Methods for solid-phase nucleic acid hybridization

Abstract

A method is provided for hybridizing a solution-phase nucleic acid to a solid-phase nucleic acid, the method comprising contacting the solid-phase nucleic acid with a hybridization solution comprising a solution-phase nucleic acid and a hybridization catalyst, wherein the hybridization catalyst comprises a polycationic backbone having hydrophilic side chains. This hybridization method may be used in aptamer-based methods for detecting protein targets.

Description

Technical Field

[0001] Cross-Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 583,836, filed on September 19, 2023, which is hereby incorporated by reference in its entirety for all purposes into the present specification.

[0002] The present disclosure relates generally to the field of nucleic acid hybridization, for example, to nucleic acid hybridization on a solid support.

Summary of the Invention

[0003] Nucleic acid hybridization on a solid support is an integral part of many nucleic acid-based assays, including assays performed on arrays or beads. The present disclosure provides improvements to nucleic acid hybridization on a solid support, including, for example, reduced hybridization time.

[0004] Embodiment 1. A method of hybridizing a plurality of solution-phase nucleic acids to a plurality of solid-phase nucleic acids, the method comprising contacting the solid-phase nucleic acids with a hybridization solution comprising the solution-phase nucleic acids and a hybridization catalyst, wherein the hybridization catalyst comprises a polycationic backbone having hydrophilic side chains.

[0005] The method of Embodiment 1, wherein the plurality of solution-phase nucleic acids comprises at least 100, at least 500, at least 1000, at least 2000, at least 3000, or at least 5000 different nucleic acids having different nucleic acid sequences.

[0006] The method of Embodiment 1 or Embodiment 2, wherein the plurality of solid-phase nucleic acids comprises at least 100, at least 500, at least 1000, at least 2000, at least 3000, or at least 5000 different nucleic acids having different nucleic acid sequences.

[0007] Embodiment 4. The method according to any one of Embodiments 1 to 3, wherein the solid nucleic acid is bound to an array, flow cell, or beads.

[0008] Embodiment 5. The method according to any one of Embodiments 1 to 3, wherein the solid-phase nucleic acid is bound to an array.

[0009] Embodiment 6. The method according to any one of Embodiments 1 to 5, wherein the hybridization solution contains a monovalent cationic salt in a concentration of 100 to 300 mM.

[0010] Embodiment 7. The method according to Embodiment 6, wherein the monovalent cationic salt is NaCl or sodium citrate.

[0011] Embodiment 8. The method according to any one of Embodiments 1 to 7, wherein the hybridization catalyst comprises a polylysine, polyarginine, or polyornithine backbone.

[0012] Embodiment 9. The method according to any one of Embodiments 1 to 8, wherein the hybridization catalyst comprises polyethylene glycol, polyacrylamide, polyvinyl alcohol, or a dextran side chain.

[0013] Embodiment 10. The method according to any one of Embodiments 1 to 9, wherein the hybridization catalyst comprises a polylysine main chain and polyethylene glycol (PEG) side chains.

[0014] Embodiment 11. The method according to Embodiment 10, wherein the hybridization catalyst comprises 10 to 500, or 10 to 300, or 20 to 200 lysines in the polylysine main chain, the hybridization catalyst comprises 5 to 50%, or 5 to 40%, or 5 to 30%, or 10 to 30% PEG modification, and / or the PEG comprises an average molecular weight of 1 to 20 kDa, or 2 to 10 kDa, or 2 to 8 kDa, or about 5 kDa.

[0015] Embodiment 12. The method according to any one of Embodiments 1 to 11, wherein the hybridization catalyst is present in an amount such that the ratio (N:P ratio) of the number of cations in the polycationic skeleton of the hybridization catalyst to the number of phosphates in the plurality of solution-phase nucleic acids and the plurality of solid-phase nucleic acids is 1:1 to 200:1, or 1:1 to 100:1, or 1:1 to 50:1, or 1:1 to 25:1.

[0016] Embodiment 13. The method according to any one of the prior embodiments, wherein the hybridization of the solution-phase nucleic acid to the solid-phase nucleic acid is at least 2 times, at least 3 times, at least 4 times, or at least 5 times faster in the presence of the hybridization catalyst than in the absence of the hybridization catalyst.

[0017] Embodiment 14. The method according to any one of Embodiments 1 to 13, wherein the solution-phase nucleic acid comprises RNA, DNA, genomic DNA, mitochondrial DNA, mRNA, microRNA, aptamers, and / or modified oligonucleotides.

[0018] Embodiment 15. The method according to any one of Embodiments 1 to 14, wherein the hybridization method is a step in an assay selected from next-generation sequencing, diagnostic microarrays (for detecting specific gene mutations, chromosomal abnormalities, epigenetic changes, SNPs, etc.), gene expression microarrays, microsatellite analysis, proteomics assays, and strand substitution assays.

[0019] Embodiment 16. The method according to any one of Embodiments 1 to 15, wherein the solution-phase nucleic acid is conjugated to the non-nucleic acid portion.

[0020] Embodiment 17. The method according to Embodiment 16, wherein the non-nucleic acid portion is selected from proteins, antibodies, lipids, and small molecules.

[0021] Embodiment 18. The method according to any one of Embodiments 1 to 17, wherein each of the solution-phase nucleic acids contains a barcode sequence.

[0022] Embodiment 19. The method of any one of Embodiments 1 to 18, wherein the solution-phase nucleic acid is not amplified prior to hybridization to the solid-phase nucleic acid.

[0023] Embodiment 20. The method of any one of Embodiments 1 to 18, wherein the solution-phase nucleic acid is amplified prior to hybridization to the solid-phase nucleic acid.

[0024] Embodiment 21. The method of Embodiment 20, wherein the amplification comprises PCR, RT-PCR, or qPCR.

[0025] Embodiment 22. The method of any one of Embodiments 1 to 21, wherein the solution-phase nucleic acid is an aptamer.

[0026] Embodiment 23. The method of Embodiment 22, wherein the aptamer is a slow off-rate aptamer.

[0027] Embodiment 24. The method of Embodiment 22 or Embodiment 23, wherein each aptamer comprises at least 1, at least 2, at least 3, or at least 5 C-5 modified pyrimidines.

[0028] Embodiment 25. The method of Embodiment 24, wherein each C-5 modified pyrimidine comprises a C-5 modified uracil base or a C-5 modified cytosine base.

[0029] Embodiment 26. The method of any one of Embodiments 22 to 25, wherein each aptamer is capable of binding to a protein target.

[0030] Embodiment 27. The method of any one of Embodiments 21 to 25, wherein the aptamer is eluted from an aptamer / protein complex prior to hybridization to the solid-phase nucleic acid.

[0031] Embodiment 28. The method of Embodiment 27, wherein the aptamer / protein complex is formed by contacting the aptamer with a biological sample containing a protein.

[0032] Embodiment 29. A method for detecting a plurality of protein targets in a biological sample, comprising contacting the biological sample with a plurality of aptamers, each aptamer being a nucleic acid, each aptamer specifically binding to a protein target to form a plurality of aptamer / protein complexes, the method further comprising releasing the bound aptamers from the aptamer / protein complexes into a solution to form a plurality of solution-phase nucleic acids, hybridizing the plurality of solution-phase nucleic acids to a plurality of solid-phase nucleic acids according to the method of any one of Embodiments 1 to 28, and detecting the hybridized solution-phase nucleic acids.

[0033] Embodiment 30. The method according to Embodiment 28 or Embodiment 29, wherein the biological sample is selected from plasma, serum, urine, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, sputum, tears, mucus, nasal lavage fluid, nasal aspirate, semen, saliva, peritoneal lavage fluid, ascites, cystic fluid, cerebrospinal fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate, bronchial swab, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, and cerebrospinal fluid. [Brief explanation of the drawing]

