wolf BA, et al, N Engl J Med. 1989 320: 1755-6; Wheeler CA, et al., Obstet Gynecol. 1990 75:547-9; Furuya Y, et ah, J Urol. 2001 166:213), as well as drug authentication assays being developed by our company.

[0044] Similarly, biologically relevant polymers of proteins cannot be distinguished from their soluble precursors unless there are biochemical sites that are unique to polymerized forms. For example, the major component of blood clots is fibrin, which is polymerized from fibrinogen precursors following thrombin cleavage of a small peptide fragment. Fibrinogen is present in blood at roughly 3 mg/ml, so any molecularly targeted agent or probe would have to have a significantly greater affinity for fibrin, presumably through interactions with the very small region of the fibrin protein that is altered following thrombin cleavage of fibrinogen. While several such agents have been described, and advanced in clinical trials, there is still no FDA approved molecularly targeted agent for thrombus detection. Tumors are known to contain many microdots in their tortuous vasculature (Ruoslahti E. Nat Rev Cancer. 2002 2:83-90) so clot binding imaging agents would have immediate application in tumor imaging for both research and diagnostic use.

[0045] Targeted in vivo delivery of imaging agents, drugs, gene therapies, and other agents has been frequently cited as a major area for nanotechnology to impact biomedical science. However, the approaches taken to date have been limited and complex. Most have tried to couple existing affinity reagents such as antibodies, peptides, aptamers, or small molecule ligands to nanoparticles of various types. This approach has two major difficulties: the lack of specific targeting ligands for many applications and the difficulty of making nanoparticles that are biocompatible yet retain the desired specificity. To date, the list of nanoparticles in the clinic is small and such nanoparticles are mostly iron oxide, liposomes, and albumin (e.g. Feridex, Doxil and Abraxane). None of these are targeted by anything other than their bulk physical properties and generic biomolecular interactions.

[0046] Many tumors, such as breast cancer, exhibit increased vascularization and altered vascular characteristics, and several new imaging techniques have been developed to visualize vascular properties and differentiate between malignant and benign tissue, thereby improving specificity (Heijblom M, et al, Technol Cancer Res Treat. 201 1 10:607-23). Biomolecular recognition of specific markers of tumor vasculature has been applied towards delivery of cytotoxic or diagnostic agents to tumors because tumor blood vessels express a number of cell surface and extracellular matrix proteins that normal vessels do not express or only at much reduced levels. Examples include a contrast reagent coupled to anti-avP3 antibody to image tumor vasculature and chemotherapeutic drugs coupled to av 3-binding peptides to enable drug targeting to tumors. Superparamagnetic nanoparticles coupled to a clot-binding protein were shown to home to tumor blood vessels in a mouse model and may be useful as contrast reagents for MRI and also for therapeutic applications (Simberg D, et al, Proc Natl Acad Sci U S A. 2007 104:932-6).

[0047] Currently, the primary imaging tools used to detect blood clots, or thrombi, in large vessels are CT, MR angiography or ultrasound. However, these methods are only able to detect large thrombi or occlusions in large or moderate size vessels when used with conventional contrast agents, primarily by recognizing filling defects or highlighting stenosis but without any further information. Compared to normal blood vessels, tumor vessels are tortuous, leaky, and have irregular diameters and thin walls. Molecularly targeted agents that accumulate in thrombi have been considered, but a major challenge is the paucity of molecular determinants unique to clots. The major component of a clot is fibrin. However, the precursor fibrinogen is present in blood at roughly 3mg/ml, so any molecularly targeted agent or probe would have to have a significantly greater affinity for fibrin, presumably through interactions with the very small region of the fibrin protein that is altered following thrombin cleavage of fibrinogen. Fibrin-specific peptides have been advanced into clinical trials but have not progressed to approval (Spuentrup E, et al., European radiology. 2008 18: 1995-2005; Vymazal J, et al , Investigative radiology. 2009 44:697-704).

[0048] In some embodiments, the nucleic acids and nanoparticles described herein may be used as a biomolecular affinity reagent that replaces single or bi-valent affinity with hyper-avidity. In some embodiments, rolling circle replication of circular oligonucleotide templates produces a concatemeric single strand of DNA that is composed of many copies of single strand DNA that is complementary to the template sequence. Under physiological conditions, this strand forms a nanoparticle whose size and number of template copies can be tuned by the reaction conditions. When random sequences are inserted into a template circle, massively diverse libraries can be made with each particle containing many copies of a unique sequence. As with aptamer libraries, some of these sequences may adopt secondary and tertiary structures that give rise to specific binding interactions. Unlike aptamers, those interactions need not be high affinity since the presence of many copies allows avidity to compensate if the target is also multimeric or a polymer. In fact, in Applicant's studies to date a particle whose monomer sequence can function as an aptamer in any quantifiable assay has yet to be identified.

[0049] Initial proof-of-concept experiments were performed with human dendritic cells (DC) (Steiner JM, et al, J Biotechnol. 2010 145:330-3). Particles that bound specifically to human DC and not to other mouse or human cells were obtained. Since then, particles that selectively bind to mouse pancreatic cell line panc02, human breast cancer cell line MDA-MB-231 , and primary chronic lymphocytic leukemia cells have been developed (Ruff, L. E., et ah, Nanotechnol. Rev. 3, 569-578 (2014)). In all cases the selection and binding function were confirmed in serum and plasma.

[0050] More recently, particles that bound a defined protein target were selected by screening a nanoparticle library against streptavidin coated magnetic beads (FIG. 3). After the final round of selection, four unique sequences were recovered. When regenerated as nanoparticles, these clones bound to the streptavidin coated beads and were completely inhibited by free biotin or biotin derivatives pre-incubated with the beads, suggesting they bind streptavidin at or near the biotin binding site. A biotinylated particle used as control was similarly inhibited but needed an order of magnitude higher biotin concentration for equivalent degree of competitive inhibition. When the particles were allowed to bind the streptavidin coated bead first, free biotin or its weaker analogs could competitively displace the selected particles but not the biotinylated particle control, suggesting that the selected particles were binding through a collection of relatively weak interactions, unlike the high affinity interactions in the biotinylated control. Interestingly, when the reciprocal experiment was done using free streptavidin, the inverse relationship was seen. The biotinylated control particles were completely inhibited from binding to the beads by as little as 100 nM free streptavidin, whereas the selected particles were only inhibited by 100 μΜ. Collectively, these data demonstrate that the selected nanoparticles bind through a highly multivalent collection of weak interactions. [0051] Library biopanning approaches such as phage display, SELEX, or those described herein utilize sufficient initial diversity such that there is likely to be one or more desired element among the initial population being screened. Short peptide (<7 aa) phage libraries can potentially contain every possible combination but longer peptide libraries and most nucleic acid based libraries can contain only a tiny fraction of the possible sequence combinations and the frequency of binding sequences is usually unknown. Aptamer libraries typically contain >10¹⁵ members whereas, in some embodiments, the nucleic acid or nanoparticle libraries are 10¹² or less. Despite this much lower initial diversity, binders are selected with relative ease and in much few rounds of panning, suggesting that there is a greater relative frequency of binders and that the discrimination of a "binder" from a non- binder is relatively greater. Thus, in some embodiments, the nucleic acids and nanoparticles described herein are more similar to phage displayed peptide libraries in terms of their panning characteristics, despite sampling a much smaller fraction of the possible sequence variants.

