Suppression of Targeted Aptamer Clusters

Abstract

The present disclosure relates to a method for sequencing low-abundance aptamers from an aptamer library, comprising the steps of: (i) amplifying a plurality of aptamers capable of binding to one or more target molecules in a sample by emulsion PCR to generate an aptamer library; (ii) sequencing the aptamer library; and (iii) contacting the aptamer library with a plurality of antisense oligonucleotides (ASOs) capable of targeting high-abundance aptamers within the aptamer library to form a mixture, wherein the contact between the ASO and the aptamer library results in the inactivation of high-abundance aptamers within the aptamer library.

Description

Technical Field

[0001] The present invention generally relates to a method for sequencing low-abundance aptamers from an aptamer library.

Background Art

[0002] Enriched aptamers are subjected to sequencing analysis to obtain sequence information. However, in an enriched aptamer library, a large amount of sequence information for low-abundance aptamers was not found.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

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Non - Patent Document 8

Non - Patent Document 9

Non - Patent Document 10

Non - Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide a method for sequencing low-abundance aptamers from an aptamer library.

Means for Solving the Problems

[0006] The present disclosure relates, at least in part, to a method for mining sequence information of low-abundance aptamers from an aptamer library. The aptamer library includes high-abundance aptamers (e.g., aptamers with a higher sequence frequency level than the percentage in a sequencing reaction) and low-abundance aptamers (e.g., aptamers with a lower sequence frequency level than the percentage in a sequencing reaction). In some embodiments, during a sequencing reaction, the high-abundance aptamers interfere with the effective analysis of the sequence information of the low-abundance aptamers due to few reads of the low-abundance aptamers (e.g., few reads below the detection limit). In some embodiments, the present disclosure provides a method for reducing high-abundance aptamers (e.g., by an antisense oligonucleotide (ASO) targeting the high-abundance aptamers) such that the low-abundance aptamers are effectively amplified (e.g., by emulsion PCR) and sequenced. In some embodiments, the method may be described as suppression of targeted aptamer clusters (STAC).

[0007] In one aspect, the present disclosure provides a method for sequencing low-abundance aptamers from an aptamer library, the method comprising: (i) amplifying a plurality of aptamers capable of binding to one or more target molecules in a sample by emulsion PCR to generate an aptamer library; (ii) sequencing the aptamer library; and (iii) contacting the aptamer library with a plurality of antisense oligonucleotides (ASOs) that target high-abundance aptamers in the aptamer library to form a mixture, wherein the contact between the ASO and the aptamer library results in the inactivation of the high-abundance aptamers in the aptamer library.

[0008] In some embodiments, the method further comprises steps (a)-(c): (a) contacting a plurality of candidate aptamers with a sample comprising one or more target molecules to form a composition comprising a plurality of aptamer-target molecule complexes; (b) purifying the plurality of aptamer-target molecule complexes; and (c) extracting a plurality of aptamers capable of binding to the one or more target molecules from the aptamer-target molecule complexes, thereby selecting a plurality of aptamers capable of binding to the one or more target molecules in the sample.

[0009] In some embodiments, the method further comprises repeating steps (a)-(c) and (i)-(iii), wherein the mixture obtained from step (iii) comprises the plurality of candidate aptamers for repeating step (a). In some embodiments, the method is repeated at least three times. In some embodiments, the method further comprises sequencing the aptamer library obtained from step (iii).

[0010] In some embodiments, the ASO comprises modified nucleotides.

[0011] In some embodiments, sequencing of the low-abundance aptamers from the aptamer library comprises next-generation sequencing (NGS).

[0012] In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is cultured, serum, plasma, cerebrospinal fluid (CSF), urine, amniotic fluid, bone marrow, bronchoalveolar lavage fluid, oral swab, feces, gastrointestinal fluid, liposuction sample, saliva, milk, nasal swab, peritoneal fluid, semen, sputum, synovial fluid, tears, vaginal fluid, tissue biopsy, cell lysate, vaginal fluid, tissue biopsy, or cell lysate. In some embodiments, the biological sample comprises a target molecule comprising nucleic acid, protein, polypeptide, carbohydrate, lipid, or a combination thereof. In some embodiments, the biological sample is not denatured.

[0013] In some embodiments, the high-abundance aptamers in the aptamer library are aptamers having a sequence frequency level higher than 0.05% in the sequencing reaction of step (ii). In some embodiments, the high-abundance aptamers in the aptamer library are aptamers having a sequence frequency level higher than 0.1% in the sequencing reaction of step (ii). In some embodiments, the high-abundance aptamers in the aptamer library are aptamers having a sequence frequency level higher than 0.15% in the sequencing reaction of step (ii). In some embodiments, the high-abundance aptamers in the aptamer library are aptamers having a sequence frequency level higher than 0.2% in the sequencing reaction of step (ii). In some embodiments, the high-abundance aptamers in the aptamer library are aptamers having a sequence frequency level higher than 0.5% in the sequencing reaction of step (ii).

[0014] In some embodiments, step (b) comprises subjecting the composition to electrophoresis in a first electrophoretic medium in a first direction to obtain a portion of the first electrophoretic medium comprising the aptamer-target molecule complex.

[0015] In some embodiments, step (b) further comprises subjecting a portion of the first electrophoretic medium to electrophoresis in a second electrophoretic medium in a second direction to obtain a portion of the second electrophoretic medium comprising the aptamer-target molecule complex.

[0016] In some embodiments, the first electrophoresis medium is a first agarose gel. In some embodiments, the second electrophoresis medium is a second agarose gel.

[0017] In some embodiments, the first and second electrophoresis media contain sodium ions, potassium ions, lithium ions, ammonium ions, or any combination thereof, at a concentration between 100 mM and 200 mM. In some embodiments, the sodium ions are in the form of sodium chloride.

[0018] In some embodiments, the first and second electrophoresis media contain magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof, at a concentration of 10 mM or less. In some embodiments, the first and second electrophoresis media contain magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof, at a concentration between 0.5 mM and 2 mM. In some embodiments, the first and second electrophoresis media contain magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof, at a concentration of 1 mM. In some embodiments, the magnesium ions are in the form of magnesium chloride.

[0019] In some embodiments, step (iii) further includes cutting out a portion of the first electrophoresis medium containing the aptamer - target molecule complex from the remaining portion of the first electrophoresis medium. In some embodiments, a portion of the first electrophoresis medium is sized to fit into a well in the second electrophoresis medium for performing electrophoresis in the second direction.

[0020] In some embodiments, the electrophoresis in the first direction and the second direction is performed at a temperature between 10 °C and 20 °C.

[0021] In some embodiments, the plurality of candidate aptamers are single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA, or peptides. In some embodiments, the plurality of candidate aptamers are single-stranded DNA (ssDNA).

[0022] In some embodiments, each of the plurality of candidate aptamers comprises modified nucleotides. In some embodiments, each of the plurality of candidate aptamers comprises one or more 5-trifluoroamino-uracils instead of thymine.

[0023] In some embodiments, each of the plurality of candidate aptamers is labeled. In some embodiments, each of the plurality of candidate aptamers is fluorescently labeled.

[0024] In some embodiments, the method further comprises, prior to step (c), cutting out a portion of the second electrophoretic medium containing the aptamer-target molecule complex from the remainder of the second electrophoretic medium, and extracting the aptamer-target molecule complex from the portion of the second electrophoretic medium.

[0025] In some embodiments, the method further comprises denaturing and reconstituting the aptamer library prior to contacting the ASO with the aptamer library.

[0026] In some embodiments, the method further comprises denaturing and reconstituting the aptamer library after contacting the ASO with the aptamer library.

Advantages of the Invention

[0027] The present invention provides a method for sequencing low-abundance aptamers from an aptamer library. This method enables data mining and deeper analysis of the aptamer library.