[0034] [Figure 1-1] The following are some specific exemplary 5-position modified uridines and cytidines that may be incorporated into aptamers. [Figure 1-2] The following are some specific exemplary 5-position modified uridines and cytidines that may be incorporated into aptamers. [Figure 1-3] The following are some specific exemplary 5-position modified uridines and cytidines that may be incorporated into aptamers. [Figure 1-4] The following are some specific exemplary 5-position modified uridines and cytidines that may be incorporated into aptamers. [Figure 1-5]The following are some specific exemplary 5-position modified uridines and cytidines that may be incorporated into aptamers. [Figure 2-1] The following shows certain exemplary modifications that may be present at the 5-position of uridine. The chemical structures of the C-5 modifications include an exemplary amide bond linking the modification to the 5-position of uridine. The 5-position moieties shown contain two phenyl groups covalently bonded to each other. Examples of the 5-position moieties shown include phenylbenzyl moieties (e.g., BPE, PBnd, DBM), 4-phenoxybenzyl moieties (e.g., POP), diphenylpropyl moieties (e.g., DPP), and benzhydryl moieties (e.g., BH). [Figure 2-2] The following shows certain exemplary modifications that may be present at the 5-position of uridine. The chemical structures of the C-5 modifications include an exemplary amide bond linking the modification to the 5-position of uridine. The 5-position moieties shown contain two phenyl groups covalently bonded to each other. Examples of the 5-position moieties shown include phenylbenzyl moieties (e.g., BPE, PBnd, DBM), 4-phenoxybenzyl moieties (e.g., POP), diphenylpropyl moieties (e.g., DPP), and benzhydryl moieties (e.g., BH). [Figure 3-1] The following shows certain exemplary modifications that may be present at the 5-position of cytidine. The chemical structures of the C-5 modifications include an exemplary amide bond linking the modification to the 5-position of cytidine. The 5-position moieties shown contain two phenyl groups covalently bonded to each other. Examples of the 5-position moieties shown include phenylbenzyl moieties (e.g., BPE, PBnd, DBM), 4-phenoxybenzyl moieties (e.g., POP), diphenylpropyl moieties (e.g., DPP), and benzhydryl moieties (e.g., BH). [Figure 3-2]The following shows certain exemplary modifications that may be present at the 5-position of cytidine. The chemical structures of the C-5 modifications include an exemplary amide bond linking the modification to the 5-position of cytidine. The 5-position moieties shown contain two phenyl groups covalently bonded to each other. Examples of the 5-position moieties shown include phenylbenzyl moieties (e.g., BPE, PBnd, DBM), 4-phenoxybenzyl moieties (e.g., POP), diphenylpropyl moieties (e.g., DPP), and benzhydryl moieties (e.g., BH). [Figure 4-1] The following describes certain exemplary modifications that may be present at the 5-position of uridine. The chemical structures of the C-5 modifications include exemplary amide bonds linking the modifications to the 5-position of uridine. Examples of 5-position moieties shown include benzyl moieties (e.g., Bn, PE, and PP), naphthyl moieties (e.g., Nap, 2Nap, NE), butyl moieties (e.g., iBu), fluorobenzyl moieties (e.g., FBn), tyrosyl moieties (e.g., Tyr), 3,4-methylenedioxybenzyl (e.g., MBn), morpholino moieties (e.g., MOE), benzofuranyl moieties (e.g., BF), indole moieties (e.g., Trp), and hydroxypropyl moieties (e.g., Thr). [Figure 4-2] The following describes certain exemplary modifications that may be present at the 5-position of uridine. The chemical structures of the C-5 modifications include exemplary amide bonds linking the modifications to the 5-position of uridine. Examples of 5-position moieties shown include benzyl moieties (e.g., Bn, PE, and PP), naphthyl moieties (e.g., Nap, 2Nap, NE), butyl moieties (e.g., iBu), fluorobenzyl moieties (e.g., FBn), tyrosyl moieties (e.g., Tyr), 3,4-methylenedioxybenzyl (e.g., MBn), morpholino moieties (e.g., MOE), benzofuranyl moieties (e.g., BF), indole moieties (e.g., Trp), and hydroxypropyl moieties (e.g., Thr). [Figure 4-3]The following describes certain exemplary modifications that may be present at the 5-position of uridine. The chemical structures of the C-5 modifications include exemplary amide bonds linking the modifications to the 5-position of uridine. Examples of 5-position moieties shown include benzyl moieties (e.g., Bn, PE, and PP), naphthyl moieties (e.g., Nap, 2Nap, NE), butyl moieties (e.g., iBu), fluorobenzyl moieties (e.g., FBn), tyrosyl moieties (e.g., Tyr), 3,4-methylenedioxybenzyl (e.g., MBn), morpholino moieties (e.g., MOE), benzofuranyl moieties (e.g., BF), indole moieties (e.g., Trp), and hydroxypropyl moieties (e.g., Thr). [Figure 5-1] The following shows certain exemplary modifications that may be present at the 5-position of cytidine. The chemical structures of the C-5 modifications include exemplary amide bonds linking the modifications at the 5-position of cytidine. Examples of 5-position moieties shown include benzyl moieties (e.g., Bn, PE, and PP), naphthyl moieties (e.g., Nap, 2Nap, NE, and 2NE), and tyrosyl moieties (e.g., Tyr). [Figure 5-2] The following shows certain exemplary modifications that may be present at the 5-position of cytidine. The chemical structures of the C-5 modifications include exemplary amide bonds linking the modifications at the 5-position of cytidine. Examples of 5-position moieties shown include benzyl moieties (e.g., Bn, PE, and PP), naphthyl moieties (e.g., Nap, 2Nap, NE, and 2NE), and tyrosyl moieties (e.g., Tyr). [Figure 6] This document provides an overview of dilution sets for biological samples, corresponding capture reagent sets for each dilution, and an overall overview of two sequential catch systems (Catch-1 and Catch-2). Three different dilution groups may be prepared from a biological sample containing a Z% dilution or DIL3, a Y% dilution or DIL2, and an X% dilution or DIL1, where Z is greater than Y and Y is greater than X (or Z is a greater dilution than Y, and Y is a greater dilution than X). Each dilution has a specific corresponding set of capture reagents that bind to a particular protein set (A3 for DIL1, A2 for DIL2, and A1 for DIL3). [Figure 7]We provide summaries of three different plasma dilutions, prepared as 0.005% dilution (DIL1), 0.5% dilution (DIL2), and 20% dilution (DIL3), and measure the relative high, moderate, and low abundances of proteins, respectively. Furthermore, the aptamer sets for DIL1, DIL2, and DIL3 were A1, A2, and A3, respectively. Of the 5,272 distinct aptamers in total, the A3 group contained 4,271 distinct aptamers (or approximately 81% of the total number of aptamers), the A2 group contained 828 distinct aptamers (or approximately 16% of the total number of aptamers), and the A1 group contained 173 distinct aptamers (or approximately 3% of the total number of aptamers). The three different dilution sets were sequentially passed from the Catch-1 step to the Catch-2 step of the assay. [Figure 8] This document provides an overview of dilution sets for biological samples, corresponding capture reagent sets for each dilution, and an overall overview of two catch systems (Catch-1 and Catch-2). Two different dilution groups may be prepared from a biological sample containing a Z% dilution or DIL4 of the biological sample and an X% dilution or DIL1 of the biological sample, where Z is greater than X (or Z is a dilution greater than X). Each dilution has its own specific set of corresponding capture reagents that bind to a particular protein set (A3 for DIL1 and A1 for DIL4). The two different dilution sets were sequentially advanced from the Catch-1 step to the Catch-2 step of the assay. [Figure 9] This specification shows plots of signal intensity from hybridization of SOMAmers on Soma array plates at various N:P ratios (ratio of the number of amines on the catalyst to the number of phosphates on the nucleic acid backbone) in the presence of the hybridization catalyst ("Magic Buffer") provided herein, against hybridization in the absence of the hybridization catalyst (x-axis). [Figure 10] This specification shows plots of the frequencies of the coefficients of variation of approximately 7,000 SOMAmers hybridized on a Soma array plate, with and without the hybridization catalyst provided herein. [Figure 11] This specification shows plots of the frequency of the coefficient of variation of hybridization for sequences on a Soma array plate that were not complementary to any SOMAmer in the assay, with or without the hybridization catalyst provided herein. [Figure 12] This demonstrates the reproducibility of SOMAmer hybridization on a Soma array plate in the presence of a hybridization catalyst. [Figure 13-1] This shows the hybridization of high-concentration SOMAmers on a Soma array plate in the presence of a hybridization catalyst after two weeks. [Figure 13-2] This shows the hybridization of high-concentration SOMAmers on a Soma array plate in the presence of a hybridization catalyst after 4 weeks. [Figure 14-1] This shows the hybridization of low-concentration SOMAmers on a Soma array plate in the presence of a hybridization catalyst after two weeks. [Figure 14-2] This shows the hybridization of low-concentration SOMAmers on a Soma array plate in the presence of a hybridization catalyst after 4 weeks. [Figure 15] This specification demonstrates the hybridization of SOMAmer into a custom CGH slide microarray (Agilent, part number G4885A) in and without the hybridization catalyst provided herein (19-hour incubation time). [Figure 16] This shows a plot of the measured coefficient of variation (CV) of the cumulative distribution function of SOMAmer. The leftward shift of the blue curve (using the magic buffer) indicates that the CV is lower compared to the SOP buffer. [Modes for carrying out the invention]

[0035] Unless otherwise specified, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9), Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9), and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0036] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this disclosure pertains. The singular forms “a,” “an,” and “the” include plural references unless otherwise explicitly indicated in their content. “A or B” means A, or B, or A and B. It should be further understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for illustrative purposes only.

[0037] Furthermore, it should be understood that the ranges provided herein are abbreviated representations of all values ​​within that range. For example, the range 1 to 50 should be understood to include any number, combination of numbers, or subrange (and fractions thereof, unless otherwise explicitly stated by the context) in the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. Any concentration range, percentage range, ratio range, or integer range should be understood to include any integer within the listed range, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated. Similarly, any numerical ranges listed herein relating to any physical characteristics, such as polymer subunits, size, or thickness, should be understood to include any integer within the listed range, unless otherwise indicated. Where used herein, “approximately” or “essentially consisting of” means ±20% of the indicated range, value, or structure, unless otherwise indicated. Where used herein, the terms “include” and “comprise” are open-ended and used synonymously.

[0038] Similar or equivalent methods and materials may be used in the implementation or testing of this disclosure, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including the definition of terms, shall prevail. In addition, the materials, methods, and examples are illustrative and not intended to limit the scope of this disclosure.

[0039] As used herein, the term “nucleotide” refers to ribonucleotides or deoxyribonucleotides, or modified forms thereof, and their analogues. Nucleotides include species such as purines (e.g., adenine, hypoxanthine, guanine, and their derivatives and analogues), as well as pyrimidines (e.g., cytosine, uracil, thymine, and their derivatives and analogues). As used herein, the term “cytidine” is generally used to refer to ribonucleotides, deoxyribonucleotides, or modified ribonucleotides containing a cytosine base, unless otherwise specifically indicated. The term “cytidine” includes 2'-modified cytidines, such as 2'-fluoro, 2'-methoxy, etc. Similarly, the term “modified cytidine” or a specific modified cytidine also refers to ribonucleotides, deoxyribonucleotides, or modified ribonucleotides (2'-fluoro, 2'-methoxy, etc.) containing a modified cytosine base, unless otherwise specifically indicated. The term "uridine" is generally used to refer to ribonucleotides, deoxyribonucleotides, or modified ribonucleotides containing a uracil base, unless otherwise specified. The term "uridine" includes 2'-modified uridines, such as 2'-fluoro and 2'-methoxy. Similarly, the term "modified uridine" or a specific modified uridine also refers to ribonucleotides, deoxyribonucleotides, or modified ribonucleotides (such as 2'-fluoro and 2'-methoxy) containing a modified uracil base, unless otherwise specified.

[0040] As used herein, the terms “C-5 modified carboxamide cytidine,” “cytidine-5-carboxamide,” “5-position modified cytidine,” or “C-5 modified cytidine” refer to cytidine having a carboxyamide (-C(O)NH-) modification at the C-5 position, including, but not limited to, those portions (R') illustrated herein. See, for example, Figures 1, 3, and 5. Examples of C-5 modified carboxamide cytidines include 5-(N-((1,1'-biphenyl)-4-yl)ethyl)-2'-deoxycytidine (referred to as "BPEdC" and shown in Figure 3), 5-(N-4-phenylbenzyl)-2'-deoxycytidine (referred to as "PBnddC" and shown in Figure 3), 5-(N-4-phenoxybenzyl)-2'-deoxycytidine (referred to as "POPdC" and shown in Figure 3), and 5-(N-3,3-di Phenylpropyl)-2'-deoxycytidine (referred to as "DPPdC" and shown in Figure 3), 5-(N-3-phenylbenzyl)-2'-deoxycytidine (referred to as "DBMdC" and shown in Figure 3), 5-(N-benzhydryl)-2'-deoxycytidine (referred to as "BHdC" and shown in Figure 3), 5-(N-benzylcarboxamide)-2'-deoxycytidine (referred to as "BndC" and shown in Figure 5), 5-(N-2-phenyl Ethylcarboxamide)-2'-deoxycytidine (referred to as "PEdC" and shown in Figure 5), 5-(N-3-phenylpropylcarboxamide)-2'-deoxycytidine (referred to as "PPdC" and shown in Figure 5), 5-(N-1-naphthylmethylcarboxamide)-2'-deoxycytidine (referred to as "NapdC" and shown in Figure 5), 5-(N-2-naphthylmethylcarboxamide)-2'-deoxycytidine (referred to as "2NapdC" and Examples include, but are not limited to, 5-(N-1-naphthyl-2-ethylcarboxamide)-2'-deoxycytidine (referred to as "NEdC" and shown in Figure 5), 5-(N-2-naphthyl-2-ethylcarboxamide)-2'-deoxycytidine (referred to as "2NEdC" and shown in Figure 5), and 5-(N-tyrosylcarboxamide)-2'-deoxycytidine (referred to as "TyrdC" and shown in Figure 5).In some embodiments, C5-modified cytidines can be incorporated into oligonucleotides by polymerase (e.g., KOD DNA polymerase), for example, in their triphosphate form.

[0041] The chemical modifications of C-5 modified cytidine described herein may also be combined, either alone or in any combination, with 2' sugar modifications, modifications with extracyclic amines, and substitutions of 4-thiocytidine.

[0042] As used herein, the terms “C-5 modified uridine” or “5-position modified uridine” refer to uridine (typically deoxyuridine) having a carboxyamide (-C(O)NH-) modification at the C-5 position, as shown in Figure 1, for example. In some embodiments, C5-modified uridines can be incorporated into oligonucleotides by polymerase (e.g., KOD DNA polymerase), for example, in their triphosphate form. Non-limiting and exemplary 5-position modified uridines include: 5-(N-((1,1'-biphenyl)-4-yl)ethyl)-2'-deoxyuridine (BPEdU), 5-(N-4-phenylbenzyl)-2'-deoxyuridine (PBnddU), 5-(N-4-phenoxybenzyl)-2'-deoxyuridine (POPdU), 5-(N-3,3-diphenylpropyl)-2'-deoxyuridine (DPPdU), 5-(N-3-phenylbenzyl)-2'-deoxyuridine (DBMdU), 5-(N-benzhydryl)-2'-deoxyuridine (BHdU), 5-(N-benzylcarboxyamide)-2'-deoxyuridine (BndU), 5-(N-benzylcarboxamide)-2'-O-methyluridine, 5-(N-benzylcarboxamide)-2'-fluorouridine, 5-(N-phenethylcarboxamide)-2'-deoxyuridine (PEdU), 5-(N-thiophenylmethylcarboxamide)-2'-deoxyuridine (ThdU), 5-(N-isobutylcarboxyamide)-2'-deoxyuridine (iBudU), 5-(N-tyrosylcarboxamide)-2'-deoxyuridine (TyrdU), 5-(N-3,4-methylenedioxybenzylcarboxamide)-2'-deoxyuridine (MBndU), 5-(N-4-fluorobenzylcarboxyamide)-2'-deoxyuridine (FBndU), 5-(N-3-phenylpropylcarboxamide)-2'-deoxyuridine (PPdU), 5-(N-imidizolylethylcarboxamide)-2'-deoxyuridine (ImdU), 5-(N-isobutylcarboxamide)-2'-O-methyluridine, 5-(N-isobutylcarboxyamide)-2'-fluorouridine, 5-(N-tryptaminocarboxamide)-2'-deoxyuridine (TrpdU), 5-(NR-threoninylcarboxamide)-2'-deoxyuridine (ThrdU), 5-(N-tryptaminocarboxamide)-2'-O-methyluridine, 5-(N-tryptaminocarboxamide)-2'-fluorouridine, 5-(N-[1-(3-trimethylammonium)propyl]carboxamide)-2'-deoxyuridine chloride, 5-(N-naphthylmethylcarboxamide)-2'-deoxyuridine (NapdU), 5-(N-naphthylmethylcarboxamide)-2'-O-methyluridine, 5-(N-naphthylmethylcarboxamide)-2'-fluorouridine, 5-(N-[1-(2,3-dihydroxypropyl)]carboxamide)-2'-deoxyuridine), 5-(N-2-naphthylmethylcarboxamide)-2'-deoxyuridine (2NapdU), 5-(N-2-naphthylmethylcarboxamide)-2'-O-methyluridine, 5-(N-2-naphthylmethylcarboxamide)-2'-fluorouridine, 5-(N-1-naphthylethylcarboxamide)-2'-deoxyuridine (NEdU), 5-(N-1-naphthylethylcarboxamide)-2'-O-methyluridine, 5-(N-1-naphthylethylcarboxamide)-2'-fluorouridine, 5-(N-2-naphthylethylcarboxamide)-2'-deoxyuridine (2NEdU), 5-(N-2-naphthylethylcarboxamide)-2'-O-methyluridine, 5-(N-2-naphthylethylcarboxamide)-2'-fluorouridine, 5-(N-3-benzofuranylethylcarboxamide)-2'-deoxyuridine (BFdU), 5-(N-3-benzofuranylethylcarboxamide)-2'-O-methyluridine, 5-(N-3-benzofuranylethylcarboxamide)-2'-fluorouridine, 5-(N-3-benzothiophenylethylcarboxamide)-2'-deoxyuridine (BTdU), 5-(N-3-benzothiophenylethylcarboxamide)-2'-O-methyluridine, and 5-(N-3-benzothiophenylethylcarboxamide)-2'-fluorouridine.