[0052] In some embodiments, the nucleic acids and nanoparticles described herein may be generated by rolling circle replication of a circular oligonucleotide template. Particle size can be roughly tuned by the rate of the reaction, adjusted via dNTP concentration and the reaction time, although creating a precise number of copies may be difficult. At smaller copy numbers the presence of the template circle could also affect the overall particle structure. Furthermore, while the rolling circle method is rapid and straightforward for discovery purposes, in some embodiments, such as production of nucleic acids or nanoparticles destined for in vivo use in humans, other methods may be beneficial. Therefore, at least two synthetic methods have been designed in which the particles will be directly assembled from their monomer components. In one example method (FIG. 4, panel A), adapters are added to the ends of the synthesized monomer sequence that form stem loops encoding Type II restriction sites. Removal of the adapter from either end leaves a ligate-able end allowing precise formation of dimers that themselves contain protective adapters on both ends. The process can be iterated to produce larger concatemers of any size. Tetramers from a monomer starting oligonucleotide have been created (FIG. 4, panel B). [0053] In another approach, Click chemistry is used in place of DNA ligase to fuse monomers or concatemers. The monomer oligo is prepared with an amino group on one end and a thiol on the other. The amino group is converted to an alkyne using the succinimidyl ester DIBO alkyne reagent from Molecular Probes or the thiol converted to an azide with iodoacetamide azide. The functionalized monomers are combined for a copper- free Click reaction. The bio-orthogonal nature of the Click reaction ensures that the non- functionalized ends as well as the rest of the oligonucleotide are unaffected by the reaction. As with the molecular biology based scheme above, this process can be iterated and any size concatemer potentially produced.

[0054] The molecular biology based scheme creates a continuous DNA strand, similar to that produced by rolling circle replication. For certain secondary amplification methods, such as hyperbranching, that is beneficial and would not be possible with the chemical approach. Conversely, the Click chemistry approach can be used to incorporate other molecules besides DNA into the continuous strand, including RNA, peptides, small molecules, or other polymers, as long as the moiety can be dual functionalized for the Click reaction or synthesized on or in an oligonucleotide. Thus, it will be appreciated that, in some embodiments, the present nucleic acids or nanoparticles may have one or more molecules other than a DNA base within the continuous strand. In each scheme, hybrid nucleic acids or nanoparticles that consist of concatemers of different composition can be produced, potentially overcoming a drawback that was observed with hybrid templates (i.e. AAAAA can be coupled to BBBBB to produce AAAAABBBBB rather than ABABABABAB that results from an AB template by rolling circle). In fact, any pattern of any number of moieties can be produced. This can be readily tested directly, for example, using one of the cell binding particles previously recovered and the streptavidin binder shown in FIG. 3.

[0055] The nucleic acids or nanoparticles produced by rolling circle are estimated to contain several hundred copies, and particles of roughly 50 copies have also been produced with no loss of binding. Furthermore, no loss in binding activity has been observed for nanoparticles that have been denatured when hybridizing fluorescent or biotinylated probes, suggesting that the key secondary structures are not exclusively consequent to the rolling circle process. To validate that concatemers produced by these synthetic schemes function equivalently to nanoparticles made by rolling circle replication, each scheme will be used to produce an exponential series of concatemer sizes, from 2 to 128 copies, of the streptavidin binding nanoparticle sequence D8 (FIG. 3 (binding data) and FIG. 9 (sequence and motif)). In addition, the dose response to competitive inhibition and displacement with biotin or its weaker analogs will be tested across the different sized particles. These results will inform the design of immunoassays.

[0056] Immunoassay development - The data shown for the streptavidin binding, rituximab binding, and avastin binding nanoparticles illustrate several of the unique properties engendered by high avidity/low affinity type binding. The ability of a nucleic acid or nanoparticle to bind its aggregated or polymerized target even in the presence of large amounts of soluble target is a very useful feature in immunoassays since it can, in principle, both obviate the high dose hook effect as well as eliminate the need for a washing step in between sample and nucleic acid or nanoparticle application to a primary capture surface or bead. Both are illustrated in the data shown in FIG. 3, panel C (streptavidin binding in the presence of free streptavidin), FIG. 5, panel A (Rituximab binding in the presence of free Rituximab titration), FIG. 7 (illustration), and FIG. 14 (Rituximab and Avastin-binding in the presence of free Rituximab or Avastin with no wash step).

[0057] An anticipated limitation of using nanoparticles as a secondary detection reagent in traditional sandwich immunoassay formats was the prediction that the dynamic range would be fairly limited, based on the assumption that the density of analyte captured by the primary antibody would need to achieve a minimum threshold for nanoparticles to find sufficient avidity, and that once this threshold was passed nanoparticle saturation would rapidly follow. However, this concern has surprisingly not been supported by the data. In a representative fluorescent sandwich assay (FIG. 5, panel A), not only was there no high dose hook effect, but the fluorescent signal from bound nanoparticles was log linear over a three- log concentration range. This surprising but impressive result suggests a different model of nanoparticle binding in which the analyte is collected into patches that collectively stabilize the interactions with a nanoparticle. Coupled with the multitude of detection formats available with the present nucleic acids and nanoparticles, including PCR and isothermal DNA amplification as well as fluorescent and colorimetric methods, these results suggest that these nucleic acids and nanoparticles may offer greater sensitivity, dynamic range, and ease of use with less background than traditional secondary antibody reagents.

[0058] A strategy has been developed to sequence nanoparticle populations on the MiSeq platform from Illumina. A given pool of nanoparticles were amplified by tagged primers that add the sequencing primer sites as well as a tag for multiplexing on the sequencer. This technique was used to evaluate a selection on primary leukemia cells as well as several controls and technical replicates (FIG. 6). A total of 32 different nanoparticle pools were prepared and sequenced simultaneously, yielding -100,000 complete sequences per pool with excellent concordance between both technical and biological replicates.

[0059] High dose hook effect in lateral flow - As discussed above, the high dose hook effect is a particularly acute problem for lateral flow immunoassays (LFA). In other work, LFA has been developed for therapeutic monoclonal antibody authentication. These LFAs show a strong high dose hook effect (FIG. 1). To demonstrate the superiority of the present nucleic acids or nanoparticles in this format, methods to attach colloidal or nano- particulate gold to rituximab binding nucleic acids or nanoparticles are optimized. The gold can be attached directly to the particle or to an oligonucleotide that hybridizes to the particle. The latter method is how fluorescent labels are routinely attached.