Brief Description of the Drawings

[0028]

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Mode for Carrying Out the Invention

[0029] The present disclosure relates, at least in part, to a method for mining sequence information of low-abundance aptamers from an aptamer library. The aptamer library includes high-abundance aptamers (e.g., aptamers with a higher sequence frequency level than the percentage in the sequencing reaction) and low-abundance aptamers (e.g., aptamers with a lower sequence frequency level than the percentage in the sequencing reaction). In some embodiments, during the sequencing reaction, the high-abundance aptamers interfere with the effective analysis of the sequence information of the low-abundance aptamers due to the few reads of the low-abundance aptamers (e.g., few reads below the detection limit). In some embodiments, the present disclosure provides a method for reducing high-abundance aptamers (e.g., by an antisense oligonucleotide (ASO) that targets the high-abundance aptamers) so that the low-abundance aptamers can be effectively amplified (e.g., by emulsion PCR) and sequenced.

[0030] As used herein, the term "aptamer" refers to an oligonucleotide (e.g., a single-stranded DNA (ssDNA) molecule or a single-stranded RNA (ssRNA) molecule) that can specifically bind to a target molecule. In some embodiments, the aptamer is a single-stranded DNA aptamer. In some embodiments, the aptamer comprises between 20 and 60 nucleotides, between 25 and 55 nucleotides, between 30 and 50 nucleotides, between 35 and 45 nucleotides, between 20 and 50 nucleotides, between 20 and 40 nucleotides, between 25 and 40 nucleotides, between 20 and 30 nucleotides, between 30 and 40 nucleotides, between 30 and 60 nucleotides, between 40 and 60 nucleotides, or between 50 and 60 nucleotides. In some embodiments, target molecules of the aptamer include proteins, peptides, carbohydrates, small molecules, toxins, and cells (e.g., live cells). Aptamers bind to the aptamer's target with high affinity, selectivity, and specificity (see, e.g., Non-Patent Documents 1 and 2). The binding of an aptamer is determined by the aptamer's tertiary structure rather than its primary sequence. Target recognition and aptamer binding involve three-dimensional, shape-dependent interactions, as well as hydrophobic interactions, base stacking, and intercalation. Aptamers offer advantages over antibodies because they can be fully manipulated in vitro, are readily generated by chemical synthesis, have desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.

[0031] The concept of in vitro evolution of aptamers was introduced in 1990 and named in vitro molecular evolution method, i.e., SELEX (Non-Patent Documents 3 to 7). SELEX combines the rules of combinatorial library screening with in vitro evolution to enrich aptamers (e.g., DNA aptamers) against a wide range of target molecules. The SELEX process involves three interconnected steps: (i) repeated incubation of a plurality of candidate aptamers with one target molecule to bind high-affinity aptamers, (ii) separation of high-affinity binders from low-affinity binders and / or non-binders, and (iii) amplification of high-affinity binders using polymerase chain reaction (PCR). This process is repeated until high-affinity aptamers are enriched in the selection pool.

[0032] In some embodiments, after each round consisting of selection and amplification, the resulting aptamers can be further analyzed, for example, by a binding assay, a diversity assay, or sequencing. In conventional SELEX, the final aptamer library after selection is cloned, and 30 to 100 representatives are sequenced by Sanger sequencing. In this regard, the accurate identification of candidate aptamers is an important point for the success of the overall selection. The aptamer isolation process yields a nucleic acid library in which sequences that specifically bind to the target are enriched. In conventional methods, the resulting enriched nucleic acid pool is cloned, and 30 to 100 clones are then Sanger sequenced for the purpose of determining a small number of aptamer candidates for further detailed characterization. Cluster analysis helps in the identification of aptamer candidates. Usually, the most abundant species, i.e., the representatives of the largest cluster among the sequenced pools, are considered potential aptamers. Many aptamers have been isolated and characterized using this most common approach. In the last decade, next-generation sequencing (NGS) technology has evolved into a common method for high-throughput aptamer sequencing. In some embodiments, the aptamers obtained from each selection round are sequenced by next-generation sequencing (NGS). However, in some cases, the most highly abundant aptamer sequences identified by NGS may not show the best binding to the target, probably due to PCR bias (Non-Patent Document 8). Furthermore, in some embodiments, sequence information for low-level aptamers may not be found due to few reads below the detection level.

[0033] I. Suppression of Targeted Aptamer Clusters The present disclosure provides, at least in part, a method for suppressing a targeted aptamer cluster (e.g., a high-abundance aptamer having a higher sequence frequency level than the percentage in a sequencing reaction) to increase the sequence frequency of a low-abundance aptamer so that it can be detected. In some embodiments, the methods provided herein enable data mining and deeper analysis of aptamer libraries.

[0034] In some aspects, the present disclosure provides a method for sequencing low-abundance aptamers from an aptamer library, the method comprising: (i) amplifying a plurality of aptamers capable of binding to one or more target molecules in a sample by emulsion PCR to generate an aptamer library; (ii) sequencing the aptamer library; and (iii) contacting the aptamer library with a plurality of antisense oligonucleotides (ASOs) that target high-abundance aptamers in the aptamer library to form a mixture, wherein the contact between the ASO and the aptamer library results in inactivation of the high-abundance aptamers in the aptamer library.

[0035] In some embodiments, the plurality of aptamers capable of binding to one or more target molecules in a sample can be a plurality of aptamers enriched for the target molecule by any suitable known method, e.g., by conventional SELEX or any variant thereof, e.g., the SELEX method described in Non-Patent Document 9.

[0036] In some embodiments, a plurality of aptamers capable of binding to one or more target molecules in a sample are amplified to form an aptamer library before being sequenced. In some embodiments, the plurality of aptamers includes different amounts of aptamers (i.e., sequence information of the sequences). In some embodiments, aptamers that bind to the target molecule with high affinity are present in a greater amount than aptamers that bind to the target molecule with low affinity among the plurality of aptamers capable of binding to the target molecule. In some embodiments, it is important to retain the sequence information during amplification so that the sequencing step can accurately identify the high-abundance aptamers in the aptamer library. In some embodiments, if any amplification method (e.g., PCR) can preserve the sequence information in the plurality of aptamers capable of binding to one or more target molecules (e.g., biomolecules) after creating the aptamer library by amplification, this amplification method can be utilized by the methods described herein.

[0037] In some embodiments, the present disclosure is based, at least in part, on the discovery that amplification of a plurality of aptamers capable of binding to one or more target molecules in a sample by emulsion PCR retains information on the sequence of the correct sequence. As used herein, the term "emulsion PCR" refers to a PCR reaction performed in aqueous droplets emulsified in the oil phase of a water-in-oil emulsion. In some embodiments, the aqueous droplets function as miniaturized "reactors" for each PCR reaction, are physically separated from each other, and do not involve the exchange of macromolecules, especially PCR products. Individual DNA molecules are compartmentalized into different reaction droplets of these molecules, allowing amplification of these molecules independently of each other. In emulsion PCR, the formation of non-productive chimeras and other by-products is avoided, and the overall amplification bias is reduced (see, for example, Non-Patent Documents 10 and 11). In some embodiments, the emulsion PCR reaction is prepared by mixing a PCR solution with an emulsion oil. In some embodiments, the emulsion oil contains an oil (e.g., mineral oil). In some embodiments, the emulsion oil contains an oil (e.g., mineral oil) at a concentration between 90% and 98%, between 91% and 97%, between 92% and 96%, between 93% and 95%, between 94% and 96%, between 90% and 95%, between 91% and 96%, between 92% and 96%, between 93% and 96%, between 94% and 97%, or between 95% and 96%. In some embodiments, the emulsion oil contains 95.05% oil (e.g., mineral oil). In some embodiments, the emulsion PCR reaction is vigorously shaken to form water-in-oil emulsion droplets. In some embodiments, prior to performing the amplification step, a pre-run of the emulsion PCR is performed in a fixed number of cycles, and the PCR products are analyzed to select an appropriate PCR cycle that results in a single population of PCR products (e.g., shown as a single band evident by DNA electrophoresis). In some embodiments, the emulsion PCR is performed for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more cycles to amplify a plurality of aptamers.