[0043] As used herein, the terms “modified,” “modified,” and any variations thereof, when used in relation to oligonucleotides, mean that at least one of the four constituent nucleotide bases (i.e., A, G, T / U, and C) of the oligonucleotide is an analogue or ester of a naturally occurring nucleotide. In some embodiments, modified nucleotides confer nuclease resistance to the oligonucleotide. Additional modifications include skeletal modifications, methylation, and combinations of rare base pairings, such as isobases isocytidine and isoguanidine. Modifications may also include 3' and 5' modifications, such as capping. Other modifications include substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as modifications by uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and modifications by charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), modifications by intercalators (e.g., acridine, psoralens, etc.), modifications containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), modifications containing alkylating agents, and modifications by modified bonds (e.g., alpha-anomeric nucleic acids, etc.). Furthermore, any of the hydroxyl groups normally present on the sugar of a nucleotide may be replaced with a phosphonate group or a phosphate group, protected with a standard protecting group, or activated to prepare additional binding to additional nucleotides or to a solid support. The 5' and 3' terminal OH groups may be phosphorylated or replaced with amines, organic capping groups of about 1 to about 20 carbon atoms, polyethylene glycol (PEG) polymers in the range of about 10 to about 80 kDa in one embodiment, PEG polymers in the range of about 20 to about 60 kDa in another embodiment, or other hydrophilic or hydrophobic biopolymers or synthetic polymers.

[0044] As used herein, “nucleic acid,” “oligonucleotide,” and “polynucleotide” are interchangeable terms used to refer to polymers of nucleotides, including DNA, RNA, DNA / RNA hybrids, and modified forms of these types of nucleic acids, oligonucleotides, and polynucleotides, which involve the binding of various entities or parts to nucleotide units at arbitrary positions. The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” include triple-helical molecules in addition to double-stranded or single-stranded molecules. Nucleic acid, oligonucleotide, and polynucleotide are broader terms than aptamer, and therefore the terms nucleic acid, oligonucleotide, and polynucleotide include polymers of nucleotides that are aptamers, but are not limited to aptamers.

[0045] Polynucleotides also contain analogous forms of ribose or deoxyribose sugars generally known in the art, such as 2'-O-methyl, 2'-O-allyl, 2'-O-ethyl, 2'-O-propyl, 2'-O-CH2CH2OCH3, 2'-fluoro, 2'-NH2 or 2'-azide, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars, such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs and debasic nucleoside analogs, such as methylriboside. As described herein, one or more phosphodiester bonds may be replaced with alternative linking groups. These alternative linking groups include phosphates P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR X 2 ("Amidate"), P(O)R X , P(O)OR X ', replaced by CO or CH2 ("formacetal"), in the formula, each R X or R XEmbodiments include those in which ' is independently H, or optionally contains an ether (-O-) bond and is a substituted or unsubstituted alkyl (C1-C20), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl alkyl (C1-C20), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl alkyl group. Not all bonds in the polynucleotide need to be identical. Substitutions of sugars, purines, and pyrimidines in similar forms may be advantageous in designing the final product, as may alternative skeletal structures such as a polyamide skeleton.

[0046] Polynucleotides may also contain analogues of carbocyclic sugars, α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acrylic acid analogues, and debasic nucleoside analogues such as methyl riboside.

[0047] If present, modifications to the nucleotide structure may be introduced before or after polymer assembly. The nucleotide sequence may be interposed by non-nucleotide components. After polymerization, polynucleotides may be further modified, for example, by complexing with labeling components.

[0048] As used herein, the term “at least one nucleotide” means, when referring to the modification of a nucleic acid, one, some, or all of the nucleotides in the nucleic acid, indicating that the presence of any or all of A, C, T, G, or U in the nucleic acid may or may not be modified.

[0049] As used herein, “nucleic acid ligand,” “aptamer,” “SOMAmer,” “modified aptamer,” and “clone” are interchangeable and refer to non-naturally occurring nucleic acids that have a desired effect on a target molecule. Desired effects include, but are not limited to, binding to a target, catalytically altering a target, reacting with a target in a way that modifies or alters the target or its functional activity, covalent binding to a target (as in suicide inhibitors), and promoting a reaction between a target and another molecule. In one embodiment, the effect is specific binding affinity to a target molecule, such a target molecule being a three-dimensional chemical structure other than a polynucleotide that binds to the aptamer by a mechanism independent of Watson / Crick base pairing or triple helix formation, and the aptamer is not a nucleic acid with a known physiological function to which the target molecule is bound. An aptamer for a given target comprises a nucleic acid identified from a candidate mixture of nucleic acids, and the aptamer is a ligand for the target by a method comprising: (a) contacting the candidate mixture with the target so that nucleic acids having a higher affinity for the target compared to other nucleic acids in the candidate mixture can be separated from the remainder of the candidate mixture; (b) separating the high-affinity nucleic acids from the remainder of the candidate mixture; and (c) amplifying the high-affinity nucleic acids to produce a ligand-enriched mixture of nucleic acids, thereby identifying the aptamer of the target molecule. While affinity interactions are recognized as a matter of degree, in this context, the “specific binding affinity” of an aptamer for its target means that the aptamer binds to that target with a generally much higher degree of affinity than the aptamer would bind to other non-target components in the mixture or sample. An “aptamer,” “SOMAmer,” or “nucleic acid ligand” is one type or copy set of one type of nucleic acid molecule having a specific nucleotide sequence. An aptamer may contain any preferred number of nucleotides. An "aptamer" refers to two or more sets of such molecules. Different aptamers may have the same or different numbers of nucleotides. Aptamers may be DNA or RNA, and may be single-stranded, double-stranded, or may contain double-stranded or triple-stranded regions.In some embodiments, the aptamers are prepared using SELEX processes as described herein or known in the art.

[0050] As used herein, “SOMAmer” or slow off-rate modified aptamer refers to an aptamer having improved off-rate properties. SOMAmers can be produced using the improved SELEX method described in U.S. Patent No. 7,947,447, entitled “Method for Generating Aptamers with Improved Off-Rates”. In some embodiments, slow off-rate aptamers (including aptamers containing at least one nucleotide having hydrophobic modifications) have off-rates (t1 / 2) of ≥20 min, ≥30 min, ≥60 min, ≥90 min, ≥120 min, ≥150 min, ≥180 min, ≥210 min, or ≥240 min.

[0051] As used herein, aptamers comprising two different 5-position modified pyrimidines or C-5 modified pyrimidines may also be referred to as “double-modified aptamers,” “aptamers having two modified bases,” “aptamers having two base modifications” or “double-modified” aptamers, or “aptamers having two modified bases,” all of which may be used interchangeably. The same terminology may also be used for aptamer libraries or aptamer libraries. Thus, in some embodiments, an aptamer comprises two different 5-position modified pyrimidines, and the two different 5-position modified pyrimidines may be NapdC and NapdU, NapdC and PPdU, NapdC and MOEdU, NapdC and TyrdU, NapdC and ThrdU, PPdC and PPdU, PPdC and NapdU, PPdC and MOEdU, PPdC and TyrdU, PPdC and ThrdU, NapdC and 2NapdU, NapdC and T Selected from rpdU, 2NapdC and NapdU, as well as 2NapdC and 2NapdU, 2NapdC and PPdU, 2NapdC and TrpdU, 2NapdC and TyrdU, PPdC and 2NapdU, PPdC and TrpdU, PPdC and TyrdU, TyrdC and TyrdU, TrydC and 2NapdU, TyrdC and PPdU, TyrdC and TrpdU, TyrdC and TyrdU, and TyrdC and TyrdU. In some embodiments, the aptamer comprises at least one modified uridine and / or thymidine and at least one modified cytidine, wherein the at least one modified uridine and / or thymidine is modified at position 5 by a moiety selected from naphthyl moiety, benzyl moiety, fluorobenzyl moiety, tyrosyl moiety, indole moiety, morpholino moiety, isobutyl moiety, 3,4-methylenedioxybenzyl moiety, benzothiophenyl moiety, and benzofuranyl moiety, and the at least one modified cytidine is modified at position 5 by a moiety selected from naphthyl moiety, tyrosyl moiety, and benzyl moiety.In certain embodiments, the portion is covalently bonded to the 5-position of the base via a linker containing a group selected from amide linkers, carbonyl linkers, propynyl linkers, alkyne linkers, ester linkers, urea linkers, carbamate linkers, guanidine linkers, amidine linkers, sulfoxide linkers, and sulfone linkers. See Figure 1 for further exemplary linkers that may be used to covalently bond the portion to the 5-position of the pyrimidine.

[0052] As used herein, “hydrophobic group” and “hydrophobic moiety” are interchangeable herein and refer to any uncharged group or moiety, the majority of which are hydrogen and carbon atoms, the group or moiety having a small dipole, and / or being repelled by water. These groups or moieties may include aromatic hydrocarbons or planar aromatic hydrocarbons. Methods for determining whether a molecule (or group or moiety) is hydrophobic are well known in the art and include calculation methods in addition to empirically obtained methods. An exemplary method is described in Zhu Chongqin et al. (2016) Characterizing hydrophobicity of amino acid side chains in a protein environment via measuring contact angle of a water nanodroplet on planar peptide network. Proc. Natl. Acad. Sci., 113(46) pgs. 12946-12951. As disclosed herein, exemplary hydrophobic parts include, but are not limited to, groups I, II, III, IV, V, VII, VIII, IX, XI, XII, XIII, XV, and XVI in Figure 1. Further exemplary hydrophobic parts include those shown in Figures 2-5 (e.g., BPE, PBnd, POP, DPP, DBM, BH, Bn, Nap, PE, PP, iBu, 2Nap, Try, NE, MBn, BF, BT, Trp).

[0053] As used herein, aptamers containing a single type of 5-position modified pyrimidine or C-5 modified pyrimidine may be referred to as “single-modified aptamers,” “aptamers having a single modified base,” “aptamers having a single base modification,” or “aptamers that are single-base modified,” and all of these may be used interchangeably. Libraries of aptamers or aptamer libraries may also use the same terminology. As used herein, “protein” is used synonymously with “peptide,” “polypeptide,” or “peptide fragment.” A “purified” polypeptide, protein, peptide, or peptide fragment is substantially free of cellular material or other contaminating proteins from cells, tissues, or cell-free sources from which the amino acid sequence is obtained, or, if chemically synthesized, substantially free of chemical precursors or other chemicals.

[0054] In certain embodiments, the aptamer comprises a first 5-position modified pyrimidine and a second 5-position modified pyrimidine, wherein the first 5-position modified pyrimidine contains a tyrosyl moiety at position 5 of the first 5-position modified pyrimidine, and the second 5-position modified pyrimidine contains a naphthyl or benzyl moiety at position 5 of the second 5-position modified pyrimidine. In related embodiments, the first 5-position modified pyrimidine is uracil. In related embodiments, the second 5-position modified pyrimidine is cytosine. In related embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the uracil in the aptamer is modified at position 5. In related embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cytosine in the aptamer is modified at position 5.