[0060] The present nucleic acids and nanoparticles are potential contrast, drug delivery or therapeutic agents but their behavior in vivo may require characterization or optimization. In vivo selections will also be performed to identify particles that selectively accumulate in tumors. Fibrin clot binding particles have been identified (FIG. 8) and will be tested as imaging agents for tumors. After 5 rounds of selection the population was enriched for specific binding particles. A number of clones were sequenced and most were unique, though the majority contained an extended motif enriched in C and T bases. The particles bound the beads even in the presence of physiological concentration of free fibrinogen. Particles tested on clots formed in vitro by incubating human plasma with thrombin and divalent cations also showed good binding with an average enrichment of > 10-fold over a non-specific particle (FIG. 8). Nucleic acids and nanoparticles

[0061] Some embodiments of the methods and compositions provided herein include a non-naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences wherein the nucleic acid selectively binds to a target molecule when the target molecule is bound to a surface relative to the target molecule not bound to a surface, for example when the particle is in solution, or suspended in a fluid, such as a liquid. In some embodiments, the target molecule can be associated with the surface via covalent bonds, or non-covalent bonds. In some embodiments, non-covalent bound can include protein-protein interactions, and nucleic acid base pairing. In some embodiments, the target molecule can be associated with a surface through intermediate non-target molecules. In some embodiments, individual concatemeric sequences can have low affinity for the target molecule associated with the surface and wherein the concatemeric sequences, in multimeric form, have high avidity for the target molecule associated with the surface. In some embodiments, a bead comprises the surface. In some embodiments, the surface is a planar.

[0062] In some embodiments, the nucleic acid comprises a nanoparticle. As used herein, a "nanoparticle" refers to a particle that is less than or equal to 100, 200, 300, or 400 nm in diameter. In some embodiments, the nanoparticle comprises a tertiary structure. Without wishing to be bound to any one theory, in some embodiments, the tertiary structure of a nanoparticle provides properties of the nanoparticle in which individual concatemeric sequences can have low affinity for the target molecule associated with the surface and wherein the concatemeric sequences, in multimeric form, have high avidity for the target molecule associated with the surface.

[0063] In some embodiments, binding of the nanoparticle to the target molecule bound to a surface compared to binding of the nanoparticle to the target molecule not bound to the surface can be increased at least 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100- fold, 1000-fold, and a range between any two of the foregoing numbers.

[0064] In some embodiments, the nucleic acid may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a combination of RNA and DNA. In some embodiments, the nucleic acid may be DNA fragments, RNA fragments, chromosomes, structural or regulatory genes or any other nucleic acid in either single- or double-stranded form. In some embodiments, the nucleic acid can include artificial nucleotides, nucleic acid analogs, or a combination of artificial and analogous nucleic acids. In some embodiments, the nucleic acid can include fluorescent nucleotides (e.g., fluorescently-labeled dCTP).

[0065] In some embodiments, the nucleic acid has a length that is more than 0.5 kb, 1 kb, 2 kb, 5 kb, 10 kb, 20 kb, 50 kb, 100 kb, 200 kb, 500 kb, and a range between any two of the foregoing numbers.

[0066] In some embodiments, the nucleic acid has a number of copies of the concatemeric sequences that is at least about 10, 50, 100, 300, 500, 1000, 1200, 1500, 2000, 3000, 5000 copies, and a range between any two of the foregoing numbers. In some embodiments, the nucleic acid comprises copies the concatemeric sequences in a range of from about 10 to about 300 copies, from about 10 to about 500 copies, from about 10 to about 1200 copies, from about 10 to about 1500 copies, from about 10 to about 3000 copies.

[0067] In some embodiments, the nucleic acid comprises a sequence selected from the group consisting of a siR A, a reporter gene, a nucleic acid encoding a therapeutic protein, and a CpG sequence. In some embodiments, the nucleic acid comprises a consensus motif found in several different nucleic acids. In some embodiments, the consensus motif comprises: A/C, C/T, G/C, A, C, G/A, C, A/C which is also MWSACRCM (SEQ ID NO:09).

[0068] In some embodiments, the nucleic acid is associated with a therapeutic or diagnostic agent. For example, a non-naturally occurring nucleic acid as described herein may be associated with a therapeutic agent (e.g., a drug, a probiotic, an antibiotic, a prebiotic, or a combination thereof) useful for the treatment, amelioration, and/or prevention of one or more conditions and/or one or more adverse side-effects resulting from treatment of a condition. In some embodiments, the therapeutic agent comprises a drug. For example, a non-naturally occurring nucleic acid as described herein may be associated with one or more drugs (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 drugs). The drug may be, for example, a drug already available, a drug in development, a drug in clinical trials, a drug previously available but withdrawn from the market, an available or withdrawn drug under consideration for treating a new indication, and combinations thereof. In some embodiments, the drug comprises a chemotherapeutic agent. In some embodiments, the diagnostic agent comprises an in vivo imaging agent. In some embodiments, the in vivo imaging agent can include a fluorescent label, a radioisotope, or a combination thereof.

[0069] In some embodiments, the target molecule comprises a peptide or protein. In some embodiments, the protein comprises an antibody. In some embodiments, the target molecule comprises a molecule differentially expressed in individuals with a disease. In some embodiments, the target molecule comprises a molecule differentially expressed in tumors. In some embodiments, the target molecule comprises a marker expressed on tumor vasculature. Molecules differentially expressed in tumors and markers expressed on tumor vasculature include alpha fetoprotein (AFP), AL , B2M, CA 15-3, CA27-29, CA 19-9, CA- 125, calcitonin, calretinin, carcinembryonic antigen, CD34, CD99MIC 2, CD1 17, chromogranin, cytokeratin, desmin, epithelial membrane antigen (EMA), Factor VIII, CD31 , FL1, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45, human chorionic gonadotropin (hCG), inhibin, keratin, CD 16, CD56, TCRa , CD3, CD4, CD8, TCRy5,MART-l , Myo Dl , muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase (FLAP), prostate-specific antigen, P I PRC (CD45), S I 00 protein, smooth muscle actin (SMA), synaptophysin, thyroglobulin, thyroid transcription factor- 1 , tumor M2-P , and vimentin. In some embodiments, the target molecule comprises a molecule found in blood clots. Molecules found in blood clots include Factor I, Factor II, Factor III, Factor IV, Factor V, Factor VI, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, fibrin, von Willebrand factor, prekallikrein, high-molecular-weight kininogen (HMWK), fibronectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, protein Z-related protease inhibitor (ZPI), plasminogen, alpha 2-antiplasmin, tissue plasminogen activator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI 1 ), plasminogen activator inhibitor-2 (PAI2), and cancer procoagulant. In some embodiments, the target molecule is fibrin.

[0070] In some embodiments, the nucleic acid selectively binds to the target molecule when the target molecule is bound to a lateral flow immunoassay support. In some embodiments, the nucleic acid selectively binds to the target molecule when the target molecule is bound to a bead. Kits

[0071] Some embodiments of the methods and compositions provided herein include a kit comprising a surface adapted to bind a target molecule and any one of the foregoing non-naturally occurring nucleic acid, in which the nucleic acid selectively binds to the target molecule when said target molecule is bound to a surface relative to said target molecule in solution. In some embodiments, the surface is planar. In some embodiments, a bead comprises the surface. In some embodiments, a lateral flow assay comprises the surface.