[0038] In some embodiments, the method further includes sequencing the aptamers within the aptamer library generated from emulsion PCR amplification. As used herein, the term "sequencing" refers to the process of determining the order of nucleotides of a given nucleic acid fragment with respect to a nucleic acid. Methods for sequencing nucleic acids (e.g., aptamers) are known in the art and include, but are not limited to, basic sequencing (e.g., Maxam-Gilbert sequencing), chain termination sequencing (e.g., Sanger sequencing), large scale sequencing and de novo sequencing (e.g., shotgun sequencing), next generation sequencing (e.g., single molecule real time sequencing), ion torrent sequencing, pyrosequencing, sequencing by synthesis (e.g., MiSeq), combinatorial probe anchor synthesis, sequencing by ligation (SOLiD sequencing), nanopore sequencing, GenapSys sequencing, or chain termination (Sanger sequencing), long read sequencing, short read sequencing methods (e.g., massively parallel signature sequencing (MPSS), polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, microfluidics systems). In some embodiments, the aptamers are sequenced by next generation sequencing (e.g., MiSeq).

[0039] In some embodiments, the sequencing data of the aptamer library (e.g., NGS data) includes regions of high frequency corresponding to high-abundance aptamers in the aptamer library and regions of low frequency corresponding to low-abundance aptamers in the aptamer library. In some embodiments, the high-abundance aptamers are aptamers having a sequence frequency level higher than 0.05%, higher than 0.06%, higher than 0.07%, higher than 0.08%, higher than 0.09%, higher than 0.10%, higher than 0.11%, higher than 0.12%, higher than 0.13%, higher than 0.14%, higher than 0.15%, higher than 0.16%, higher than 0.17%, higher than 0.18%, higher than 0.19%, higher than 0.20%, higher than 0.21%, higher than 0.22%, higher than 0.23%, higher than 0.24%, higher than 0.25%, higher than 0.26%, higher than 0.27%, higher than 0.28%, higher than 0.29%, higher than 0.30%, higher than 0.31%, higher than 0.32%, higher than 0.33%, higher than 0.34%, higher than 0.35%, higher than 0.36%, higher than 0.37%, higher than 0.38%, higher than 0.39, higher than 0.40%, higher than 0.41%, higher than 0.42%, higher than 0.43%, higher than 0.44%, higher than 0.45%, higher than 0.46%, higher than 0.47%, higher than 0.48%, higher than 0.49, higher than 0.50%, or exceeding it. In some embodiments, the sequence information of the low-abundance aptamers is below the detection limit, and thus the sequence information of these aptamers cannot be obtained. In some embodiments, the high-abundance aptamers interfere with the efficient sequencing analysis of the low-abundance aptamers.

[0040] The present disclosure is based, at least in part, on the discovery that sequence information of low-abundance aptamers can be obtained in a subsequent selection round by removing high-abundance aptamers from an aptamer library. In some embodiments, the methods provided herein further include contacting an aptamer library with a plurality of antisense oligonucleotides (ASOs) that target high-abundance aptamers within the aptamer library to form a mixture. In some embodiments, the ASOs that target high-abundance aptamers within the aptamer library are designed according to sequence information of the high-abundance aptamers obtained from a sequencing reaction.

[0041] As used herein, the term "antisense oligonucleotide (ASO)" refers to an oligomeric compound, at least a portion of which is at least partially complementary to an aptamer to which the antisense oligonucleotide hybridizes, and such hybridization results in at least one antisense activity (e.g., inactivation of the aptamer).

[0042] In some embodiments, the ASO targeting the aptamer is designed to cause a conformational change in the aptamer such that the aptamer can no longer bind to its target molecule. In some embodiments, the ASO targeting the aptamer comprises a complementary region to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of the aptamer. In some embodiments, the ASO targeting the aptamer comprises a complementary region to any one of the highly abundant aptamers in the aptamer library. In some embodiments, the ASO targeting the aptamer comprises a complementary region to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of the nucleotide sequence of any one of the highly abundant aptamers in the aptamer library. In some embodiments, the ASO targeting the aptamer comprises a complementary region to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of the aptamer, excluding at least 1, at least 2, at least 3, at least 4, or at least 5 mismatches.

[0043] ASOs can be of various, different lengths. In some embodiments, the oligonucleotide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, the ASO is 25-30 nucleotides in length, 8-50 nucleotides in length, 8-40 nucleotides in length, 8-30 nucleotides in length, 10-15 nucleotides in length, 10-20 nucleotides in length, 15-25 nucleotides in length, or 21-23 nucleotides in length.

[0044] In some embodiments, for the purposes of the present disclosure, an ASO specifically hybridizes to (e.g., has complementarity to) an aptamer if the binding of the ASO to the aptamer mRNA interferes with the normal function of the aptamer, resulting in a loss of activity (e.g., inhibition of binding of the aptamer to its target molecule), and there is a sufficient degree of complementarity to avoid non-specific binding under conditions where it is desirable to avoid non-specific binding to non-target aptamers, such as under appropriate stringency conditions under which the assay is performed. Thus, in some embodiments, the ASO can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to consecutive nucleotides of the aptamer. In some embodiments, the ASO need not be 100% complementary to a continuous region of the aptamer in order to be capable of specifically hybridizing to the aptamer or to be specific for the aptamer.

[0045] In some embodiments, one or more of the thymine bases (T) in any one of the ASOs may optionally be uracil bases (U), and / or one or more of the Us may optionally be Ts.

[0046] The antisense oligonucleotides described herein may be modified, for example, to include modified sugar moieties, modified internucleotide linkages, modified nucleobases, modified nucleotides, and / or (e.g., and) combinations thereof.

[0047] In some embodiments, the antisense oligonucleotides described herein include at least one nucleoside modified at the 2'-position of the sugar. In some embodiments, the oligonucleotide includes at least one 2'-modified nucleoside. In some embodiments, all of the nucleosides within the oligonucleotide are 2'-modified nucleosides.

[0048] In some embodiments, the antisense oligonucleotides described herein include one or more non-bicyclic 2'-modified nucleosides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamide (2'-O-NMA) modified nucleosides. In some embodiments, the antisense oligonucleotide includes one or more 2'-O-methoxyethyl (2'-MOE) modified nucleosides. In some embodiments, each of the nucleosides of the antisense oligonucleotide is a 2'-O-methoxyethyl (2'-MOE) modified nucleoside. In some embodiments, the antisense oligonucleotides described herein include one or more 2'-4' bicyclic nucleosides, in which the ribose ring includes a bridging moiety connecting two atoms within the ring, such as a methylene (LNA) bridge, an ethylene (ENA) bridge, or an (S)-constrained ethyl (cEt) bridge, connecting the 2'-O atom to the 4'-C atom. Examples of LNA are described in International Publication No. WO 2008 / 043753, published on April 17, 2008, with the title "RNA Antagonist Compounds For The Modulation Of PCSK9", the contents of which are incorporated herein by reference in their entirety.Examples of ENA are provided in International Publication No. WO 2005 / 042777, published May 12, 2005, entitled "APP / ENA Antisense"; Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al., Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8:144-149, 2006; and Horie et al., Nucleic Acids Symp. Ser (Oxf), 49:171-172, 2005, the disclosures of which are incorporated herein by reference in their entirety. Examples of cEt are provided in U.S. Patent No. 7,101,993, U.S. Patent No. 7,399,845, and U.S. Patent No. 7,569,686, each of which is incorporated herein by reference in its entirety.