[0055] Those skilled in the art of nucleic acid hybridization will recognize that factors commonly used to give or control the stringency of hybridization include formamide concentration (or other chemical denaturing reagents), salt concentration (i.e., ionic strength), hybridization temperature, surfactant concentration, pH, and the presence or absence of chaotrope. The optimal stringency of a probe / target sequence combination is often found by the well-known technique of fixing some of the aforementioned stringency factors and then determining the effect of varying a single stringency factor. Except for the fact that PNA hybridization is entirely independent of ionic strength, the same stringency factors can be modulated to control the stringency of PNA hybridization to nucleic acids. The optimal stringency of an assay may be experimentally determined by testing each stringency factor until the desired degree of discrimination is achieved.

[0056] As used herein, the terms “hybridization,” “hybridizing,” “binding,” and similar terms may be used interchangeably in the context of nucleotide sequences. The ability of two nucleotide sequences to hybridize with one another is based on the degree of complementarity between the two sequences and the proportion of similarly matched complementary nucleotide pairs. The more nucleotides in a given sequence that are complementary to another sequence, the more stringent the conditions for hybridization can be, and the more specific the binding of the two sequences becomes. High stringency can be achieved by increasing the temperature, increasing the ratio of cosolvents, decreasing the salt concentration, etc. While hybridization of complementary Watson / Crick base pairs of probe and target materials on a microarray is generally preferred, non-Watson / Crick base pairings can also occur during hybridization.

[0057] Conventional hybridization solutions and hybridization processes are described in J. Sambrook, Molecular Cloning: A Laboratory Manual (above) and are incorporated herein by reference. Hybridization conditions typically include (1) a high ionic strength solution, (2) a controlled temperature, and (3) the presence of carrier DNA, a surfactant, and a divalent cation chelating agent, all of which are known in the art.

[0058] As used herein, “array” includes any one-dimensional, two-dimensional, or three-dimensional arrangement of an addressable region having one or more specific chemical moieties associated with that region (e.g., biopolymers such as peptide nucleic acid molecules, peptides, or polynucleotide sequences), where one or more chemical moieties are immobilized on the surface of the region. “Immobilized” means that one or more moieties stably associate with the substrate surface within the region so that they do not separate from the region under the conditions under which the array is used, for example, under hybridization and washing and stripping conditions. As is known in the art, one or more moieties may be covalently or non-covalently bonded to the surface of the region. For example, each region may extend three-dimensionally if the substrate is porous, but may have no substantial three-dimensional measurement (thickness) if the substrate is non-porous. An array may contain more than 10, more than 100, more than 1,000, more than 10,000, or more than 100,000 features in a region less than 20 cm or less than 10 cm. For example, a feature may have a width (i.e., diameter in the case of a circular spot) in the range of about 10 μm to about 1.0 cm. In other embodiments, each feature may have a width in the range of about 1.0 μm to about 1.0 mm, for example, about 5.0 μm to about 500 μm, and about 10 μm to about 200 μm. Non-circular features may have a regional range equivalent to the range of circular features having the aforementioned width (diameter) range. A given feature consists of a chemical moiety, e.g., a peptide nucleic acid molecule, a peptide, or a nucleic acid, that binds to (e.g., hybridizes with) a target molecule (e.g., a target nucleic acid or aptamer) so that the given feature corresponds to a specific target.

[0059] The terms “biological sample,” “sample,” and “test sample” are interchangeable herein and refer to any material, biological fluid, tissue, or cell obtained from or otherwise derived from an individual, environment, animal, or food sample. These include blood (including whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, and serum), sputum, tears, mucus, nasal lavage fluid, nasal aspirate, exhaled breath, urine, semen, saliva, peritoneal lavage fluid, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, lymph, papillary aspirate, bronchial aspirate (e.g., bronchoalveolar lavage fluid), bronchial swabs, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, and cerebrospinal fluid. These also include all of the experimentally separated fractions mentioned above. For example, a blood sample may be fractionated into serum, plasma, or fractions containing specific types of blood cells, such as red blood cells or white blood cells (leukocytes). In some embodiments, the sample may be a combination of samples from an organism, such as a combination of tissue and liquid samples. The term “biological sample” also includes materials containing homogenized solid material from, for example, fecal samples, tissue samples, or tissue biopsies. The term “biological sample” also includes materials derived from tissue culture or cell culture. Any preferred method for obtaining a biological sample may be used, exemplary methods including, for example, venotomy, swabbing (e.g., cheek swabs), and fine-needle aspiration cytology procedures. Exemplary tissues that can be fine-needled include lymph nodes, lungs, lung lavage fluid, BAL (bronchial alveolar lavage fluid), thyroid gland, breast, pancreas, and liver. Samples may also be collected by, for example, microdissection (e.g., laser capture microdissection (LCM) or laser microdissection (LMD)), bladder lavage, smear (e.g., PAP smear), or tubal lavage. "Biological samples" obtained from or derived from an organism include any such sample that has been processed in any preferred manner after being obtained from the organism.

[0060] "Target" or "target molecule" or "target" as used herein refers to any compound on which nucleic acids can act in a desired or intended manner. In various embodiments, such as proteomics assays, target molecules may be, but are not limited to, proteins, peptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, viruses, pathogens, toxic substances, substrates, metabolites, transition state analogs, cofactors, inhibitors, drugs, pigments, nutrients, growth factors, cells, tissues, or any part or fragment of any of the foregoing. Substantially, virtually any chemical or biological effector may be a suitable target. Molecules of any size can function as targets. Targets may also be modified in certain ways to increase the likelihood or intensity of interaction between the target and the nucleic acid. Targets may also include any slight changes to a particular compound or molecule, for example, in the case of proteins, slight changes in the amino acid sequence, disulfide bond formation, glycosylation, lipidization, acetylation, phosphorylation, or any other operation or modification, such as complexing with a labeling component that does not substantially alter the identity of the molecule. A “target molecule” or “target” is one type or set of copies of a molecule or multimolecular structure that can bind to an aptamer. A “target molecule” or “target” refers to two or more sets of such molecules. Embodiments of the SELEX process where the target is a peptide are described in U.S. Patent No. 6,376,190, entitled “Modified SELEX Processes Without Purified Protein.” In some embodiments, the target is a protein.

[0061] The phrases “oligonucleotides bound to the surface of a solid support” or “probes bound to a solid support” refer to oligonucleotides immobilized on the surface of a solid substrate, which can have various configurations, e.g., sheets, beads, particles, slides, wafers, webs, fibers, tubes, capillaries, microfluidic channels or reservoirs, or other structures. In certain embodiments, the oligonucleotide collections used herein are present, for example, in the form of arrays, on the surface of the same planar support. Immobilization of oligonucleotides onto a substrate or surface can be achieved by well-known techniques generally available in the literature. See, for example, ACPease, et al., Proc. Nat. Acad. Sci, USA, 91:5022-5026 (1994), Z. Guo, et al., Nucleic Acids Res, 22, 5456-65 (1994), and M. Schena, et al., Science, 270, 467-70 (1995) (each incorporated herein by reference).

[0062] The synthesis of polynucleotides is described in detail, for example, Caruthers, Science 230:281-285, 1985; Itakura et al., Ann. Rev. Biochem. 53:323-356; Hunkapillar et al., Nature 310:105-110, 1984; and “Synthesis of Oligonucleotide Derivatives in Design and Targeted Reaction of Oligonucleotide Derivatives”, CRC Press, Boca Raton, Fla., pages 100 et seq.; U.S. Patents No. 4,458,066, No. 4,500,707, No. 5,153,319, No. 5,869,643, EP0294196, and elsewhere. While the phosphoramidite and phosphite triester approaches are the most widely used, other approaches include the phosphodiester approach, the phosphotryester approach, and the H-phosphonate approach. The substrate is typically functionalized to bind to the first deposited monomer. Preferred techniques for functionalizing substrates containing such binding sites are described, for example, in Southern, EM, Maskos, U., and Elder, JK, Genomics, 13, 1007-1017, 1992. For array fabrication, different monomers and activators may be deposited at different addresses on the substrate during any given cycle, so that different features of the completed array have different desired biopolymer sequences. For in situ fabrication of polynucleotide arrays, one or more further intermediate steps, such as conventional oxidation, capping, and washing steps, may be required in each cycle (these steps may also be performed in a flooding procedure).

[0063] A computer program may be used to perform one or more steps of any of the methods disclosed herein. Another aspect of this disclosure is a computer program product including a computer-readable storage medium on which the computer program is stored, which, when loaded into a computer, performs or assists in performing any of the methods disclosed herein.

[0064] One aspect of this disclosure is the product of any of the methods disclosed herein, i.e., assay results, which may be evaluated at the test site or, if desired, transported to another location for evaluation and communication with relevant parties in a remote location. As used herein, “remote location” means a location physically different from the location where the results are obtained. Thus, the results may be sent to a different room, a different building, a different area of ​​a city, a different city, etc. The data may be transmitted by any preferred means, e.g., facsimile, mail, next-day delivery, email, FTP, voicemail, etc.

[0065] "Communicating" information means the transmission of data representing that information as electrical signals over a suitable communication channel (e.g., a private or public network). "Transferring" an item means any means of moving the item from one place to another, either by physical transport or by other means (if possible), and, at least in the case of data, by physical transport of the medium holding the data or the medium on which the data is transmitted.

[0066] Exemplary surface hybridization catalysts As used herein, “surface hybridization catalyst” or “hybridization catalyst” refers to any material that catalyzes the hybridization of a solution-phase oligonucleotide, such as an aptamer (e.g., SOMAmer), to a solid-phase oligonucleotide, which is an oligonucleotide bound to a solid support such as an array. In some embodiments, the hybridization catalyst is hydrophilic and cationic. In some embodiments, the hybridization catalyst comprises a main chain (or “backbone”) and side chains bound to the main chain, often forming a bottlebrush structure. In some embodiments, the hybridization catalyst comprises a polymer cationic amino acid as the main chain and polyethylene glycol (PEG) as the side chain. Non-limiting examples of such hybridization catalysts include catalysts containing polylysine (PLL), polyarginine, or polyornithine as the main chain. In some embodiments, the hybridization catalyst comprises a neutral and water-soluble side chain. Non-limiting examples of such side chains include polyethylene glycol, polyacrylamide, polysaccharides, polyvinyl alcohol, dextran, and their derivatives. Side chains can be attached to various positions on the main chain. As a non-limiting example, the hybridization catalyst polylysine-grafted polyethylene glycol (PLL-g-PEG) may contain a PEG side chain attached to the amine portion of the polylysine main chain. Alternatively, polylysine-grafted polyethylene glycol (PLL-g-PEG) may contain a PEG side chain attached to the alkyl portion of the polylysine main chain.

[0067] The molecular weight of the hybridization catalyst can be controlled in various embodiments by the length of the main chain, the length of the side chains, or the graft density of the side chains. For example, PLL200-g10-PEG5k contains 200 lysine groups per molecule with 10% PEG modification, or 20 PEG groups per molecule, each PEG group having a molecular weight of 5 kDa. The following table shows an example of a PLL-g-PEG hybridization catalyst, the number of lysine groups per molecule, the percentage of PEG modification, and the number of PEG groups per molecule. The table also shows the number of amines per molecule, which is equal to the number of lysine groups per molecule minus the number of PEG groups per molecule, and this is used to calculate the amount of hybridization catalyst to use based on the desired N:P ratio, as described herein. [Table 1]

[0068] In some embodiments, the hybridization catalyst contains 10 to 500, 10 to 300, or 20 to 200 lysines in the polylysine backbone. In some embodiments, the hybridization catalyst contains 5 to 50%, 5 to 40%, 5 to 30%, or 10 to 30% PEG modification. In some embodiments, the PEG has an average molecular weight of 1 to 20 kDa, 2 to 10 kDa, 2 to 8 kDa, or about 5 kDa.

[0069] This method allows the use of various hybridization catalysts, including those with varying main chain lengths, side chain lengths, and graft densities. The amount of selected hybridization catalyst used in the hybridization reaction can be calculated for the desired N:P ratio provided herein. These factors are not particularly limited and can be appropriately selected and prepared by methods known to those skilled in the art.