Libraries of nanoparticles

[0072] Some embodiments of the methods and compositions provided herein include a library of nanoparticles comprising at least two populations of nucleic acids. In some embodiments, each of the at least two populations comprise the nucleic acids and/or nanoparticles provided herein. In some embodiments, the library has a population of nucleic acids less than about 10¹⁵ nucleic acids, 10¹⁴ nucleic acids, 10¹² nucleic acids, 10¹⁰ nucleic acids, 10⁸ nucleic acids 10⁵ nucleic acids, 10³ nucleic acids, 10² nucleic acids, and a range between any two of the foregoing numbers. In some embodiments, the percentage of nucleic acids in a library comprising the same sequence is greater than 5%, 10%, 20%, 50%, 70%, 90%, 95%, 99%, and a range between any two of the foregoing numbers.

Liposomes and pharmaceutical compositions

[0073] Some embodiments of the methods and compositions provided herein include a liposome comprising a nucleic acid or nanoparticles provided herein. Some embodiments of the methods and compositions provided herein include a liposome comprising a library of nucleic acids or nanoparticles provided herein. Some embodiments of the methods and compositions provided herein include a pharmaceutical composition comprising a nucleic acid or nanoparticles provided herein. Some embodiments of the methods and compositions provided herein include a pharmaceutical composition comprising a library of nucleic acids or nanoparticles provided herein. Preparing nucleic acids and nanoparticles

[0074] Some embodiments of the methods and compositions provided herein include a method of making a non-naturally occurring nucleic acid provided herein. Some methods include performing rolling circle amplification on a circular template comprising a sequence complementary to the sequence to be concatemerized. In some embodiments, the rolling circle amplification on a circular template is performed for a period that is at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 60 minutes, at least 90 minutes, at least 100 minutes, at least 1 10 minutes, at least 120 minutes, at least 180 minutes, at least 240 minutes, and a range between any two of the foregoing numbers.

[0075] Some embodiments also include selecting the nucleic acid comprising a continuous strand of concatemeric sequences wherein the nucleic acid selectively binds to a target molecule associated with a surface.

[0076] Some embodiments also include amplifying the selected nucleic acid. In some embodiments, the amplification comprises a polymerase lacking 5 '-3' exonuclease activity. In some embodiments, the polymerase comprises the Stoffel fragment of Taq polymerase,

[0077] Some embodiments also include asymmetrically amplifying the amplified nucleic acid, thereby generating a single-stranded linear template. In some embodiments, asymmetric amplification includes use of a single primer.

[0078] Some embodiments also include circularizing a linear template, thereby preparing a circular template comprising a sequence complementary to the sequence to be concatemerized.

[0079] Some embodiments also include repeating the foregoing method with the circular template comprising a sequence complementary to the sequence to be concatemerized. In some embodiments, the method is repeated at least three times, four times, five times, six times, seven times, eight times, nine times, ten times, and a range between any two of the foregoing numbers.

[0080] Some embodiments of the methods and compositions provided herein include a method of making a non-naturally occurring nucleic acid provided herein which includes performing a ligation reaction to generate the concatemeric sequences. In some embodiments, prior to performing the ligation reaction to generate the concatemeric sequences, a nucleic acid comprising a first and second hairpin having a single-stranded region therebetween can be obtained. In some embodiments, the first hairpin comprises a first restriction site and the second hairpin comprises a second restriction site, wherein the cleaved first and second restriction sites are capable of being ligated to one another. In some embodiments, the first and second cleavage sites are different. In some embodiments, the first and second restriction sites are selected from the group consisting of Faul and BtsCI. Some embodiments also include cleaving the first and second cleavage sites, thereby obtaining a first and second nucleic acid. Some embodiments also include ligating the first nucleic acid to the second nucleic acid, thereby obtaining a nucleic acid comprising a hairpin at each end and having a single-stranded region therebetween. Some embodiments also include repeating the foregoing method with the obtained nucleic acid comprising a hairpin at each end and having a single-stranded region therebetween. In some embodiments, the method is repeated at least three times, four times, five times, six times, seven times, eight times, nine times, ten times, and a range between any two of the foregoing numbers.

[0081] Some embodiments of the methods and compositions provided herein include a method of making a non-naturally occurring nucleic acid provided herein which includes performing a Click chemistry reaction to generate the concatemeric sequences. Some embodiments include azide alkyne Huisgen cycloaddition (Kolb., HC, et ah, (2003) Drug Discovery Today 8: 1 128-1 137; Rosovtsev VV., et ah, (2002) Angewandte Chemie International Edition 41 : 2596-2599; and Tornoe CW., et al., (2002) J Org Chem 67:3057- 64).

[0082] In some embodiments, a population of single stranded nucleic acids having a 5' amine moiety and 3' thiol moiety is obtained. Some embodiments also include converting the 5' amine moiety amine to a 5' alkyne moiety in a first subpopulation of the single stranded nucleic acids, thereby obtaining a first subpopulation of single stranded nucleic acids having a 5' alkyne moiety and 3' thiol moiety. Some embodiments also include converting the 3' thiol moiety amine to a 3' azide moiety in a second subpopulation of the single stranded nucleic acids, thereby obtaining a second subpopulation of single stranded nucleic acids having a 5' amine moiety and 3' azide moiety. Some embodiments also include joining the 5' alkyne moiety of the nucleic acids of the first subpopulation to the 3' azide moiety of the nucleic acids of the second subpopulation, thereby obtaining a obtaining a population of single stranded nucleic acids having a 5' amine moiety and 3' thiol moiety and concatemeric sequences therebetween. Some embodiments also include repeating the foregoing method with the obtained population of single stranded nucleic acids having a 5' amine moiety and 3' thiol moiety and concatemeric sequences therebetween. In some embodiments, the method is repeated at least three times, four times, five times, six times, seven times, eight times, nine times, ten times, and a range between any two of the foregoing numbers.

[0083] Some embodiments of the methods and compositions provided herein include a method of determining whether a sample contains a target molecule. Some such embodiments include binding the target molecule to a surface; and contacting the bound target molecule with a non-naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences wherein the nucleic acid selectively binds to the target molecule when the target molecule is bound to a surface relative to the target molecule in solution. In some embodiments, the target molecule is bound to the surface via an antibody. In some embodiments, the target molecule is bound directly or indirectly to the surface. In some embodiments, the surface comprises a lateral flow immunoassay support.

[0084] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," has," with", "such as", or variants thereof, are used in either the specification and/or the claims, such terms are not limiting and are intended to be inclusive in a manner similar to the term "comprising." The term "about," as used herein, generally refers to a range that is 15% greater than or less than a stated numerical value within the context of the particular usage. For example, "about 10" would include a range from 8.5 to 1 1.5. EXAMPLES

Example 1 : High dose hook effect in a lateral flow immunoassay (LFA)

[0085] This Example illustrates the high dose hook effect. FIG. 1 , panel A provides a schematic of a LFA for monoclonal antibody authentication. The test line and anti- kappa control lines as well as the anti-IgG control lines should appear when the test antibody (Herceptin) is present. FIG. 1, panel B shows that prototype LFAs show a strong high dose hook effect (arrows) above 100 μg/ml, giving rise to false negative results.