[0049] In some embodiments, the antisense oligonucleotide comprises a modified nucleoside disclosed in one of the following U.S. patents or published patent applications, the entire contents of each of which are incorporated herein by reference: U.S. Patent No. 7,399,845, entitled "6-Modified Bicyclic Nucleic Acid Analogs", issued July 15, 2008; U.S. Patent No. 7,741,457, entitled "6-Modified Bicyclic Nucleic Acid Analogs", issued June 22, 2010; U.S. Patent No. 8,022,193, entitled "6-Modified Bicyclic Nucleic Acid Analogs", issued September 20, 2011; U.S. Patent No. 7,569,686, entitled "Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs", issued August 4, 2009; U.S. Patent No. 7,335,765, entitled "Novel Nucleoside And Oligonucleotide Analogues", published February 26, 2008; U.S. Patent No. 7,314,923, entitled "Novel Nucleoside And Oligonucleotide Analogues", issued January 1, 2008; U.S. Patent No. 7,816,333, entitled "Oligonucleotide Analogues And Methods Utilizing The Same", issued October 19, 2010; and U.S. Patent Application Publication No. 2011 / 0009471, entitled "Oligonucleotide Analogues And Methods Utilizing The Same", published February 17, 2015, now U.S. Patent No. 8,957,201.

[0050] In some embodiments, the antisense oligonucleotide may have phosphorothioate or other modified internucleotide linkages. In some embodiments, the antisense oligonucleotide comprises phosphorothioate internucleotide linkages. In some embodiments, the antisense oligonucleotide comprises a phosphorothioate internucleotide linkage between at least two nucleotides. In some embodiments, the antisense oligonucleotide comprises phosphorothioate internucleotide linkages between all nucleotides. For example, in some embodiments, the antisense oligonucleotide comprises modified internucleotide linkages at the first, second, and / or (e.g., and) third internucleotide linkages at the 5' end or 3' end of the nucleotide sequence.

[0051] Examples of phosphorus-containing linkages that can be used include, but are not limited to, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, methylphosphonate and other alkylphosphonates including 3'-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'-aminophosphoramidate and aminoalkyl phosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, and boranophosphate having a normal 3'-5' linkage, 2'-5' linkage analogs of these boranophosphates, and boranophosphates having an opposite polarity in which adjacent nucleoside unit pairs are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. See U.S. Patent No. 3,687,808, U.S. Patent No. 4,469,863, U.S. Patent No. 4,476,301, U.S. Patent No. 5,023,243, U.S. Patent No. 5,177,196, U.S. Patent No. 5,188,897, U.S. Patent No. 5,264,423, U.S. Patent No. 5,276,019, U.S. Patent No. 5,278,302, U.S. Patent No. 5,286,717, U.S. Patent No. 5,321,131, U.S. Patent No. 5,399,676, U.S. Patent No. 5,405,939, U.S. Patent No. 5,453,496, U.S. Patent No. 5,455,233, U.S. Patent No. 5,466,677, U.S. Patent No. 5,476,925, U.S. Patent No. 5,519,126, U.S. Patent No. 5,536,821, U.S. Patent No. 5,541,306, U.S. Patent No. 5,550,111, U.S. Patent No. 5,563,253, U.S. Patent No. 5,571,799, U.S. Patent No. 5,587,361, and U.S. Patent No. 5,625,050.

[0052] In some embodiments, the method includes designing a plurality of ASOs that target a plurality of high-abundance aptamers (e.g., aptamers having an array frequency level higher than 0.1%). In some embodiments, contacting the plurality of ASOs that target the plurality of high-abundance aptamers results in inactivation of the high-abundance aptamers such that the aptamers can no longer bind to the target molecule. In some embodiments, contacting the plurality of ASOs that target the plurality of high-abundance aptamers results in inactivation of the high-abundance aptamers such that the aptamers can no longer bind to the target molecule. In some embodiments, contacting the plurality of ASOs that target the plurality of high-abundance aptamers results in a decrease or knockdown of the high-abundance aptamers within the aptamer library. In some embodiments, contacting the plurality of ASOs that target the plurality of high-abundance aptamers results in a decrease or knockdown of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the high-abundance aptamers within the aptamer library.

[0053] In some embodiments, the aptamer library is denatured and reconfigured to promote the formation of the 3D structure of the aptamer. In some embodiments, denaturation of the aptamer library includes heating the aptamer library at a temperature sufficient to disrupt the 3D structure of the aptamer for a certain period of time. In some embodiments, denaturation of the aptamer library includes heating the aptamer library at a temperature of 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C, or higher for at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, or longer. In some embodiments, denaturation of the aptamer library includes heating the aptamer library at 95°C for 3 minutes. In some embodiments, the denatured aptamer is reconfigured to form the correct 3D structure. In some embodiments, reconstitution of the aptamer within the aptamer library includes cooling the aptamer library at a certain temperature to a certain temperature over a certain period of time to reform the 3D structure of the aptamer. In some embodiments, the methods described herein include contacting the ASO with the aptamer library prior to the step of denaturing the aptamer library. Contact between the ASO and the aptamer library prior to denaturation results in a more potent knockdown efficiency but does not result in a higher non-specific knockdown. In some embodiments, the methods described herein include contacting the ASO with the aptamer library after the step of reconstituting the aptamer library. Contact between the ASO and the aptamer library prior to denaturation results in a higher specific knockdown.

[0054] In some embodiments, the method described herein includes the following steps: contacting a plurality of candidate aptamers with a sample containing one or more target molecules to form a composition comprising a plurality of aptamer-target molecule complexes; purifying the plurality of aptamer-target molecule complexes; and extracting from the aptamer-target molecule complexes a plurality of aptamers that can bind to one or more of the target molecules, further comprising the step of enriching a plurality of aptamers that can bind to one or more target molecules in the sample.

[0055] As used herein, the term "aptamer-target molecule complex" refers to a molecular complex formed between an aptamer and the aptamer's target molecule via specific binding. As used herein, the term "specific binding" refers to the ability of a molecule (e.g., an aptamer) to bind a binding partner (e.g., a target molecule) with an affinity or binding strength that allows the molecule to be used to distinguish the binding partner from appropriate controls in a binding assay or other binding context. With respect to an aptamer, the term "specific binding" refers to the ability of the aptamer to bind a target molecule with an affinity or binding strength that allows the aptamer to be used to distinguish the specific target molecule from other molecules when compared to one or more appropriate reference target molecules. In some embodiments, an aptamer specifically binds a target molecule if it has a K for binding the target molecule of at least about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, or less. D When present, the aptamer specifically binds the target molecule.

[0056] As used herein, the term "candidate aptamer" refers to a pool of random aptamers or random oligonucleotides that can be aptamers, which are subjected to the methods described herein for the enrichment of aptamers that can bind to one or more target molecules (e.g., biomolecules) in a sample (e.g., a biological sample).

[0057] The starting pool of candidate aptamer sequences contains a random core sequence of a species 20 - 60 nt in length. In some embodiments, the core sequence is flanked by regions (e.g., primer binding sites) used for re - amplification of the library. In some embodiments, it is important to have a high diversity in the first candidate aptamer pool due to the possibility of the presence of target - binding aptamers in the first candidate aptamers.

[0058] In some embodiments, in the generation of the first plurality of candidate aptamers, the nucleotide ratios are optimized at, for example, A:C:G:T of 1.0:1.0:1.0:1.0, 1.5:1.5:1.0:1.2, 1.30:1.25:1.45:1.00, or 1.50:1.25:1.15:1.00. In some embodiments, the plurality of candidate aptamers contain at least 10 14 at least 10 15 at least 10 16 at least 10 17 at least 10 18 at least 10 19 at least 10 20 or more candidate aptamers.