[0070] Exemplary surface hybridization This method may be applied to any method involving the hybridization of solution-phase nucleic acids to solid-phase nucleic acids. As used herein, “solution-phase nucleic acid” refers to any type of nucleic acid present in solution and not bound to a solid support, including but not limited to RNA, DNA, modified oligonucleotides, aptamers, and genomic DNA. Solution-phase nucleic acids may be conjugated to non-nucleic acid moieties such as proteins, antibodies, hormones, and small molecules, but are not limited to these. As used herein, “solid-phase nucleic acid” refers to any type of nucleic acid described above bound to one or more solid supports, including but not limited to three-dimensional solid supports such as beads or other particles, and two-dimensional solid supports such as planar arrays.

[0071] A “solid support” refers to any substrate having a surface to which molecules may be directly or indirectly bound, either covalently or non-covalently. A solid support may include any substrate material capable of providing physical support to a capture element or probe, such as nucleic acid, bound to its surface. The material is generally able to withstand conditions associated with the binding of the capture element or probe to the surface and any subsequent procedures, handling, or processing faced during the performance of the assay. The material may be naturally occurring, synthetic, or a modified version of a naturally occurring material. Suitable solid support materials include silicon, silicon wafer chips, graphite, mirrors, laminates, films, ceramics, plastics (polymers, e.g., poly(vinyl chloride), cycloolefin copolymers, agarose gels or beads, polyacrylamide, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), polytetrafluoroethylene (PTFE or Teflon®), nylon, poly(vinyl butyrate), etc.), germanium, gallium arsenide, gold, silver, Langmuir Blodgett films, flow-through chips, etc., which may be used alone or in combination with other materials. Additional rigid materials, such as glass, may be considered, including silica and, for example, glass available as bioglass. Other materials that may be used include porous materials such as controlled pore glass beads, crosslinked beads Sepharose® or agarose resin, or crosslinked bisacrylamide and azalactone copolymers. Other types of beads include nanoparticles, polymer beads, solid core beads, paramagnetic beads, or microbeads. Any other materials known in the art may also be considered, for example, having one or more functional groups incorporated on the surface of the material, such as amino, carboxyl, thiol, or hydroxyl functional groups.

[0072] The materials used for solid supports can take on a variety of configurations, ranging from simple to complex. Solid supports can have one of numerous shapes, including strips, plates, disks, rods, particles, beads, tubes, and wells (microtiters). Solid supports may be porous or non-porous, magnetic, paramagnetic, or non-magnetic, polydisperse or monodisperse, hydrophilic or hydrophobic. Solid supports may also be in the form of a gel or slurry of densely packed (as in a column matrix) or loosely packed particles. In some embodiments, solid supports are planar arrays, beads, or flow cells.

[0073] In some embodiments, a DNA hybridization array or chip is used to hybridize each solution-phase nucleic acid to a specific probe or a set of specific probes immobilized on a slide or chip, such as an Agilent array, Illumina BeadChip array, NimbleGen array, or a custom-printed array. Each specific probe is complementary to a sequence on the solution-phase nucleic acid. The complementary sequence may be a specific hybridization tag incorporated into the solution-phase nucleic acid, or a portion of the solution-phase nucleic acid, or the entire solution-phase nucleic acid. The solution-phase nucleic acid is added to a hybridization buffer containing a hybridization catalyst provided herein and hybridized as described herein. In some embodiments, the hybridization reaction is carried out for up to 20 hours, or up to 15 hours, or up to 10 hours, or up to 8 hours, or up to 6 hours, or about 1 to 10 hours, or about 1 to 8 hours, or about 1 to 6 hours, or about 2 to 10 hours, or about 2 to 8 hours, or about 2 to 6 hours, or about 4 hours. The array is washed and then scanned with a fluorescence slide scanner to generate an image of the aptamer hybridization intensity for each feature of the array. Image segmentation and quantification are achieved using image processing software such as ArrayVision. In some embodiments, the hybridization reaction includes at least 100, at least 500, at least 1000, at least 2000, at least 3000, or at least 5000 different solution-phase nucleic acids having different nucleic acid sequences, and / or at least 100, at least 500, at least 1000, at least 2000, at least 3000, or at least 5000 different solid-phase nucleic acids having different nucleic acid sequences.

[0074] In one embodiment, addressable microbeads having a specific DNA probe complementary to the solution-phase nucleic acid described above are used for hybridization. The microbeads may be addressable using a specific fluorescent dye, such as in Luminex bead technology, or using barcode labeling, such as in Illumina VeraCode technology, or a laser-powered transponder. In one embodiment, the solution-phase nucleic acid is added to a hybridization buffer containing a hybridization catalyst provided herein and hybridized as described herein. The solution is then processed on a Luminex instrument that counts individual bead types and quantifies the fluorescence signal. In another embodiment, VeraCode beads are brought into contact with the solution-phase nucleic acid in a hybridization buffer containing a hybridization catalyst described herein, and then deposited on a grid-covered surface and scanned using a slide scanner for identification and fluorescence quantification. In some embodiments, the hybridization reaction includes at least 100, at least 500, at least 1000, at least 2000, at least 3000, or at least 5000 different solution-phase nucleic acids having different nucleic acid sequences, and / or microbeads bound to at least 100, at least 500, at least 1000, at least 2000, at least 3000, or at least 5000 different solid-phase nucleic acids having different nucleic acid sequences.

[0075] In some embodiments, solution-phase nucleic acids may be amplified and optionally tagged before hybridization. Standard PCR amplification, including but not limited to quantitative PCR (qPCR) or RT-PCR, may be used. Such amplification can be performed before hybridization.

[0076] In various embodiments, the methods provided herein involve hybridizing a plurality of solution-phase nucleic acids into a plurality of solid-phase nucleic acids. These plurality of solution-phase nucleic acids are at least 100, at least 200, at least 1000, at least 2000, at least 3000, or at least 5000 different nucleic acids, i.e., at least 100, at least 200, at least 1000, at least 2000, at least 3000, or at least 5000 different nucleic acids having different nucleic acid sequences. As used herein, “different nucleic acid sequences” means that each of the at least 100, at least 200, at least 1000, at least 2000, at least 3000, or at least 5000 different nucleic acids is different from each of the other nucleic acids of the at least 100, at least 200, at least 1000, at least 2000, at least 3000, or at least 5000 different nucleic acids by at least one nucleic acid base in its respective sequence. This includes, for example, nucleic acids of different lengths and / or different sequences.

[0077] In some embodiments, the response time can be shortened by promoting faster hybridization and / or increasing the amount of hybridization by including a hybridization catalyst when hybridizing solution-phase nucleic acids to solid-phase nucleic acids. In some embodiments, the detection limit can be lowered by including a hybridization catalyst when hybridizing solution-phase nucleic acids to solid-phase nucleic acids. "Faster" hybridization, in some embodiments, means that the same amount or degree of hybridization occurs in a shorter period of time and / or an increase in the amount or degree of hybridization occurs in the same period of time.

[0078] In some embodiments, the amount of hybridization catalyst to be included in a surface hybridization reaction is determined by the ratio of the number of cations in the hybridization catalyst ("N") to the total number of phosphates in the solution-phase nucleic acid and the solid-phase nucleic acid ("P"). Surprisingly, the inventors have found that when determining the amount of hybridization catalyst to be included in a hybridization reaction, the number of phosphates in the solid-phase nucleic acid, as well as in the solution-phase nucleic acid, should be considered. Therefore, in the case of hybridization to a planar microarray, for example, the number of phosphates in the nucleic acid bound to the microarray is calculated and added to the number of phosphates in the solution-phase nucleic acid, and the amount of hybridization catalyst is determined accordingly. 13 An exemplary calculation for a microarray hybridization reaction, including the probe density and with the hybridization catalyst PLL200-g20-PEG5k, is as follows: [Table 2-1] [Table 2-2]

[0079] As shown above, when calculating the desired N:P ratio, including only solution-phase oligonucleotides results in a hybridization catalyst concentration of only 25% of the calculated concentration (approximately 2.7 μM vs. 11 μM). In other words, this method takes into account the number of phosphates in the solid-phase nucleic acid in addition to the phosphates in the solution-phase nucleic acid. Surprisingly, the inventors discovered that when calculating the amount of hybridization catalyst needed to achieve the desired N:P ratio, including only phosphates in the solution-phase nucleic acid can result in an insufficient concentration of the hybridization catalyst, potentially leading to insufficient improvement in hybridization. Surprisingly, the inventors found that including phosphates in the solid-phase nucleic acid when determining the amount of hybridization catalyst to include significantly improves the hybridization rate and / or degree.

[0080] Exemplary assay involving solid-phase hybridization In various embodiments, the hybridization catalyst may be used in one or more hybridization steps in any assay method involving hybridization of solution-phase nucleic acids to solid-phase nucleic acids, such as any assay method including multiplex solid-phase hybridization. Non-limiting exemplary assays that may include multiplex solid-phase hybridization include next-generation sequencing, diagnostic microarrays (for detecting specific gene mutations, chromosomal abnormalities, epigenetic changes, SNPs, etc.), gene expression microarrays, microsatellite analysis, and strand substitution assays.

[0081] In various embodiments, solution-phase nucleic acids are polynucleotides, including but not limited to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and modified oligonucleotides (which may be based on DNA, RNA, and / or analogs thereof, including double-stranded polynucleotides). It is understood that the term “solution-phase nucleic acid” does not refer to or infer a specific length of nucleic acid. The term “nucleic acid” encompasses polynucleotides and oligonucleotides of any length and sequence. In various embodiments, solution-phase nucleic acids may be genomic DNA, cDNA, RNA (including mRNA, microRNA, tRNA, etc.), mixed RNA / DNA, modified oligonucleotides, or aptamers. Solution-phase nucleic acids may be conjugated to non-nucleic acid portions such as proteins, antibodies, lipids, and small molecules, but are not limited to these. In some such embodiments, the solution-phase nucleic acid is a barcode.

[0082] The multiple solution-phase nucleic acids described herein refer to at least two solution-phase nucleic acids with different sequences. Solution-phase nucleic acids may include any type of nucleic acid suitable for use in the processes of this disclosure, such as solution-phase nucleic acids that can hybridize to solid-phase nucleic acids. Solution-phase nucleic acids in a particular embodiment may include DNA (e.g., complementary DNA (cDNA), genomic DNA (gDNA), mitochondrial DNA (mtDNA), etc.), RNA (e.g., messenger RNA (mRNA), small interfering RNA (siRNA), ribosomal RNA (rRNA), tRNA, etc.), DNA or RNA analogs (e.g., including base analogs, sugar analogs, and / or non-native skeletons), modified oligonucleotides, and / or aptamers. Nucleic acids may be in any form useful in the hybridization assays provided herein (e.g., linear, cyclic, supercoiled, single-stranded, double-stranded, etc.). In certain embodiments, nucleic acids may be plasmids, phages, autonomous replicating sequences (ARS), centromeres, artificial chromosomes, chromosomes, cells, cell nuclei, or cytoplasm of cells, or derived therefrom.

[0083] In some embodiments, a hybridization catalyst is used in one or more hybridization steps of a solid-phase sequencing assay. In some embodiments, the sequencing assay may be a next-generation sequencing (NGS) assay or a massively parallel signature sequencing (MPSS) assay. The solution-phase nucleic acid may be DNA or RNA. In some embodiments, the DNA library may be sequenced from a DNA or RNA starting sample. In some such embodiments, the DNA or RNA may be fragmented to prepare the library. An adapter ligation step may be applied to the cDNA before the PCR step with primers that enable subsequent universal PCR. PCR is performed with target-specific primers complementary to the universal PCR primers to amplify one or more genes of interest. In some embodiments, amplifying the cDNA library with universal primers facilitates a particular hybridization-based capture method. In some embodiments, the target primers may be used with a tag and / or in combination with the universal primers. In some embodiments, multiplex PCR may be used.

[0084] Following PCR, the amplified nucleic acids may be sequenced in the hybridization step of the sequencing reaction using a surface hybridization catalyst described herein. Sequencing may include dual-index sequencing, which increases the number of samples per run. Sequencing may include whole-genome sequencing, genotyping, gene expression and / or transcriptome profiling, and / or detection of epigenetic changes. Sequencing may be performed using a sequencing platform, such as a high-throughput sequencing platform like NGS or MPSS. Commercially available high-throughput sequencing platforms include those from Illumina, Qiagen, or ThermoFisher Scientific.