Example 2: Selection method for DNA nanoparticles that bind to target coated beads

[0086] This Example illustrates an embodiment for preparing nanoparticles against targets bound to beads. As illustrated in FIG. 2, a 100 base library with a 60 base random region flanked by two 20 base primer sites is ligated and amplified with rolling circle amplification to produce nanoparticles. The nanoparticles are incubated with the target cells and washed. Remaining nanoparticles are amplified by PCR with a polymerase that lacks 5'- 3' exonuclease activity, such as the Stoffel fragment of Taq polymerase, and then asymmetrically by PCR (using only one primer) to generate an excess of the template strand. The template strand is re-ligated and the cycle repeated.

Example 3: Selection and characterization of streptavidin binding nanoparticles

[0087] This Example illustrates binding properties of nanoparticles. Nanoparticles were prepared against streptavidin-coated beads as shown in FIG.2. Nanoparticles were measured using fluorescently-labeled probes to nanoparticles sequences. FIG. 3, panel A shows binding to streptavidin-coated beads with nanoparticles generated with 1 , 2, 3, or 5 rounds (Rd) of selection, probe-only, library from which the nanoparticles were generated, and positive control (biotinylated library). FIG. 3, panel B shows staining of 4 selected streptavidin clones on streptavidin beads and BSA beads, negative control clone (GlOneg), and biotinylated positive control clone (GlObio) also shown.

[0088] FIG. 3, panel C shows a free streptavidin competition titration of SA-D7 and SA-D8 clones and GlObio positive control in which nanoparticles were pre-incubated with varying concentrations of free streptavidin, then streptavidin-coated beads were added. Binding of the biotinylated control (G l Obio) to streptavidin-coated beads decreased as the concentration of streptavidin in solution increased. In contrast, nanoparticles continued to bind streptavidin-coated beads decreased as the concentration of streptavidin in solution increased.

[0089] In FIG. 3, panel D, streptavidin beads were pre-incubated with 100 bases oligo the same sequence as the concatemer that makes up SA-D8, an irrelevant (control) 100 bases oligo, or buffer, then nanoparticles were added. Binding of the nanoparticles to streptavidin-coated beads was not inhibited by competing oligos in solution

[0090] In FIG. 3, panel E, a free biotin/desthiobiotin/2-iminobiotin competition titration was performed by pre-incubating streptavidin beads with biotin and biotin derivatives, then adding nanoparticles. Results were graphed as percent inhibition. In FIG. 3, panel F, a biotin/desthiobiotin/2-iminobiotin competitive release was performed by staining streptavidin beads with nanoparticles, then adding biotin/biotin derivative (or buffer for baseline). Results were graphed as percent released from beads.

Example 4: Synthetic construction of nanoparticles

[0091] This example illustrates embodiments of methods for preparing nanoparticles. FIG. 4, panel A shows a schematic of a ligation process using restriction sites in short hairpins. Only a 5'-digested oligo (Faul) can be ligated to a 3'-digested oligo (BtsCI), via a biotinylated, 40 bases linker. The linker includes 20 bases of non-specific sequence on the 5' end, which allows the linker to be unzipped from the ligated product with the complementary oligo following purification via the biotin or gel extraction. This process is then repeated to produce larger oligos. FIG. 4, panel B shows agarose gels of 5'-digested and 3 '-digested oligos that are subsequently ligated (left panel), purification of ligated product from unligated/undigested oligos via streptavidin bead pulldown and elution via temperature (middle panel), and 5'- and 3'-digestion of 2-copy oligo and subsequent ligation to 4-copy oligo. FIG. 4, panel C shows a schematic of ligation process using click chemistry. An oligo with 5' amine and 3' thiol is synthesized; the core 100 bases oligo is the same as in FIG. 4, panel A. In one reaction, the amine is converted to alkyne; in another reaction, the thiol is converted to azide. The click reaction will only join the azide to the alkyne, forming a 2-copy oligo. The process is repeated to form oligos with more copies.

Example 5: Wide dynamic range and no high dose hook effect in nanoparticle sandwich assay

[0092] In FIG. 5, panel A, protein G beads were incubated with varying concentrations of either the humanized monoclonal antibody rituximab, serving as a model analyte, or Avastin (bevacizumab) together with a fluorescently labeled rituximab binding nanoparticle. After incubation and final washing, the fluorescence on the beads was measured. FIG. 5, panel B shows a model of how the observed nanoparticle detection range differs from initial prediction as well as traditional sandwich immunoassay. Traditional sandwich immunoassay loses signal at high analyte concentrations whereas nanoparticles are insensitive to excess free analyte. The unexpected result that nanoparticles are linear over a wide concentration range and achieves high sensitivity suggests an alternate model for the interaction with analyte.

Example 6: High-throughput sequencing of nanoparticle libraries during cell based selection

[0093] This example illustrates increased selection of certain nanoparticle sequences as the number of rounds of selection increases. FIG. 6, panel A shows global changes in particle frequency distribution as the selection against leukemia cells proceeds. Most sequences in the initial library were unique, as expected. As the selection proceeds, an increase in the fraction of sequences with greater population frequency is observed. FIG. 6, panel B is a technical control replicate. The same sample was independently amplified in duplicate and the population frequencies compared. FIG. 6, panel C is a biological control replicate. The 2nd round of selection was performed in duplicate and independently amplified for sequencing.

Example 7: Nanoparticles overcome high dose hook effect in LFA

[0094] As shown in FIG. 7, traditional LFA sandwich assays, such as those shown in FIG. 1 , suffer from loss of signal at high analyte concentrations since the excess analyte saturates both the capture agent immobilized on the test line and the secondary detecting agent on the colloidal gold. In contrast, nanoparticles have increased binding to the analyte that is available for multivalent interaction once captured by the primary test line capture agent, compared to binding the analyte in solution.

Example 8: Selection of clot binding nanoparticles

[0095] This example illustrates certain characteristics for nanoparticles prepared against fibrinogen-coated beads. A nanoparticle library was prepared and screened against fibrinogen-coated beads as shown in FIG. 2. In FIG. 8, panel A, the population of particles from each round was fluorescently labeled with probe, and the bound particles measured on fibrinogen-coated beads. In FIG. 8, panel B, individual clones were regenerated as nanoparticles and tested for binding on fibrinogen- or BSA- coated beads (as well as other proteins not shown). The selected particles only bound to fibrinogen. FIG. 8, panel C, shows selected particles were not inhibited from binding to fibrinogen-coated beads by 4 mg/ml free fibrinogen. Fib3 is a non-binding clone that was a "hitchhiker" in the original selection i.e. it bound to other bound clones. G 10 and VI 1 are random particles. In FIG. 8, panel D, at least 10 clones were sequenced from each of 3 independent selections against fibrinogen-coated beads. A C/T rich motif was observed in the majority of the particle sequences. In FIG. 8, panel E, selected particles bound to clots made from thrombin treated plasma. An irrelevant control particle was included, and the ratio of the selected to unselected control particle, as measured by quantitative PCR after washing the clot, was determined.