[0059] In some embodiments, the candidate aptamer is an initial pool of unselected, random aptamers or random oligonucleotides that can be aptamers. In some embodiments, the candidate aptamer is a plurality of aptamers that can bind to one or more target molecules (e.g., biomolecules) in a sample (e.g., a biological sample) obtained as a result of the final enrichment round using the methods described herein. In some embodiments, the plurality of candidate aptamers are DNA or RNA. In some embodiments, the candidate aptamer is single-stranded DNA (ssDNA). In some embodiments, the plurality of candidate aptamers are folded into their proper tertiary structure to bind the target molecule. In some embodiments, the plurality of candidate aptamers include modified nucleotides. Modified nucleotides have been described previously; see, e.g., Non-Patent Document 12. In some embodiments, each of the plurality of candidate aptamers includes at least one modified nucleotide. In some embodiments, each of the plurality of candidate aptamers includes a modified nucleotide (e.g., a modified nucleotide that can be used as a substrate by DNA polymerase). Modified nucleotides are well known in the art (see, e.g., Patent Document 1 and Non-Patent Documents 13 and 14).In some embodiments, each of the plurality of candidate aptamers includes, but is not limited to, for example, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueuosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutosine, pseudouracil, queuosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl ester of uracil-5-oxyacetic acid, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, 2,6-diaminopurine, or 5-trptamino-uracil. In some embodiments, the modified nucleotide is a 2'-modified nucleotide. For example, a nucleotide modified at 2' is a 2'-deoxy, 2'-fluoro, 2'-O-methoxyethyl, 2'-amino, and 2'-aminoalkoxy modified nucleotide. In some embodiments, each of the plurality of candidate aptamers includes, instead of thymine, but is not limited to, one or more 5-trptamino-uracil. In some embodiments, at least one thymine in each of the plurality of candidate aptamers is replaced by 5-trptamino-uracil. In some embodiments, all thymines in each of the plurality of candidate aptamers are replaced by 5-trptamino-uracil.

[0060] In some embodiments, each of the plurality of candidate aptamers includes a detectable label. The detectable label may facilitate the detection of an aptamer-target molecule (e.g., a biomolecule) complex. In some embodiments, the detectable label is a protein that can cause a change in colorimetric analysis. In some embodiments, the protein that can cause a change in colorimetric analysis is alkaline phosphatase, horseradish peroxidase, or luciferase. In some embodiments, the detectable label is a fluorescent molecule. In some embodiments, the fluorescent molecule includes, but is not limited to, a fluorescent dye, and the fluorescent dye is TYE665, lucifer yellow, dansyl, TruRed, fluorescein, Cy2, Cy3, Cy7, TRITC, X-rhodamine or Texas red, green fluorescent protein (GFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), mCherry, mTurquoise2, or mOrange. In some embodiments, each of the plurality of candidate aptamers is labeled with a phosphate group at the 5' end. In some embodiments, each of the plurality of candidate aptamers is labeled with a hydroxyl group at the 3' end of the candidate aptamer. In some embodiments, each of the plurality of candidate aptamers includes one or more detectable labels. In some embodiments, each of the plurality of candidate aptamers includes a TYE665 fluorophore at the 5' end.

[0061] In some embodiments, a plurality of candidate aptamers are prepared in a selection buffer before contacting the target molecule (e.g., a biomolecule) in a sample (e.g., a biological sample). In some embodiments, the selection buffer contains Nonidet-P40. In some embodiments, the selection buffer contains Nonidet-P40 at a concentration between 0.001% and 0.01%. In some embodiments, the selection buffer contains Nonidet-P40 at a concentration of 0.005%. In some embodiments, the selection buffer contains a salt (e.g., magnesium ions) that can stabilize the tertiary structure of the aptamer. In some embodiments, in some embodiments, the selection buffer contains magnesium ions at a concentration between 0.1 mM and 10 mM, between 1 mM and 10 mM, between 5 mM and 10 mM, between 8 mM and 10 mM, between 6 mM and 8 mM, between 2 mM and 8 mM, between 0.1 mM and 5 mM, between 0.5 mM and 4.5 mM, between 1 mM and 4 mM, between 2 mM and 3 mM, between 0.5 mM and 2 mM, between 0.6 mM and 1.5 mM, between 0.7 mM and 1.3 mM, between 0.8 mM and 1.2 mM, between 0.9 mM and 1.1 mM, between 0.6 mM and 1.2 mM, between 0.6 mM and 1.2 mM, between 0.7 mM and 1.2 mM, between 0.8 mM and 1.2 mM, between 0.9 mM and 1 mM, between 0.8 mM and 1 mM, between 0.9 mM and 1.5 mM, between 0.9 mM and 1.2 mM, or between 1 mM and 2 mM. In some embodiments, the selection buffer contains magnesium at a concentration of 1 mM. In some embodiments, the magnesium ions are in the form of magnesium chloride.In some embodiments, the plurality of candidate aptamers are prepared in a selection buffer at a concentration between 50 nM and 8000 nM, between 60 nM and 7000 nM, between 50 nM and 6000 nM, between 50 nM and 8000 nM, between 50 nM and 8000 nM, between 50 nM and 8000 nM, between 50 nM and 5000 nM, between 60 nM and 4000 nM, between 70 nM and 3000 nM, between 80 nM and 2000 nM, between 90 nM and 1000 nM, between 100 nM and 1000 nM, between 50 nM and 100 nM, between 100 nM and 200 nM, between 200 nM and 500 nM, between 500 nM and 1000 nM, between 1000 nM and 2000 nM, between 2000 nM and 3000 nM, between 3000 nM and 4000 nM, between 4000 nM and 5000 nM, between 1000 nM and 5000 nM, between 1000 nM and 2500 nM, or between 2500 nM and 5000 nM.

[0062] In some embodiments, the enrichment of the plurality of aptamers is also referred to as the selection of the plurality of aptamers. The terms "enrich," "enrichment," or "enrichment process" are used interchangeably with "select," "selection," or "selection process," respectively. In some embodiments, the enrichment of the plurality of aptamers includes selecting aptamers that can bind to one or more target molecules (e.g., biomolecules) from aptamers that do not bind to one or more target molecules (plural) (e.g., biomolecules). In some embodiments, the enrichment of the plurality of aptamers includes selecting aptamers that have a higher affinity for one or more target molecules (e.g., biomolecules) than aptamers that have a lower affinity for one or more target molecules (plural) (e.g., biomolecules).

[0063] In some embodiments, the methods described herein include contacting a plurality of candidate aptamers with a sample. In some embodiments, the sample includes one or more target molecules (e.g., proteins, nucleic acids, toxins, and / or small molecules). In some embodiments, the sample includes one target molecule. In some embodiments, the sample includes two or more target molecules.

[0064] In some embodiments, the methods described herein include contacting a plurality of candidate aptamers with a complex sample (e.g., a biological sample) that includes a plurality of different target molecules. In some embodiments, the complex sample includes a plurality of different target molecules of the same type (e.g., the complex sample contains a plurality of different proteins). In some embodiments, the complex sample contains a plurality of different types of target molecules (e.g., the complex sample contains proteins, nucleic acids, small molecules, toxins, etc.). In some embodiments, the complex sample contains different types of target molecules, and each type of target molecule further contains different individual target molecules (e.g., the complex sample contains a plurality of different proteins, a plurality of different nucleic acids, a plurality of different small molecules, and a plurality of different toxins, etc.). Examples of complex samples include, but are not limited to, biological samples (e.g., biological fluids such as serum), environmental samples (e.g., samples obtained from rivers, lakes, ponds, soil, air, outer space, etc.), manufacturing samples (e.g., samples obtained from bioreactors, samples obtained from HPLC flow-through fluids, samples containing intermediates of small molecule drugs, etc.). In some embodiments, the complex sample is a biological sample. As used herein, the term "biological sample" refers to a sample obtained from a biological subject.