[0085] In some embodiments, the hybridization catalysts provided herein can be used for RNA sequencing, such as RNA-Seq. RNA for use in sequencing assays can be isolated from a sample using standard extraction methods. Briefly, in some embodiments, the RNA-Seq method uses reverse transcriptase to convert RNA to cDNA using either random primers or oligo(dT) primers. The RNA is fragmented or poly(a)-selected and then fragmented, followed by random priming, oligo(T) priming, or random hexamer priming. First and second strand cDNAs are synthesized, and the cDNA is amplified for sequencing. The sample is then sequenced using the hybridization catalysts described herein in the hybridization step of the sequencing reaction. Additional types of RNA sequencing may also be used with the surface hybridization catalysts described herein.

[0086] In some embodiments, the hybridization catalysts provided herein may be used in assays involving strand substitution in which one of the nucleic acids in a strand substitution assay is bound to a solid surface. In such assays, a first oligonucleotide bound to a solid surface hybridizes to a second oligonucleotide. A third oligonucleotide is introduced into a hybridization buffer containing the hybridization catalyst, and the third oligonucleotide can hybridize to the first oligonucleotide and substitute for (and thus release) the second oligonucleotide. An exemplary assay involving strand substitution on a solid surface is described, for example, in PCT Publication WO2022 / 060728A1.

[0087] In some embodiments, the hybridization assays provided herein are carried out in a hybridization buffer containing a hybridization catalyst. Any suitable hybridization buffer may be used, to which a desired amount of hybridization catalyst may be added. In some embodiments, the hybridization buffer contains a monovalent salt. In some embodiments, the hybridization buffer contains sodium citrate, and the monovalent salt is sodium. The sodium citrate concentration may be in the range of 10 mM to 1 M, or 50 to 500 mM, or 50 to 400 mM, or 100 to 300 mM. The hybridization buffer may further contain a surfactant such as the nonionic surfactant polysorbate 20 or Tween 20. The concentration of polysorbate 20 or Tween 20 may be in the range of 0.1 wt% to 2 wt% or 0.1 wt% to 1 wt%. In some embodiments, the hybridization buffer contains a metal chelating agent such as EDTA. In some embodiments, the assays described herein are carried out in Agilent hybridization buffer (Agilent, part number 5190-0403) to which a hybridization catalyst has been added.

[0088] Exemplary proteomics assay Multiplex aptamer assays in solution-based targeted interaction and separation steps are described, for example, in U.S. Patents No. 7,855,054 and 7,964,356 and PCT application PCT / US2013 / 044792. In one embodiment, a multiplex assay is described in Example 1 of this specification.

[0089] In a multiplex assay format where multiple target proteins are measured by multiple capture reagents, the ability of a particular capture reagent to measure a particular target protein may be limited by the natural variation in the abundance of different target proteins (for example, high abundances of a target protein may saturate the assay, interfering with or reducing the ability of an assay to measure low abundances of a target protein). To address this variation in biological samples, aptamer reagents may be divided into at least two distinct groups (DIL1 capture reagents and DIL2 capture reagents), preferably three distinct groups (A3-DIL1 capture reagents, A2-DIL2 capture reagents, and A1-DIL3 capture reagents) based on the abundance of their respective protein targets in the biological sample. Each of the capture reagent groups A1, A2, and A3 has a different set of aptamers, each of which has a specific affinity for the target protein. Biological samples are diluted into two (Dilution 1 or DIL1 and Dilution 2 or DIL2), preferably three different dilution groups (Dilution 1 or DIL1, Dilution 2 or DIL2 and Dilution 3 or DIL3), based on their relative concentrations detected by the protein target capture reagents, to prepare individual test samples. Thus, the biological samples are diluted into high, medium, and low abundance target protein dilution groups, with the least abundant protein target being measured in the lowest dilution group and the most abundant protein target being measured in the highest dilution group. The capture reagents for each dilution group of the capture reagents are incubated together (for example, A3 set of aptamers is incubated with the test sample in Dilution 1 or DIL1, A2 set of aptamers is incubated with the test sample in Dilution 2 or DIL2, and A1 set of aptamers is incubated with the test sample in Dilution 3 or DIL3). The total number of aptamers for A1, A2, and A3 may be 4,000, 4,500, or 5,000 or more.

[0090] Figure 6 provides an overview example of a biological sample dilution set, a corresponding capture reagent set for each dilution, and an overall overview of two sequential catch systems (Catch-1 and Catch-2). Three different dilution groups may be prepared from a biological sample containing a Z% dilution or DIL3, a Y% dilution or DIL2, and an X% dilution or DIL1, where Z is greater than Y and Y is greater than X (or Z is a greater dilution than Y, and Y is a greater dilution than X). Each dilution has a specific corresponding set of capture reagents that bind to a particular protein set (A3 for DIL1, A2 for DIL2, and A1 for DIL3).

[0091] In some embodiments, the disclosure describes methods for performing aptamer and photoaptamer-based multiplexed assays aimed at quantifying one or more target molecules that may be present in a test sample, and the aptamer (or photoaptamer) can be separated from the aptamer-target affinity complex (or photoaptamer-target covalent complex) for final detection using a hybridization-based nucleic acid detection method comprising a hybridization catalyst provided herein.

[0092] As used herein, “Catch-1” refers to the splitting of an aptamer-target affinity complex or an aptamer-target covalent complex. The objective of Catch-1 is to remove substantially all components in the test sample that do not associate with the aptamer. Removing most of such components can improve target tagging efficiency by removing non-target molecules from the target tagging step, which is typically used for Catch-2 capture, and may lead to a reduction in assay background. In one embodiment, the tag is bound to the aptamer by attaching the tag to the aptamer either before the assay, during assay preparation, or during the assay. In one embodiment, the tag is a freeable tag. In one embodiment, the freeable tag includes a cleavable linker and the tag. As described above, the tagged aptamer may be captured on a solid support, which contains a capture element suitable for the tag. The solid support may then be washed as described herein before equilibration with the test sample to remove all unwanted material (Catch-0).

[0093] As used herein, “Catch-2” refers to the splitting of an aptamer-target affinity or aptamer-target covalent complex based on the capture of the target molecule. The purpose of the Catch-2 step is to remove free or non-complexed aptamers from the test sample before detection and any quantification. Removal of free aptamers from the sample enables detection of the aptamer-target affinity or aptamer-target covalent complex by any suitable nucleic acid detection technique. When using Q-PCR for detection and any quantification, removal of free aptamers is necessary for accurate detection and quantification of the target molecule.

[0094] In one embodiment, the target molecule is a protein or peptide, and the free aptamer is separated from the aptamer-target affinity (or covalent) complex (and the remainder of the test sample) using a complex containing a reagent that can be incorporated into the protein (and peptide) and the protein (or peptide), such as an aptamer-target affinity (or covalent) complex. The tagged protein (or peptide) and the aptamer-target affinity (or covalent) complex may be immobilized on a solid support to enable the separation of the protein (or peptide) and the aptamer-target affinity (or covalent) complex from the free aptamer. Such tagging may include, for example, a biotin moiety that can be incorporated into the protein or peptide.

[0095] In one embodiment, the Catch-2 tag is bound to a protein (or peptide) by chemically conjugating the tag to the target either before the assay, during assay preparation, or during the assay. In one embodiment, the Catch-2 tag is a releaseable tag. In one embodiment, the releaseable tag includes a cleavable linker and tag. However, it is generally not necessary to release the protein (or peptide) from the Catch-2 solid support. As described above, the tagged target may be captured on a second solid support, the solid support containing a capture element suitable for the target tag. The solid support is then washed with a variety of buffer solutions, including buffer solutions containing organic solvents and buffer solutions containing salts and / or surfactants containing salts and / or surfactants.

[0096] After washing the second solid support, the aptamer-target affinity complex is then subjected to a dissociation step in which the complex is broken down to produce free aptamers, while the target molecule generally remains bound to the solid support by binding interactions between the capture element and the target capture tag. The aptamer can be released from the aptamer-target affinity complex by any method that breaks down the structure of either the aptamer or the target. This may be achieved by washing the support-bound aptamer-target affinity complex in a high-salt buffer that dissociates the non-covalently bound aptamer-target complex. The eluted free aptamers are collected and detected. In another embodiment, high or low pH is used to break down the aptamer-target affinity complex. In another embodiment, high temperature is used to dissociate the aptamer-target affinity complex. In another embodiment, any combination of the above methods may be used. In another embodiment, proteolytic digestion of the protein portion of the aptamer-target affinity complex is used to release the aptamer component.

[0097] An example of a proteomics affinity assay (multiplex assay) is described, for example, in PCT Publication WO2019 / 246289.

[0098] In the case of aptamer-target covalent complexes, the release of the aptamer for subsequent quantification is achieved using a cleavable linker in the aptamer construct. In another embodiment, a cleavable linker in the target tag results in the release of the aptamer-target covalent complex.

[0099] The released aptamers can be quantified using the hybridization-based methods described herein. In some embodiments, the released aptamers are quantified on an Agilent 8×15k microarray slide in Agilent HiRPM hybridization buffer containing an amount of hybridization catalyst calculated as described herein.

[0100] After hybridization, the microarray slide may be imaged using a microarray scanner (such as the Agilent G2565CA microarray scanner system from Agilent Technologies). The resulting TIFF images may be processed using Agilent feature extraction software version 10.7.3.1 with the GE1_107_Sep09 protocol.

[0101] As described above, one objective of proteomics assays is to convert protein signals into aptamer signals. As a result, the amount of aptamers collected / detected may indicate and be directly proportional to the amount of bound target molecules and the amount of target molecules in the sample. In addition to the embodiments of the detection methods described below, other detection methods are known to those skilled in the art.

[0102] Some detection methods require explicit labeling incorporated into the aptamer before detection. In these embodiments, the label, such as a fluorescent or chemiluminescent dye, may be incorporated into the aptamer either during or after synthesis using standard techniques for nucleic acid synthesis. Radiolabeling may be incorporated either during or after synthesis using standard enzymatic reactions with appropriate reagents. Labeling may also be performed after catch-2 splitting and elution by using suitable enzymatic techniques. For example, using primers with the label described above, the label will be incorporated into the amplified product of the eluted aptamer by PCR. When gel techniques are used for quantification, different sized mass labels may also be incorporated using PCR. These mass labels may also incorporate different fluorescent or chemiluminescent dyes for further multiplexing capability. Labeling may also be indirectly added to the aptamer by using a specific tag incorporated into the aptamer either during or after synthesis, and then adding a probe that associates with the tag and holds the label. In addition to those described above, labeling may include, for example, enzymes used in standard assays for colorimetric analysis readings. These enzymes act in combination with enzyme substrates and include, for example, enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase (AP). Labels may also include substances or compounds that are electrochemical functional groups for electrochemical detection.

[0103] For example, as described above, before contacting the test sample, the aptamer contains phosphorus-32 ( 32The aptamers may be labeled with radioactive isotopes such as P). Using any one of the four basic assays and their variations as described above, aptamer detection may be easily achieved by quantifying the radioactivity on the second solid support at the end of the assay. The radioactivity value will be directly proportional to the amount of target in the original test sample. Similarly, by labeling the aptamers with fluorescent dyes as described above before contacting the test sample, direct and simple fluorescence readings on the second solid support are possible. Likewise, chemiluminescent labeling or quantum dots may be used for direct readings from the second solid support without requiring aptamer elution.

[0104] For the multiplexed detection of aptamers released from the second solid support of Catch-2, a single fluorescent dye incorporated into each aptamer as described above can be used in conjunction with a quantification method that enables identification of the aptamer sequence along with quantification at the aptamer level. Such methods include, but are not limited to, DNA chip hybridization, microbead hybridization, next-generation sequencing, and CGE analysis. [Examples]

[0105] The following examples are provided to better illustrate some embodiments of the present invention. However, they should not be construed as limiting the broad scope of the invention. Those skilled in the art can readily adopt the principles underlying this discovery and design a variety of compounds without departing from the spirit of the invention.

[0106] Example 1. Multiplexed aptamer analysis of a sample This example describes a multiplex aptamer assay that can be used for the analysis of samples and controls.

[0107] Multiplex aptamer assay method All steps of the multiplex aptamer assay should be performed at room temperature unless otherwise instructed.