Example 9: Structure and sequence motif of streptavidin nanoparticles

[0096] This example illustrates that certain nanoparticles against streptavidin include a certain sequence motif. FIG. 9, panel A shows the structures of the 100 bases oligo that made up the 4 selected streptavidin nanoparticles (mFold). Motif is highlighted in boxes. FIG. 9, panel B shows the motif from the 4 particles. FIG. 9, panel C provides an alignment of the motif. Example 10: Streptavidin nanoparticle staining on different streptavidin beads

[0097] In FIG. 10, panel A, SA-D8 and biotinylated SA-D8 were stained on streptavidin sepharose beads and fluorescence was compared to streptavidin magnetic beads, probe only is also shown. In FIG. 10, panel B, SA-D8 and biotinylated SA-D8 were stained on streptavidin PMMA beads and fluorescence was compared to streptavidin magnetic beads, probe only is also shown.

Example 1 1 : Streptavidin nanoparticle binding by size and time

[0098] This example illustrates that binding characteristics of nanoparticles can be determined by factors such as length of time for synthesis, and concentration of nucleotide reagents. FIG. 1 1 , panel A shows binding of streptavidin nanoparticles made by alteration of time and/or dNTP dilution. Nanoparticles were made with 3 nmol dNTPs for 30 min at 30°C (the standard conditions used throughout), or 93.8 pmol dNTPs for 30 min or 7.5 min at 30°C. A control nanoparticle from a different library was also made for each of these conditions and used as an internal control in the subsequent PCR. This control was mixed with the SA-D7, SA-D8, G l Obio, or G l Oneg and included in the staining. The stained streptavidin beads and nanoparticle mixes were then analyzed by PCR and quantitated with a standard for the appropriate library (a plasmid containing the 100 bp template used to make nanoparticles). The ratio of the bound particles (streptavidin nanoparticle ontrol nanoparticle) to total particles (streptavidin nanoparticle ontrol nanoparticle) is graphed.

[0099] FIG. 1 1, panel B shows dissociation of streptavidin nanoparticles over time. Nanoparticles were made with standard conditions and used to stain streptavidin magnetic beads. Extensive washing was done to remove unbound nanoparticles. The stained beads were then incubated in 10 ml PBS 1 % BSA 10 mM MgCb for 35 days. Aliquots were taken every week of the total sample (supernatant plus beads) and supernatant only (beads were removed by magnet). PCR was done on all samples/timepoints and % unbound was graphed (Nanoparticles in supernatant/nanoparticles in total * 100%). At day 21, a biotin knockoff was also done (filled symbols), in which excess biotin was added to an aliquot of total sample, incubated for 30 min, then beads were removed via magnet to obtain supernatant only. Example 12: ituximab-specific and herceptin-specific nanoparticle competitive titration and competitive release

[0100] This example illustrates binding properties of rituximab-specific nanoparticles and herceptin-specific nanoparticles. FIG. 12, panel A shows a free rituximab competition titration of 3Ritl nanoparticles. Nanoparticles were pre-incubated with varying concentrations of free rituximab or free whole human IgG, then rituximab beads were added. FIG. 12, panel B shows a free peptide competition titration performed by pre-incubating rituximab beads with free rituximab-specific, BSA-conjugated peptide or BSA-conjugated irrelevant peptide, then adding nanoparticles. FIG. 12, panel C shows free peptide competitive release done by staining rituximab beads with nanoparticles, then adding rituximab-specific, BSA-conjugated peptide or BSA-conjugated irrelevant peptide. Percent release was determined by PCR of supernatant after incubation with free peptide and centrifugation of beads. Beads stained with nanoparticles prior to addition of peptide were amplified by PCR to determine maximum signal. FIG. 12, panels D, E and F show experiments with herceptin and nanoparticles specific for herceptin which were performed the same as rituximab (FIG. 12, panels A, B and C)

Example 13: Sandwich assay with protein G, rituximab, and 3Ritl nanoparticles

[0101] Protein G was incubated with different concentrations of rituximab, polyclonal human IgG, or a mix of rituximab and polyclonal human IgG for 1 hr. The rituximab + IgG samples are graphed as amount of rituximab in the sample (all samples had the same total amount of antibody). FIG. 13. Fluorescently-labeled 3Ritl nanoparticles were then added to the sample for 2hr, then samples were washed and fluorescence measured.

Example 14: Sandwich assay in the presence of excess amounts of free antibody

[0102] Protein G was incubated with different concentrations of rituximab or avastin, and incubated for 1 hr. Alexa Fluor647-labeled 3Ritl nanoparticles, Aval nanoparticles, MJneg nanoparticles, or Alexa Fluor488-labeled anti-kappa human light chain antibody were then added and incubated for an additional 2hr, then washed and measured for fluorescence. For anti-kappa light chain samples only: Rituximab and avastin samples <1 - fold free antibody were diluted with mouse IgG2b κ to equal 1 -fold total antibody. This was done to bind all free protein G sites before addition of anti-kappa antibody so it would not bind non-specifically. Results are summarized in FIG. 14.

Example 15: Effect of buffer composition on the binding capacity of particles

[0103] To further define the properties of the nanoparticles, the effect of buffer composition on the binding capacity of the particles was evaluated. SA-D7, SA-D8, GlOneg, and G l Obio were selected for testing. The following conditions were assessed: MgCl₂ concentration, NaCl concentration, physiologic buffers, and biologic buffers. For MgCl₂concentration, binding was observed in >5 mM MgCl₂, with no adverse effects up to 40 mM MgCl₂; no effect on binding was observed for 0-300 mM NaCl (FIG. 15, panels A- B). Binding was observed in all physiologic buffers tested, with some variability in intensity (FIG. 15, panel C). High levels of fluorescence were observed in MES, HEPES, bicine, CAPSO, carbonate, sodium phosphate, PBS, and TBS, and lower fluorescence signal (but still above G l Oneg signal) was observed in PIPES, citrate, and water. Finally, particle binding was tested in 1 .1 %, 3.3%, 10%, and 30% urine, FBS, and human serum (FIG. 15, panel D). SA-D8 bound streptavidin beads in all conditions except 30% urine, while SA-D7 did not bind in 10% urine or 30% of any biologic buffer. Overall, these results indicate the DeNAno particles bind in a variety of buffers and conditions, and perhaps unsurprising, are suited for the conditions they were selected in— namely 10 mM MgCl₂, 150 mM NaCl, and tris or sodium phosphate buffer. Additionally, the particles' ability to bind in biologic buffers demonstrates potential for diagnostic or in vivo use.

Example 16: Imaging of particles

[0104] The dominant nanoparticle clone, SA-D8, was imaged using atomic force microscopy (AFM, FIG. 16, panel A) and transmission electron microscopy (TEM, FIG. 16, panels B-C). For TEM, both 'standard' (30 min/3nmol dNTP/30°C) and 'medium' (30 min/93.8pmol dNTP/30°C) particles were made (the same size as FIG 1 1, panel A, left and middle). In all cases, discreet 'balls' of DNA were observed, with a diameter of 100-250 nm (AFM), 75 nm (standard nanoparticle/TEM) or, 58 nm (medium nanoparticle/TEM), similar in size to previous reports using nanoparticle tracking system (Ruff, L. E., et al.,. Nanotechnol. Rev. 3, 569-578 (2014)) The disparity between TEM and AFM size of standard nanoparticle is most likely due to the compactness of the imaged nanoparticle, which is partly determined by the amount of salt present in the sample and the preparation method.