[0065] Examples of biological samples include, but are not limited to, whole blood, interstitial fluid, skin, lymph, bile, serum, plasma, cerebrospinal fluid (CSF), urine, amniotic fluid, bone marrow, bronchoalveolar lavage fluid, oral swabs, feces, gastrointestinal fluid, liposuction samples, saliva, milk, nasal swabs, peritoneal fluid, semen, sputum, synovial fluid, tears, vaginal fluid, tissue biopsies, autopsy samples, cells or cell lysates, cultured cells, tissue samples (e.g., tissue samples derived from human, non-human animals, plants, insects, fungi), or in vivo endothelial cells. In some embodiments, the biological sample comprises a target molecule (e.g., a biomolecule), such as a nucleic acid (e.g., DNA and RNA), protein, peptide, lipid, polysaccharide, proteoglycan, and glycolipid. In some embodiments, the biological sample is serum. In some embodiments, the biological sample is obtained from a human subject. In some embodiments, the biological sample is obtained from a non-human subject. Examples of non-human subjects include, but are not limited to, monkeys, mice, rats, rabbits, goats, sheep, dogs, birds, and fish. In some embodiments, the subject is a healthy subject. In some embodiments, the subject is a subject suffering from a disease, suspected of suffering from a disease, or at risk of developing a disease.

[0066] In some embodiments, the target molecules (e.g., biomolecules) in a sample (e.g., a biological sample) are denatured prior to contact with a plurality of candidate aptamers. In some embodiments, the target molecules (e.g., biomolecules) in a sample (e.g., a biological sample) are not denatured prior to contact with a plurality of candidate aptamers. As used herein, the terms “denature,” “denaturing,” or “denatured” refer to subjecting a sample (e.g., a biological sample) to conditions that disrupt linkages (e.g., disulfide bridges), bonds (e.g., hydrogen bonds, ionic bonds, etc.), and / or interactions (e.g., hydrophobic interactions) within one or more target molecules (e.g., biomolecules such as proteins and / or nucleic acids) in the sample that render the target molecules in their native state (e.g., biomolecules such as proteins) into a highly ordered structure. In some embodiments, a denatured sample includes a sample in which the biomolecules (e.g., proteins or nucleic acids) in the sample have lost their quaternary, tertiary, and secondary structures, and thus retain only their primary structures (e.g., linear amino acid sequences or nucleic acid sequences), such that the biomolecules do not retain their respective structures and / or assigned functions. In some embodiments, a denatured sample includes a sample in which the biomolecules (e.g., proteins or nucleic acids) have been degraded into fragments, such that the biomolecules no longer retain their structures and / or assigned functions. Thus, a denatured sample includes biomolecules (e.g., proteins or nucleic acids) that do not retain their assigned functions (e.g., binding ability, biological activity, etc.). Conversely, a non-denatured sample is a sample that contains target molecules (e.g., biomolecules) that retain their native three-dimensional structures and their respective assigned functions. Methods for denaturing target molecules in a sample are known in the art and include, for example, heating, treatment with an alkali, an acid, urea, or a surfactant, or vigorous shaking. In some embodiments, the target molecules (e.g., biomolecules) are in their native three-dimensional structures.In some embodiments, contacting a plurality of candidate aptamers with a target molecule (e.g., a biomolecule) in its native three-dimensional structure is advantageous in that the selected aptamer may bind to the target molecule (e.g., a biomolecule) of the aptamer under conditions other than those under which the aptamer is selected (e.g., in vivo in a subject). One of ordinary skill in the art will understand that denaturation involves subjecting the target molecule (e.g., a biomolecule) in a sample (e.g., a biological sample) to conditions (e.g., heating, sonication, incubation in the presence of a surfactant, etc.) sufficient to cause some change in the native or natural structure of the target molecule (e.g., a biomolecule, such as a protein, DNA, RNA, toxin, or small molecule) such that the target molecule can no longer perform its assigned function. The term "undenatured" as used herein refers to maintaining a structure of the target molecule (e.g., a biomolecule) in a sample (e.g., a biological sample) sufficient to perform its assigned function. In some embodiments, the candidate aptamers of the present disclosure can be contacted with an undenatured biological sample. In some embodiments, the candidate aptamers of the present disclosure can be contacted with a sample containing biomolecules (e.g., proteins, DNA, RNA, toxins, or small molecules) that retain their quaternary, tertiary, and secondary structures, as well as their respective assigned functions.

[0067] The present disclosure recognizes the difficulty of concentrating aptamers that can bind to target molecules in their native three-dimensional structures from a complex sample (e.g., a biological sample). The present disclosure also identifies the disadvantages of using one-dimensional electrophoresis (1D electrophoresis) to concentrate aptamer-target molecule (e.g., biomolecule) complexes and / or performing electrophoresis under mild conditions (e.g., low salt conditions). For example, although not wishing to be bound by any particular theory, 1D electrophoresis cannot separate aptamer-target molecule complexes from aptamers that are bound to target molecules by non-specific binding. With respect to an aptamer, as used herein, the term "non-specific binding" refers to the ability of the aptamer to bind to a molecule with an affinity or binding strength that does not allow the aptamer to be used to distinguish that molecule from other molecules when compared to one or more appropriate reference target molecules. In some embodiments, an aptamer is non-specifically bound to a molecule if it has a K -6 for binding molecules of 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M, 10 -1 M, or greater than that. D

[0068] In addition, the conditions of 1D electrophoresis may not be sufficient to disrupt non-specific binding between a particular aptamer and a target molecule. The present disclosure has thus sought to concentrate multiple aptamers that can bind to one or more biomolecules in a biological sample by concentrating aptamer-target molecule complexes by two-dimensional electrophoresis (2D electrophoresis). 2D electrophoresis can be performed under salt conditions that are sufficient to disrupt non-specific interactions between aptamers and target molecules but not sufficient to disrupt specific binding between an aptamer and its target molecule. Thus, the present disclosure enables the simultaneous concentration of multiple aptamers that can bind to multiple target molecules (e.g., biomolecules) in a complex sample (e.g., a biological sample). ​In some embodiments, the sample (e.g., a biological sample) is not diluted before being contacted with a plurality of candidate aptamers. In some embodiments, the sample (e.g., a biological sample) is diluted before being contacted with a plurality of candidate aptamers. In some embodiments, the biological sample is diluted at a ratio between 1:1000 and 1:1, between 1:900 and 1:2, between 1:800 and 1:2, between 1:700 and 1:2, between 1:600 and 1:2, between 1:500 and 1:2, between 1:400 and 1:2, between 1:300 and 1:2, between 1:200 and 1:2, between 1:100 and 1:2, between 1:50 and 1:2, between 1:25 and 1:2, between 1:10 and 1:2, between 1:5 and 1:2, between 1:500 and 1:50, between 1:500 and 1:100, between 1:500 and 1:200, between 1:200 and 1:100, between 1:200 and 1:50, between 1:200 and 1:10, between 1:100 and 1:50, between 1:100 and 1:10, between 1:100 and 1:5, or between 1:100 and 1:2 before contacting the biological sample with the plurality of candidate aptamers. In some embodiments, the biological sample is serum, and the serum is diluted at a ratio between 1:500 and 1:10, between 1:500 and 1:50, between 1:500 and 1:100, between 1:300 and 1:10, between 1:300 and 1:50, between 1:300 and 1:100, between 1:300 and 1:200, between 1:200 and 1:100, between 1:200 and 1:150, between 1:200 and 1:50, between 1:250 and 1:200, or between 1:200 and 1:100, between 1:200 and 1:150, or between 1:200 before contacting the serum with the plurality of candidate aptamers. In some embodiments, the serum is diluted at a ratio of 1:200 before being contacted with the plurality of candidate aptamers. In some embodiments, the biological sample is CSF, and the CSF is diluted at a ratio between 1:50 and 1:2, between 1:40 and 1:2, between 1:30 and 1:2, between 1:20 and 1:2, between 1:10 and 1:2, between 1:5 and 1:2, between 1:4 and 1:2, between 1:3 and 1:2, between 1:20 and 1:5, or between 1:10 and 1:5 before contacting the biological sample with the plurality of candidate aptamers. In some embodiments, the biological sample is diluted in any suitable dilution buffer before contacting the biological sample with the plurality of candidate aptamers.Non-limiting examples of dilution buffers include phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline (DPBS), Hank's balanced salt solution (HBSS), and Dulbecco's modified Eagle's medium (DMEM). One of ordinary skill in the art can select an appropriate dilution buffer according to the biological sample being used.