[0108] Preparation of Aptamer Master Mix Solution The 5272 aptamers are grouped into three distinct mixtures, Dil1, Dil2, and Dil3 (corresponding to 20%, 0.5%, and 0.005% plasma or serum sample diluents, respectively). The assignment of aptamers to the mixtures is empirically determined by assaying a dilution series of plasma and serum samples matched to each aptamer and identifying the sample diluent that exhibits the maximum linear range of the signal. By separating the aptamers and mixing them with different diluents (20%, 0.5%, or 0.005%) of plasma or serum samples, the assay can be performed in 10% of the protein concentration range. 7 This allows for a doubled range. Prepare a stock solution of the aptamer master mix with 4 nM aptamers each in HE-Tween buffer (10 mM Hepes, pH 7.5, 1 mM EDTA, 0.05% Tween 20) and store frozen at -20°C. Mix 4271 aptamers into the Dil1 mixture, 828 aptamers into the Dil2 mixture, and 173 aptamers into the Dil3 mixture. Before use, dilute the stock solution in HE-Tween buffer to a working concentration of 0.55 nM for each aptamer and portion into individual aliquots. Before using the aptamer master mix to prepare the Catch-0 plate, heat and cool the working solution before use by incubating at 95°C for 10 minutes, then at 25°C for at least 30 minutes, to refold the aptamers.

[0109] Catch-0 plate preparation Combine 60 μL of streptavidin-magcepharose 10% slurry (GE Healthcare, 28-9857) with 100 μL of heated and cooled aptamer master mix. Wash the mixture once with 175 μL of assay buffer (40 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, 0.05% Tween-20), then dispense into each well of a 96-well plate (Thermo Scientific, AB-0769). Incubate the plate at 25°C for 30 minutes while shaking at 850 rpm on a ThermoMixer C shaker (Eppendorf). After incubation for 30 minutes, add 6 μL of MB block buffer (50 mM D-biotin in 50 mM Tris-HCl, pH 8, 0.01% Tween) to each well of the plate and incubate the plate for a further 2 minutes while shaking. Then, wash the plate with 175 μL of assay buffer and perform a 1-minute washing cycle on a ThermoMixer C with shaking at 850 rpm, followed by separation on a magnet for 30 seconds. After removing the washing solution, resuspend the beads in 175 μL of assay buffer and store at -20°C until use.

[0110] Catch-2 Bead Preparation Before starting the assay's robotic processing, wash the 10 mg / mL bead slurry of MyOne streptavidin C1 beads (Dynabeads, product number 35002D, Thermo Scientific) used in the Catch-2 step of the multiplex aptamer assay once in MB Prep buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA, 0.4% SDS) for 5 minutes, followed by two washes with assay buffer. After the final wash, resuspend the beads at a concentration of 10 mg / mL and dispense 75 μL of the bead slurry into each well of the Catch-2 plate. At the start of the assay, place the Catch-2 plate in the aluminum adapter and position it appropriately on the Fluent deck.

[0111] Thawing and dilution of the sample Thaw 65 μL aliquots of 100% plasma or serum samples stored at -80°C in matrix tubes by incubation at room temperature for 10 minutes. To facilitate thawing, place the tubes on top of a fan unit that circulates air through the matrix tube rack. After thawing, centrifuge the samples at 1000 × g for 1 minute and place them on a Fluent robotic deck for dilution. Prepare a 20% sample solution by transferring 35 μL of thawed sample to a 96-well plate containing 140 μL of appropriate sample diluent. Plasma sample diluents are 50 mM Hepes, pH 7.5, 100 mM NaCl, 8 mM MgCl2, 5 mM KCl, 1.25 mM EGTA, 1.2 mM benzamidine, 37.5 μM Z-block, and 1.2% Tween-20. Serum sample diluents contain 75 μM Z-block, with the other components at the same concentrations as the plasma sample diluents. Subsequently, to prepare 0.5% and 0.005% diluted samples, the samples are diluted with assay buffer using serial dilution on a Fluent robot. To prepare the 0.5% sample dilution, an intermediate dilution from the 20% sample to 4% is prepared by mixing 45 μL of the 20% sample with 180 μL of assay buffer, and then the 0.5% sample is prepared by mixing 25 μL of the 4% diluted sample with 175 μL of assay buffer. To prepare the 0.005% sample, a 0.05% intermediate dilution is prepared by mixing 20 μL of the 0.5% sample with 180 μL of assay buffer, and then the 0.005% sample is prepared by mixing 20 μL of the 0.05% sample with 180 μL of assay buffer.

[0112] Sample binding step The Catch-0 plate is prepared by immobilizing the aptamer mix onto streptavidin magnetic Sepharose beads as described above. The frozen plate is thawed at 25°C for 30 minutes and washed once with 175 μL of assay buffer. 100 μL of each sample dilution (20%, 0.5%, and 0.005%) is added to the plate containing beads with three different aptamer master mixes (Dil1, Dil2, and Dil3, respectively). The Catch-0 plate is then sealed with aluminum foil seal (Microseal'F'Foil, Bio-Rad) and placed in a 4-plate rotating shaker (PHMP-4, Grant Bio) set to 850 rpm and 28°C. The sample binding step is performed for 3.5 hours.

[0113] Multiplex aptamer assay processing on Fluent robot After the sample binding step is complete, place the Catch-0 plate in the aluminum plate adapter and then on the robotic deck. The magnetic bead washing step is performed using a temperature-controlled plate. In all robotic processing steps, except for the Catch-2 wash as described below, set the plate to a temperature of 25°C. Wash the plate four times with 175 μL of assay buffer, programming each wash cycle to shake the plate at 1000 rpm for at least 1 minute, then separate the magnetic beads for at least 30 seconds before aspirating the buffer. During the final wash cycle, prepare the tag reagent by diluting a 100-fold tag reagent (EZ-Link NHS-PEG4-biotin, product no. 21363, Thermo, 100 mM solution prepared in anhydrous DMSO) 1:100 with assay buffer and pour it into the grooved tank on the robotic deck. Add 100 μL of tag reagent to each well of the plate and incubate with shaking at 1200 rpm for 5 minutes to biotinylate the proteins captured on the bead surface. The biotinylation reaction is quenched by adding 175 μL of quench buffer (20 mM glycine in the assay buffer) to each well. The plate is allowed to stand and incubated for 3 minutes, then washed four times with 175 μL of assay buffer. The washing is performed under the same conditions as described above.

[0114] Light sectioning and kinetic challenge After the final wash of the plate, 90 μL of photocleavage buffer (2 μM oligonucleotide competitor in the assay buffer, the competitor having the nucleotide sequence 5'-(AC-Bn-Bn)7-AC-3', where Bn represents a benzyl-substituted deoxyuridine residue at position 5) is added to each well of the plate. The plate is then transferred to a photocleavage substation on a Fluent deck. The substation consists of a BlackRay light source (UVP XX series bench lamp, 365 nm) and three Bioshake 3000-T shakers (Q Instruments). The plate is irradiated for 20 minutes while shaking at 1000 rpm.

[0115] Catch-2 Bead Capture At the end of the photocleavage process, remove the buffer from the Catch-2 plate by magnetic separation and wash the plate once with 100 μL of assay buffer. Remove the photocleavage eluate containing the aptamer-protein complex from each Catch-0 plate, starting with Dilution 3 plate. Transfer all 90 μL of solution first to the Catch-1 eluate plate placed on a shaker, raise the magnet to capture all streptavidin magnetic Sepharose beads that may be attracted. Then transfer the solution to the Catch-2 plate and incubate the plate for 3 minutes with shaking at 1400 rpm at 25°C. After 3 minutes of incubation, separate the magnetic beads for 90 seconds to remove the solution from the plate and add the photocleaved Dil2 plate solution to the plate. After the same process, add the solution from Dil1 plate and incubate for 3 minutes. At the end of the 3 minutes of incubation, add 6 μL of MB block buffer to the magnetic bead suspension and incubate the beads for 2 minutes with shaking at 1200 rpm at 25°C. After incubation, transfer the plate to another shaker pre-set to 38°C. Separate the magnetic beads for 2 minutes, then remove the solution. Next, wash the Catch-2 plate four times with 175 μL of MB washing buffer (20% glycerol in assay buffer). Each washing cycle is programmed to shake the beads at 1200 rpm for 1 minute and then separate the beads on the magnet for 3.5 minutes. During the final bead separation step, set the shaker temperature to 25°C. Next, wash the beads once with 175 μL of assay buffer. In this washing step, shake the beads at 1200 rpm for 1 minute, then separate them on the magnet for 2 minutes. After the washing steps, elute the aptamers from the purified aptamer-protein complexes using elution buffer (1.8 M NaClO4, 40 mM PIPES, pH 6.8, 1 mM EDTA, 0.05% Triton X-100). Elution is performed at 25°C for 10 minutes using 75 μL of elution buffer, while shaking the beads at 1250 rpm. Transfer 70 μL of the eluate to an archive plate and separate it on a magnet to divide any magnetic beads that may be aspirated.Transfer 10 μL of eluate to a black half-area plate, dilute 1:5 with assay buffer, and use for measuring the cyanine 3 fluorescence signal to be monitored as an internal assay QC. Transfer 20 μL of eluate to a plate containing 5 μL of hybridization blocking solution (Oligo aCGH / ChIP-on-chip Hybridization Kit, high volume, Agilent Technologies 5188-5380, containing spikes of cyanine 3-labeled DNA sequences complementary to corner marker probes on the Agilent array). Remove this plate from the robotic deck and process it further for hybridization (see below). Heat seal the archive plate with the remaining eluate using aluminum foil and store at -20°C.

[0116] Alternatively, the sequential release and capture of diluted biological samples are performed along with their respective aptamer groups during the transition from the Catch-1 phase to the Catch-2 phase of the assay. The overall outlines of the two sequential dilution-catch formats and the three sequential dilution-catch formats are shown in Figures 8 and 6, respectively. In this example, the same three different dilution groups of plasma (DIL3, DIL2, and DIL1) are prepared along with the same aptamer groups (A1, A2, and A3) described above (see Figure 7). The same approach as described above is followed for the Catch-1 phase of the assay, but in this example, the different dilution groups are released separately and moved to the Catch-2 phase of the assay sequentially, rather than simultaneously (see Figure 7). More specifically, in condition 1, the DIL1 group incubated with aptamer group A1 (DIL1-A1 group) is released from Catch-1, immobilized on a second solid support (Catch-2), and washed. Next, the DIL2 group incubated with aptamer group A2 is released from catch-1, combined with the DIL1-A1 group already immobilized on catch-2, and then immobilized on the second solid support (catch-2). The DIL3 group incubated with aptamer group A3 is released from catch-1, immobilized on the second solid support (catch-2), and washed.

[0117] Standard Operating Procedure (SOP) Hybridization Manually pipette 25 μL of 2x Agilent hybridization buffer (Oligo aCGH / ChIP-on-chip Hybridization Kit, Agilent Technologies, product numbers 5188-5380) into each well of the plate containing the eluted sample and blocking buffer. Manually pipette 40 μL of this solution into each "well" of the hybridization gasket slide (Hybridization Gasket Slide - 8 microarrays per slide format, Agilent Technologies). Place custom SurePrint G3 8×60k Agilent microarray slides, containing 10 probes per array complementary to each aptamer, onto the gasket slides according to the manufacturer's protocol. Securely fix each assembly (Hybridization Chamber Kit - SureHyb compatible, Agilent Technologies) and place it in a hybridization oven at 55°C for 19 hours, rotating at 20 rpm.

[0118] Hybridization in the presence of a hybridization catalyst Manually pipette 25 μL of 2x Agilent hybridization buffer (Oligo aCGH / ChIP-on-chip Hybridization Kit, Agilent Technologies, product no. 5188-5380) containing the indicated amount of hybridization catalyst into each well of the plate containing the eluted sample and blocking buffer. Manually pipette 40 μL of this solution into each "well" of the hybridization gasket slide (Hybridization Gasket Slide - 8 microarrays per slide format, Agilent Technologies). Place custom SurePrint G3 8×60k Agilent microarray slides, containing 10 probes per array complementary to each aptamer, onto the gasket slides according to the manufacturer's protocol. Securely fix each assembly (Hybridization Chamber Kit - SureHyb compatible, Agilent Technologies) and place it in a hybridization oven at 55°C for 19 hours, rotating at 20 rpm.