Example 17: Lateral flow assay

[0105] Individuals to be tested for the presence of a target molecule are provided with a lateral flow assay kit comprising a strip made of nitrocellulose. At one end of the strip is a sample collection area made of an absorbent material. Further along the strip is provided a zone containing non-naturally occurring nucleic acids comprising a continuous strand of concatemeric sequences, where the nucleic acids selectively bind the target molecule when the target molecule is bound to a surface, relative to the target molecule in solution. This zone is arranged transverse to the long axis of the strip and can be rendered mobile by the passage of sample, from the stable dried state. Further along the strip, away from the sample collection area is a zone containing capture antibodies specific for the target molecule. This zone is also arranged transverse to the long axis of the strip and the capture antibodies specific for the target molecule are immobilized on the strip. The strip may also contain a control band to serve as a positive control to confirm when the assay has reached its conclusion and functioned correctly.

[0106] To use the kit, an individual deposits a sample (e.g., blood or urine) in the sample collection zone. The sample flows via capillary action from the zone containing non- naturally occurring nucleic acids to the zone containing capture antibodies. As the contents of the sample pass the zone containing non-naturally occurring nucleic acids, the nucleic acids are mobilized and carried in the direction of the zone containing capture antibodies. When the nucleic acids and sample reach the zone containing capture antibodies, if there is target molecule present in the sample, the target molecule will bind the capture antibodies, at which point the nucleic acids will selectively bind to the target molecule bound to the capture antibodies relative to the target molecule in solution. This complex may be visualized by labeling the nucleic acids. If there is no target molecule in solution, the nucleic acids and sample will pass through the zone containing capture antibodies without binding.

[0107] All references listed herein are incorporated by reference in their entireties.

[0108] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1 . A non-naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences wherein said nucleic acid selectively binds to a target molecule when said target molecule is bound to a surface relative to said target molecule in solution.

2. The nucleic acid of Claim 1 , wherein each of said individual concatemeric sequences has low affinity for said target molecule fixed to said surface and wherein said concatemeric sequences, in multimeric form, have high avidity for said target molecule fixed to said surface.

3. The nucleic acid of any one of Claims 1 and 2, wherein said nucleic acid comprises a nanoparticle.

4. The nucleic acid of Claim 3, wherein binding of the nanoparticle to the target molecule bound to a surface compared to binding of the nanoparticle to the target molecule in solution is at least 2-fold greater.

5. The nucleic acid of Claim 3, wherein binding of the nanoparticle to the target molecule bound to a surface compared to binding of the nanoparticle to the target molecule in solution is at least 5-fold greater.

6. The nucleic acid of Claim 3, wherein binding of the nanoparticle to the target molecule bound to a surface compared to binding of the nanoparticle to the target molecule in solution is at least 10-fold greater.

7. The nucleic acid of Claim 3, wherein binding of the nanoparticle to the target molecule bound to a surface compared to binding of the nanoparticle to the target molecule in solution is at least 100-fold greater.

8. The nucleic acid of any one of Claims 1 -7, wherein said nucleic acid comprises DNA.

9. The nucleic acid of any one of Claims 1 -8, wherein said DNA is more than 1 kb in length.

10. The nucleic acid of any one of Claims 1-9, wherein said DNA is more than 2 kb in length.

1 1. The nucleic acid of any one of Claims 1-10, wherein said DNA is more than 5 kb in length.

12. The nucleic acid of any one of Claims 1 -1 1 , wherein said DNA is more than l OO kb in length.

13. The nucleic acid of any one of Claims 1 -12, wherein said nucleic acid comprises a sequence selected form the group consisting of a siRNA, a reporter gene, a nucleic acid encoding a therapeutic protein, and a CpG sequence.

14. The nucleic acid of any one of Claims 1-13, wherein said nucleic acid is associated with a therapeutic or diagnostic agent.

15. The nucleic acid of any one of Claims 1-14, wherein said therapeutic agent comprises a drug.

16. The nucleic acid of any one of Claims 1 -14, wherein said diagnostic agent comprises an in vivo imaging agent.

17. The nucleic acid of any one of Claims 1-16, wherein said target molecule comprises a peptide or protein.

18. The nucleic acid of any one of Claims 1-17, wherein said target molecule comprises a molecule differentially expressed in individuals with a disease.

19. The nucleic acid of any one of Claims 1 -18, wherein said target molecule comprises a molecule differentially expressed in tumors.

20. The nucleic acid of any one of Claims 1 -19, wherein said target molecule comprises a marker expressed on tumor vasculature.

21. The nucleic acid of any one of Claims 1-20, wherein said target molecule comprises a molecule found in blood clots.

22. The nucleic acid of any one of Claims 1-21 , wherein said target molecule is fibrin.

23. The nucleic acid of any one of Claims 1 -22, wherein said nucleic acid comprises from about 10 to about 300 copies of said concatemeric sequences.

24. The nucleic acid of any one of Claims 1-23, wherein said nucleic acid comprises from about 10 to about 1200 copies of said concatemeric sequences.

25. The nucleic acid of any one of Claims 1-24, wherein said nucleic acid selectively binds to said target molecule when said target molecule is bound to a lateral flow immunoassay support.

26. The nucleic acid of any one of Claims 1 -25, wherein said nucleic acid selectively binds to said target molecule when said target molecule is bound to a bead.

27. The nucleic acid of any one of Claims 1 -26 comprising SEQ ID NO: 9.

28. A library of nanoparticles comprising at least two populations of nucleic acids, wherein said each of said at least two populations comprise a non-naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences wherein said nucleic acid selectively binds to a target molecule when said target molecule is bound to a surface relative to said target molecule in solution.

29. The library of Claim 28, wherein each of said individual concatemeric sequences has low affinity for said target molecule bound to said surface and wherein said concatemeric sequences, in multimeric form, have high avidity for said target molecule bound to said surface.

30. The library of any one of Claims 28 and 29, wherein said nucleic acids comprise a nanoparticle.

31. The library acid of Claim 30, wherein binding of the nanoparticle to the target molecule bound to a surface compared to binding of the nanoparticle to the target molecule in solution is 2-fold greater.

32. The library acid of Claim 30, wherein binding of the nanoparticle to the target molecule bound to a surface compared to binding of the nanoparticle to the target molecule in solution is 5-fold greater.

33. The library acid of Claim 30, wherein binding of the nanoparticle to the target molecule bound to a surface compared to binding of the nanoparticle to the target molecule in solution is 10-fold greater.

34. The library acid of Claim 30, wherein binding of the nanoparticle to the target molecule bound to a surface compared to binding of the nanoparticle to the target molecule in solution is 100-fold greater.

35. The library of any one of Claims 28-34, wherein said nucleic acids comprise

DNA.

36. The library of any one of Claims 28-35, wherein said nucleic acids are more than 1 kb in length.

37. The library of any one of Claims 28-36, wherein said nucleic acids are more than 2 kb in length.

38. The library of any one of Claims 28-37, wherein said nucleic acids are more than 5 kb in length.

39. The library of any one of Claims 28-38, wherein said nucleic acids are more than 100 kb in length.

40. The library of any one of Claims 28-39, wherein said nucleic acids comprise a sequence encoding a sequence selected from a siRNA, reporter gene, therapeutic protein, and CpG sequence.