[0069] In some embodiments, the method further includes contacting the sample (e.g., a biological sample) with a plurality of competing nucleic acids prior to contacting the sample (e.g., a biological sample) with a plurality of candidate aptamers. The competing nucleic acids are used to block non-specific binding between the aptamer and the target molecule. In some embodiments, the competing nucleic acids are a random set of unrelated nucleic acids. In some embodiments, the competing nucleic acids are salmon sperm DNA.

[0070] In some embodiments, when a plurality of candidate aptamers are contacted with a sample (e.g., a biological sample), the aptamers bind to the target molecule of the aptamer (e.g., a biomolecule) to produce a composition. In some embodiments, the composition includes an aptamer-target molecule (e.g., aptamer-biomolecule) complex. In some embodiments, the composition includes unbound aptamers. In some embodiments, the composition includes unbound target molecules (e.g., unbound biomolecules). In some embodiments, the composition includes an aptamer-target molecule (e.g., aptamer-biomolecule) complex, unbound aptamers, and / or unbound target molecules (e.g., unbound biomolecules). In some embodiments, the composition also includes aptamers that bind to biomolecules by non-specific binding.

[0071] In some embodiments, the methods described herein include separating an aptamer-target molecule (e.g., aptamer-biological molecule) complex from unbound aptamer. In some embodiments, the methods described herein include separating an aptamer-target molecule (e.g., aptamer-biological molecule) complex from unbound aptamer. In some embodiments, the methods described herein include separating an aptamer-target molecule (e.g., aptamer-biological molecule) complex from aptamers that are bound to a biological molecule by non-specific binding. In some embodiments, the method includes concentrating an aptamer-target molecule (e.g., aptamer-biological molecule complex) from a composition that also includes unbound aptamer and / or aptamers that are bound to a target molecule by non-specific binding.

[0072] In some embodiments, the step of concentrating an aptamer-target molecule (e.g., an aptamer-biological molecule complex) from a composition that also includes unbound aptamer and / or aptamer bound to the target molecule by non-specific binding involves subjecting the composition to electrophoresis. As used herein, the term "electrophoresis" refers to a technique used to separate molecules (e.g., DNA, RNA, proteins, aptamer-target molecule complexes) based on the size and charge of the molecules to be separated. An electrophoresis system includes two electrodes (an anode and a cathode) of opposite charge connected by a conductive electrophoresis medium. An electric current is used to move and separate the molecules within the electrophoresis medium. In some embodiments, a negative charge is applied, such that the molecules move towards the positive charge. Further, the electrophoresis medium typically has pores that allow smaller molecules to move faster than larger molecules. In some embodiments, the present disclosure is based on the theory that multiple aptamer-target molecule (e.g., biological molecule) complexes are of a different size than unbound aptamer (e.g., biological molecule). In some embodiments, the unbound aptamer is smaller in size than the aptamer-target molecule (e.g., biological molecule) and thus moves faster during electrophoresis. In some embodiments, the individual aptamer-target molecules (e.g., biological molecules) within the multiple aptamer-target molecule (e.g., biological molecule) complexes are of different sizes from each other. In some embodiments, the individual aptamer-target molecules (e.g., biological molecules) within the multiple aptamer-target molecule (e.g., biological molecule) complexes move at different speeds during electrophoresis. In some embodiments, the methods described herein include subjecting the composition to electrophoresis in a first electrophoresis medium in a first direction. In some embodiments, the size difference between the aptamer-target molecule (e.g., biological molecule) complex and the aptamer bound to the target molecule (e.g., biological molecule) by non-specific binding is not sufficient to separate the aptamer-target molecule (e.g., biological molecule) complex from the aptamer bound to the target molecule (e.g., biological molecule) by non-specific binding.In some embodiments, after electrophoresis in a first direction, a portion of the first electrophoretic medium contains an aptamer-target molecule complex and may also contain aptamers that are bound to a target molecule (e.g., a biomolecule) by non-specific binding.

[0073] Accordingly, in some embodiments, the present disclosure attempts to enrich a plurality of aptamers that can bind to one or more biomolecules in a biological sample by enriching an aptamer-target molecule (e.g., biomolecule) complex by 2D electrophoresis. In some embodiments, the method includes subjecting a portion of a first electrophoretic medium containing an aptamer-target molecule complex, which may also contain aptamers that are bound to a target molecule (e.g., a biomolecule) by non-specific binding, to electrophoresis in a second electrophoretic medium in a second direction. As used herein, the term "second direction" refers to a direction different from the first direction. In some embodiments, the second direction is perpendicular to the first direction. In some embodiments, the method further includes excising a portion of the first electrophoretic medium containing the aptamer-target molecule complex, which may also contain aptamers that are bound to a target molecule (e.g., a biomolecule) by non-specific binding, from the remainder of the first electrophoretic medium. In some embodiments, the method further includes aligning the excised portion of the first electrophoretic medium containing the aptamer-target molecule complex, which may also contain aptamers that are bound to a target molecule (e.g., a biomolecule) by non-specific binding, with a well in the second electrophoretic medium. Subjecting the composition to electrophoresis in two dimensions disrupts the weak non-specific binding between the aptamer and the target molecule (e.g., a biomolecule), such that these aptamers become unbound aptamers and migrate faster than the aptamer-target molecule (e.g., biomolecule) complex. In some embodiments, the aptamer-target molecule (e.g., biomolecule) complex is present in an oblique region within the second electrophoretic medium after electrophoresis in the second direction. In some embodiments, the method further includes excising a portion of the second electrophoretic medium containing the aptamer-target molecule complex from the remainder of the second electrophoretic medium.

[0074] In some embodiments, the method further includes extracting an aptamer capable of binding to one or more target molecules (e.g., biomolecules) from a portion of a second electrophoretic medium containing an aptamer-target molecule complex. In some embodiments, the aptamer is separated from the aptamer-target molecule complex during this step. Methods for extracting an aptamer from a portion of an electrophoretic medium have been described previously, for example, by commercially available kits such as Oligo Clean and Concentrator Kits by Zymo Research, QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany), Wizard SV Gel and PCR Clean Up System (Promega, Madison, WI, USA), and GENECLEAN® II Kit (MP BIOMEDICALS, Solon, OH, USA), or by the method described in Non-Patent Document 15.

[0075] In some embodiments, the electrophoresis medium includes pores that allow the movement of molecules subjected to electrophoresis. Non-limiting examples of electrophoresis media include agarose, polyacrylamide, silica matrix, or starch. In some embodiments, the first electrophoresis medium is agarose. In some embodiments, the second electrophoresis medium is agarose. In some embodiments, the first electrophoresis medium is agarose and the second electrophoresis medium is agarose. Methods for preparing agarose gels for electrophoresis are known in the art and are as described, for example, in Non-Patent Document 16. In some embodiments, the agarose gel is prepared from dry agarose powder (for example, by dissolving the agarose powder in a suitable buffer such as Tris buffer by heating and solidifying the agarose by cooling to room temperature). In some embodiments, the agarose gel is a pre-made gel purchased from a vendor. In some embodiments, the first electrophoresis medium is an agarose gel and contains agarose at any concentration between 0.5% and 3% (for example, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or 2.5%), between 1% and 2%, between 0.5% and 1%, between 1.5% and 2%, or between 2% and 2.5%).