[0119] Washing after hybridization Slide washing is performed using a Little Dipper Processor (Model 650C, Scigene). Approximately 700 mL of Wash Buffer 1 (Oligo aCGH / ChIP-on-chip Wash Buffer 1, Agilent Technologies) is poured into a large glass staining dish and used to separate microarray slides from gasket slides. Once disintegrated, the slides are quickly transferred to a slide rack in a tank containing Wash Buffer 1 on the Little Dipper. The slides are washed in Wash Buffer 1 for 5 minutes while being mixed with a magnetic stirrer. The slide rack is then transferred to a tank containing Wash Buffer 2 (Oligo aCGH / ChIP-on-chip Wash Buffer 2, Agilent Technologies) at 37°C and incubated for 5 minutes with stirring. The slide rack is slowly removed from the second tank and then transferred to a tank containing acetonitrile and incubated for 5 minutes with stirring.

[0120] Microarray imaging Microarray slides were imaged using a microarray scanner (Agilent G4900DA Microarray Scanner System, Agilent Technologies) with a 3μm resolution and 100% PMT setting in a cyanine 3-channel environment, with the 20-bit option enabled. The resulting TIFF images were processed using Agilent feature extraction software (version 10.7.3.1 or later) with the GE1_1200_Jun14 protocol.

[0121] Example 2. Surface Hybridization Solutions containing a total of approximately 7,000 aptamers (SOMAmers) (each at a concentration of 10 pM) were prepared. These solutions were incubated for 4 hours at room temperature on the surface of a SomaArray slide (containing 65,000 features and an Agilent slide containing a probe complementary to each SOMAmer of the assay) in Agilent hybridization buffer spiked with varying amounts of hybridization catalyst (PLL200-g20-PEG5k). A negative control was performed under the same assay conditions but without the hybridization catalyst. SOMAmers with N:P ratios (ratio of the number of amines on the catalyst to the number of phosphates in the nucleic acid backbone) of 1, 10, 50, and 100 were examined. The performance of the hybridization catalyst was determined by measuring the fluorescence (relative fluorescence units (RFUs)) at the homologous binding sites on the microarray surface.

[0122] Figure 9 shows the integrated results of the surface hybridization assay of the hybridization catalyst. Figure 9 shows that the signal is increased in the presence of the hybridization catalyst ("Magic Buffer") compared to the condition without the hybridization catalyst ("No Magic Buffer Control"), suggesting that the hybridization rate increases in the presence of the hybridization catalyst. Various ratios of the number of amines on the catalyst to the number of phosphates on the nucleic acid backbone (N(amine):P(phosphate) ratio) of SOMAmers were evaluated (ratios of 1, 10, 50, and 100). In Figure 9, the X axis corresponds to the fluorescence (RFU) under the uncatalyzed condition, and the Y axis corresponds to the fluorescence (RFU) under four N:P experimental conditions with the catalyst. Each dot represents a single SOMAmer. The line along X=Y indicates identical performance with zero acceleration, both with and without the catalyst. The Y offset of the line indicates an A-fold acceleration.

[0123] At an N:P ratio of 1:1, A was approximately 2. As the amount of hybridization catalyst increased, performance stabilized. A peaked at approximately 5 at N:P ratios of 10:1 and 50:1. At higher N:P ratios (N:P=100), performance decreased, with A being less than 3 in this experiment. The linearity of the data showed good agreement with the non-hybridization catalyst condition. The spread of points around the imaginary line along each N:P dataset was slightly larger than that of the self-consistent line, suggesting the possibility of some sequence dependence. However, this effect was not significant, for example, at N:P 10, the RFU spread over less than 2x. Absolute background increased somewhat at N:P 10 and 50, and decreased at N:P 100. The coefficient of variation (CV) was significantly reduced at the slide position without SOMAmer addition, where a measurement of zero was expected.

[0124] Example 3. Measurement of the coefficient of variation The performance of the hybridization catalyst was determined by measuring the fluorescence (RFU) at the homologous binding sites on the microarray surface using the experimental data described in Example 2. The assay results are shown in Figures 10 and 11.

[0125] Accelerating the hybridization of SOMAmers to the microarray surface using a hybridization catalyst increased the fluorescence per binding spot (RFU) and decreased the coefficient of variation (CV). Assays performed in the presence of the hybridization catalyst showed lower CV and lower standard deviation than assays without the catalyst (Figure 10). The CV was higher in assays performed with the hybridization catalyst in negative control experiments (Figure 11; Y axis = frequency, X axis = intensity (RFU)).

[0126] Example 4. Measurement of reproducibility We repeatedly performed hybridization of SOMAmers on SomaArray slides using a hybridization catalyst (PLL200-g20-PEG5k), demonstrating excellent data reproducibility. See Figure 12.

[0127] Example 5. Storage period of hybridization catalyst buffer A buffer solution containing the PLL200-g20-PEG5k hybridization catalyst was prepared and stored at 4°C. At a given experimental time, the SOMAmer mix was added to either the aged hybridization catalyst buffer or a newly prepared hybridization catalyst buffer at an N:P ratio of 10. The relative hybridization performance of the two solutions on the Agilent microarray surface was measured. To quantify any performance difference between the aged and fresh hybridization catalyst buffers, fluorescence (RFU) values ​​recorded with an Agilent scanner were used as a surrogate. Experiments were performed at two different SOMAmer library concentrations, approximately 10 pM and 100 fM, respectively.

[0128] As shown in Figures 13 and 14, the hybridization of SOMAmers onto the Agilent microarray surface was unaffected by the time-dependent changes in the hybridization catalyst buffer in both high and low concentration SOMAmer libraries. The hybridization catalyst buffer after 2 and 4 weeks showed the same performance. Based on this experiment, the hybridization catalyst buffer can be stored at 4°C for at least 4 weeks.

[0129] Example 6. Measurement of incubation time for the performance of hybridization catalyst buffer. The microarray hybridization time for the SomaScan® assay is typically about 19 hours. As mentioned above, in the presence of hybridization catalyst buffer, accelerated hybridization is observed on the microarray surface, and the required incubation time can be reduced from 19 hours to about 4 hours.

[0130] This experiment was designed to observe the effect of hybridization catalyst buffer when a SOMAmer library was incubated for a standard operating procedure (SOP) incubation time of 19 hours. The results of this experiment were expected to be affected by material fatigue, in contrast to a 4-hour kinetic challenge (as most SOMAmers would find hybridization partners on the surface in the early stages of the experiment, resulting in a lack of sufficient SOMAmers available in solution).

[0131] Figure 15 shows the hybridization performance of the SOMAmer library in the presence of the hybridization catalyst buffer compared to the control condition (Agilent SOP). At 19 hours of incubation, the hybridization catalyst buffer was shown to result in higher fluorescence signals, in other words, improved hybridization to the array, for virtually all SOMAmers in the library. As shown in Figure 16, improvements were observed in the 19-hour incubation experiment, similar to the improvements in CV performance observed in the 4-hour incubation assay.

Claims

1. A method for hybridizing multiple solution-phase nucleic acids to multiple solid-phase nucleic acids, comprising contacting the solid-phase nucleic acids with a hybridization solution containing the solution-phase nucleic acids and a hybridization catalyst, wherein the hybridization catalyst contains a polycationic main chain having hydrophilic side chains.

2. The method according to claim 1, wherein the plurality of solution-phase nucleic acids comprise at least 100, at least 500, at least 1000, at least 2000, at least 3000, or at least 5000 different nucleic acids having different nucleic acid sequences.

3. The method according to claim 1 or claim 2, wherein the plurality of solid-phase nucleic acids include at least 100, at least 500, at least 1000, at least 2000, at least 3000, or at least 5000 different nucleic acids having different nucleic acid sequences.

4. The method according to any one of claims 1 to 3, wherein the solid nucleic acid is bound to an array, a flow cell, or beads.

5. The method according to any one of claims 1 to 3, wherein the solid-phase nucleic acid is bound to an array.

6. The method according to any one of claims 1 to 5, wherein the hybridization solution contains a monovalent cationic salt in a concentration of 100 to 300 mM.

7. The method according to claim 6, wherein the monovalent cationic salt is NaCl or sodium citrate.

8. The method according to any one of claims 1 to 7, wherein the hybridization catalyst comprises a polylysine, polyarginine, or polyornithine main chain.

9. The method according to any one of claims 1 to 8, wherein the hybridization catalyst comprises polyethylene glycol, polyacrylamide, polyvinyl alcohol, or a dextran side chain.

10. The method according to any one of claims 1 to 9, wherein the hybridization catalyst comprises a polylysine main chain and polyethylene glycol (PEG) side chains.

11. The method according to claim 10, wherein the hybridization catalyst comprises 10 to 500, or 10 to 300, or 20 to 200 lysines in the polylysine main chain, the hybridization catalyst comprises 5 to 50%, or 5 to 40%, or 5 to 30%, or 10 to 30% PEG modification, and / or the PEG comprises an average molecular weight of 1 to 20 kDa, or 2 to 10 kDa, or 2 to 8 kDa, or about 5 kDa.

12. The method according to any one of claims 1 to 11, wherein the hybridization catalyst is present in an amount such that the ratio (N:P ratio) of the number of cations in the polycationic skeleton of the hybridization catalyst to the number of phosphates in the plurality of solution-phase nucleic acids and the plurality of solid-phase nucleic acids is 1:1 to 200:1, or 1:1 to 100:1, or 1:1 to 50:1, or 1:1 to 25:

1.

13. The method according to any one of the prior claims, wherein the hybridization of the solution-phase nucleic acid to the solid-phase nucleic acid is at least twice, at least three times, at least four times, or at least five times faster in the presence of the hybridization catalyst than in the absence of the hybridization catalyst.

14. The method according to any one of claims 1 to 13, wherein the solution-phase nucleic acid comprises RNA, DNA, genomic DNA, mitochondrial DNA, mRNA, microRNA, aptamers, and / or modified oligonucleotides.

15. The method according to any one of claims 1 to 14, wherein the hybridization method is a step in an assay selected from next-generation sequencing, diagnostic microarrays (for detecting specific gene mutations, chromosomal abnormalities, epigenetic changes, SNPs, etc.), gene expression microarrays, microsatellite analysis, proteomics assays, and strand substitution assays.

16. The method according to any one of claims 1 to 15, wherein the solution-phase nucleic acid is conjugated to a non-nucleic acid portion.

17. The method according to claim 16, wherein the non-nucleic acid portion is selected from proteins, antibodies, lipids, and small molecules.

18. The method according to any one of claims 1 to 17, wherein each of the solution-phase nucleic acids contains a barcode sequence.

19. The method according to any one of claims 1 to 18, wherein the solution-phase nucleic acid is not amplified before hybridization to the solid-phase nucleic acid.

20. The method according to any one of claims 1 to 18, wherein the solution-phase nucleic acid is amplified before hybridization to the solid-phase nucleic acid.

21. The method according to claim 20, wherein the amplification includes PCR, RT-PCR, or qPCR.

22. The method according to any one of claims 1 to 21, wherein the solution-phase nucleic acid is an aptamer.

23. The method according to claim 22, wherein the aptamer is a slow-off-rate aptamer.

24. The method according to claim 22 or claim 23, wherein each aptamer comprises at least one, at least two, at least three, or at least five C-5 modified pyrimidines.

25. The method according to claim 24, wherein each C-5 modified pyrimidine comprises a C-5 modified uracil base or a C-5 modified cytosine base.

26. The method according to any one of claims 22 to 25, wherein each aptamer can bind to a protein target.

27. The method according to any one of claims 21 to 25, wherein the aptamer is eluted from the aptamer / protein complex prior to hybridization to the solid nucleic acid.

28. The method according to claim 27, wherein the aptamer / protein complex is formed by contacting the aptamer with a biological sample containing a protein.

29. A method for detecting multiple protein targets in a biological sample, comprising contacting the biological sample with a plurality of aptamers, each aptamer being a nucleic acid, each aptamer specifically binding to a protein target to form a plurality of aptamer / protein complexes, the method further comprising releasing the bound aptamers from the aptamer / protein complexes into a solution to form a plurality of solution-phase nucleic acids, hybridizing the plurality of solution-phase nucleic acids to a plurality of solid-phase nucleic acids according to the method of any one of claims 1 to 28, and detecting the hybridized solution-phase nucleic acids.

30. The method according to claim 28 or 29, wherein the biological sample is selected from plasma, serum, urine, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, sputum, tears, mucus, nasal lavage fluid, nasal aspirate, semen, saliva, peritoneal lavage fluid, ascites, cystic fluid, cerebrospinal fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate, bronchial swab, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, and cerebrospinal fluid.

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