41 . The library of any one of Claims 28-40, wherein said nucleic acids comprise from about 10 to about 300 copies of said concatemeric sequences.

42. The library of any one of Claims 28-41, wherein said nucleic acids comprise from about 10 to about 1200 copies of said concatemeric sequences.

43. The library of any one of Claims 28-42, wherein said library comprises about 10¹² or fewer populations of nucleic acids.

44. The library of any one of Claims 28-43, wherein more than 50% of the nucleic acids of the library comprise a nucleic acid having the same sequence.

45. The library of any one of Claims 28-44, wherein more than 70% of the nucleic acids of the library comprise a nucleic acid having the same sequence.

46. The library of any one of Claims 28-45, wherein more than 90% of the nucleic acids of the library comprise a nucleic acid having the same sequence.

47. A liposome comprising the nucleic acid of any one of Claims 1-46.

48. A pharmaceutical composition comprising the nucleic acid of any one of Claims 1 -48.

49. A method of determining whether a sample contains a target molecule comprising:

binding said target molecule to a surface; and

contacting said bound target molecule with a non-naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences wherein said nucleic acid selectively binds to said target molecule when said target molecule is bound to a surface relative to said target molecule in solution.

50. The method of Claim 49, wherein said target molecule is bound to said surface via an antibody.

51. The method of any one of Claims 48 and 49, wherein said target molecule is bound directly or indirectly to said surface.

52. The method of any one of Claims 48-51, wherein said surface comprises a lateral flow immunoassay support.

53. A method of making a non-naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences wherein said nucleic acid selectively binds to a target molecule when said target molecule is bound to a surface relative to said target molecule in solution, said method comprising (a) performing rolling circle amplification on a circular template comprising a sequence complementary to the sequence to be concatemerized.

54. The method of Claim 53, wherein (a) is performed for at least 10 minutes.

55. The method of Claim 53, wherein (a) is performed for at least 20 minutes.

56. The method of Claim 53, wherein (a) is performed for at least 30 minutes.

57. The method of Claim 53, wherein (a) is performed for at least 60 minutes.

58. The method of Claim 53, wherein (a) is performed for at least 120 minutes.

59. The method of Claim 53, further comprising (b) selecting the nucleic acid comprising a continuous strand of concatemeric sequences wherein said nucleic acid selectively binds to a target molecule bound to a surface.

60. The method of Claim 59, further comprising (c) amplifying the selected nucleic acid.

61 . The method of Claim 53, wherein the rolling circle amplification is performed with a polymerase lacking 5 '-3' exonuclease activity.

62. The method of Claim 61 , wherein the polymerase comprises the Stoffel fragment of Taq polymerase,

63. The method of Claim 61 or 62, further comprising (d) asymmetrically amplifying the amplified nucleic acid, thereby generating a single-stranded linear template.

64. The method of Claim 63, further comprising (e) circularizing the linear template, thereby preparing a circular template comprising a sequence complementary to the sequence to be concatemerized.

65. The method of any one of Claims 53-64, further comprising repeating (a).

66. The method of Claim 65, wherein (a) is repeated at least three times.

67. The method of Claim 65, wherein (a) is repeated at least five times.

68. The method of any one of Claims 53-67, wherein the nucleic acid comprises the nucleic acid of any one of Claims 1-27.

69. A nanoparticle prepared by the method of any one of Claim 53-68.

70. A method of making a non-naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences wherein said nucleic acid selectively binds to a target molecule when said target molecule is bound to a surface relative to said target molecule in solution, said method comprising performing a ligation reaction to generate said concatemeric sequences.

71. The method of Claim 70, comprising, prior to performing the ligation reaction to generate said concatemeric sequences, obtaining a nucleic acid comprising a first and second hairpin having a single-stranded region therebetween.

72. The method of Claim 71 , wherein the first hairpin comprises a first restriction site and the second hairpin comprises a second restriction site, wherein the cleaved first and second restriction sites are capable of being ligated to one another.

73. The method of Claim 72, wherein the first and second cleavage sites are different.

74. The method of any one of Claims 70-73, wherein the first and second restriction sites are selected from the group consisting of Faul and BtsCI.

75. The method of any one of Claims 70-74, further comprising cleaving said first and second cleavage sites, thereby obtaining a first and second nucleic acid.

76. The method of Claim 75, further comprising ligating the first nucleic acid to the second nucleic acid, thereby obtaining a nucleic acid comprising a hairpin at each end and having a single-stranded region therebetween.

77. The method of Claim 76, further comprising repeating the method of Claim

70.

78. The method of Claim 77, wherein the method is repeated at least three times.

79. The method of any one of Claims 70-78, wherein the nucleic acid comprises the nucleic acid of any one of Claims 1-27.

80. A nanoparticle prepared by the method of any one of Claim 70-79.

81. A method of making a non-naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences wherein said nucleic acid selectively binds to a target molecule when said target molecule is bound to a surface relative to said target molecule in solution, said method comprising performing a Click chemistry reaction to generate said concatemeric sequences.

82. The method of Claim 81, comprising, prior to performing a Click chemistry reaction, obtaining a population of single stranded nucleic acids having a 5' amine moiety and 3' thiol moiety.

83. The method of Claim 82, further comprising:

converting the 5' amine moiety amine to a 5' alkyne moiety in a first subpopulation of the single stranded nucleic acids, thereby obtaining a first subpopulation of single stranded nucleic acids having a 5' alkyne moiety and 3' thiol moiety; and

converting the 3' thiol moiety amine to a 3' azide moiety in a second subpopulation of the single stranded nucleic acids, thereby obtaining a second subpopulation of single stranded nucleic acids having a 5' amine moiety and 3' azide moiety.

84. The method of Claim 83, further comprising joining the 5' alkyne moiety of the nucleic acids of the first subpopulation to the 3' azide moiety of the nucleic acids of the second subpopulation, thereby obtaining a obtaining a population of single stranded nucleic acids having a 5' amine moiety and 3' thiol moiety and concatemeric sequences therebetween.

85. The method of Claim 84, further comprising repeating the Click chemistry reaction.

86. The method of Claim 85, wherein the Click chemistry reaction is repeated at least three times.

87. The method of Claim 81 , wherein the nucleic acid comprises the nucleic acid of any one of Claims 1 -27.

88. A nanoparticle prepared by the method of any one of Claim 81-87.

89. A kit comprising a surface adapted to bind a target molecule and the non- naturally occurring nucleic acid of any one of Claims 1-27, wherein the nucleic acid selectively binds to the target molecule when said target molecule is bound to a surface relative to said target molecule in solution.

90. The kit of Claim 89, wherein the surface is planar.

91 . The kit of Claim 89, wherein a bead comprises the surface.

92. The kit of Claim 89, wherein a lateral flow assay comprises the surface.

93. A method of overcoming a high dose hook effect in a lateral flow assay comprising:

binding a target molecule to a surface of a lateral flow assay support; and contacting said bound target molecule with a non-naturally occurring nucleic acid comprising a continuous strand of concatemeric sequences,

wherein said nucleic acid selectively binds to said bound target molecule relative to said target molecule in solution.