[0076] In some embodiments, the present disclosure also considers optimal conditions (e.g., salts, temperature) for performing electrophoresis. In some embodiments, 2D electrophoresis is performed under salt conditions (e.g., an electrophoresis medium containing a certain concentration of ions) that can mitigate the electrostatic effects of biomolecules (e.g., proteins or DNA). In some embodiments, 2D electrophoresis is performed under salt conditions (e.g., an electrophoresis medium containing a certain concentration of ions) that are sufficient to disrupt non-specific interactions between aptamers and target molecules but not sufficient to disrupt specific binding between an aptamer and its target molecule. In some embodiments, the first electrophoresis medium contains a concentration of sodium ions (e.g., sodium chloride), potassium ions, lithium ions, ammonium ions, or any combination thereof that is sufficient to disrupt non-specific binding of the aptamer to the target molecule. In some embodiments, such ions include, but are not limited to, sodium ions, potassium ions, lithium ions, ammonium ions, or any combination thereof (e.g., a combination of sodium ions and potassium ions, a combination of sodium ions and lithium ions, a combination of sodium ions and ammonium ions, a combination of potassium ions and lithium ions, a combination of potassium ions and ammonium ions, a combination of lithium ions and ammonium ions, a combination of sodium ions, potassium ions, and lithium ions, a combination of sodium ions, potassium ions, and ammonium ions, a combination of potassium ions, lithium ions, and ammonium ions, or a combination of sodium ions, potassium ions, lithium ions, and ammonium ions). The term "a certain concentration of any combination thereof" as used herein refers to the total concentration of the (one or more) ions within the combination (e.g., any of the combinations described herein). For example, a combination of two ions with a concentration between X and Y refers to the total concentration of the two ions being within the range of X to Y, and a combination of three ions with a concentration between X and Y refers to the total concentration of the three ions being within the range of X to Y, and so on.The concentration can be described using any unit known in the art, such as M, mM, μM, nM, pM, g / L, g / dL, g / mL, g / μL, g / nL, mg / L, mg / dL, mg / mL, mg / μL, mg / nL, μg / L, μg / dL, μg / mL, μg / μL, μg / nL, ng / L, ng / dL, ng / mL, ng / μL, ng / nL, pg / L, pg / dL, pg / mL, pg / μL, or pg / nL. For example, if the electrophoretic medium contains a combination of ions (e.g., sodium ions, potassium ions, lithium ions, and / or ammonium ions) at a total concentration between 100 mM and 200 mM, this means that the total concentration of the combined ions is between 100 mM and 200 mM. Selecting the concentration of each ion within the combination to reach a predetermined range of total concentration is within the skill of one in the art.

[0077] In some embodiments, the first electrophoretic medium comprises sodium ions (e.g., sodium chloride), potassium ions, lithium ions, ammonium ions, or any combination thereof, at a concentration between 50 mM and 500 mM, between 80 mM and 450 mM, between 100 mM and 400 mM, between 150 mM and 350 mM, between 200 mM and 300 mM, between 100 mM and 400 mM, between 100 mM and 300 mM, between 100 mM and 200 mM, between 100 mM and 150 mM, between 150 mM and 200 mM, between 110 mM and 190 mM, between 120 mM and 180 mM, between 130 mM and 170 mM, between 140 mM and 160 mM, between 120 mM and 150 mM, between 120 mM and 160 mM, between 120 mM and 170 mM, between 120 mM and 130 mM, between 150 mM and 160 mM, between 150 mM and 170 mM, between 150 mM and 180 mM, between 150 mM and 190 mM, between 100 mM and 120 mM, between 120 mM and 130 mM, between 130 mM and 140 mM, between 140 mM and 150 mM, between 150 mM and 160 mM, between 160 mM and 170 mM, between 170 mM and 180 mM, between 180 mM and 190 mM, or between 190 mM and 200 mM. In some embodiments, the first electrophoretic medium comprises sodium ions (e.g., sodium chloride), potassium ions, lithium ions, ammonium ions, or any combination thereof, at a concentration between 100 mM and 200 mM (e.g., any concentration between 100 mM and 200 mM).In some embodiments, the second electrophoretic medium comprises sodium chloride at a concentration between 50 mM and 500 mM, between 80 mM and 450 mM, between 100 mM and 400 mM, between 150 mM and 350 mM, between 200 mM and 300 mM, between 100 mM and 400 mM, between 100 mM and 300 mM, between 100 mM and 200 mM, between 100 mM and 150 mM, between 150 mM and 200 mM, between 110 mM and 190 mM, between 120 mM and 180 mM, between 130 mM and 170 mM, between 140 mM and 160 mM, between 120 mM and 150 mM, between 120 mM and 160 mM, between 120 mM and 170 mM, between 120 mM and 130 mM, between 150 mM and 160 mM, between 150 mM and 170 mM, between 150 mM and 180 mM, between 150 mM and 190 mM, between 100 mM and 120 mM, between 120 mM and 130 mM, between 130 mM and 140 mM, between 140 mM and 150 mM, between 150 mM and 160 mM, between 160 mM and 170 mM, between 170 mM and 180 mM, between 180 mM and 190 mM, or between 190 mM and 200 mM. In some embodiments, the second electrophoretic medium comprises sodium ions (e.g., sodium chloride), potassium ions, lithium ions, ammonium ions, or any combination thereof at a concentration between 100 mM and 200 mM (e.g., any concentration between 100 mM and 200 mM).

[0078] Furthermore, in some embodiments, the 2D electrophoresis is performed under salt conditions (e.g., a certain concentration of divalent ions) sufficient to stabilize the structure of the aptamer and / or the aptamer-target molecule (e.g., biomolecule) complex. Non-limiting examples of divalent ions that can stabilize the structure of the aptamer and / or the aptamer-target molecule (e.g., biomolecule) complex include, but are not limited to, magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof. In some embodiments, the first electrophoresis medium comprises a concentration of magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof sufficient to stabilize the structure of the aptamer and / or the aptamer-target molecule (e.g., biomolecule) complex. In some embodiments, the first electrophoresis medium comprises a concentration of magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof that is 10 mM or less (e.g., less than 10 mM, less than 9 mM, less than 8 mM, less than 7 mM, less than 6 mM, less than 5 mM, less than 4 mM, less than 3 mM, less than 2 mM, less than 1 mM, or less than 0.5 mM).In some embodiments, the first electrophoretic medium comprises magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof, at a concentration between 0.1 mM and 10 mM, between 0.1 mM and 9 mM, between 0.1 mM and 8 mM, between 0.1 mM and 7 mM, between 0.1 mM and 6 mM, between 0.1 mM and 4 mM, between 0.1 mM and 3 mM, between 0.1 mM and 2 mM, between 0.1 mM and 1 mM, between 0.1 mM and 0.5 mM, between 0.5 mM and 10 mM, between 0.5 mM and 9 mM, between 0.5 mM and 8 mM, between 0.5 mM and 7 mM, between 0.5 mM and 6 mM, between 0.5 mM and 5 mM, between 0.5 mM and 4 mM, between 0.5 mM and 3 mM, between 0.5 mM and 2 mM, between 0.5 mM and 1 mM, between 1 mM and 10 mM, between 1 mM and 9 mM, between 1 mM and 8 mM, between 1 mM and 7 mM, between 1 mM and 6 mM, between 1 mM and 5 mM, between 1 mM and 3 mM, between 1 mM and 2 mM, between 2 mM and 10 mM, between 2 mM and 9 mM, between 2 mM and 8 mM, between 2 mM and 7 mM, between 2 mM and 6 mM, between 2 mM and 5 mM, between 2 mM and 4 mM, between 3 mM and 10 mM, between 3 mM and 9 mM, between 3 mM and 8 mM, between 3 mM and 7 mM, between 3 mM and 6 mM, between 3 mM and 5 mM, between 3 mM and 4 mM, between 4 mM and 10 mM, between 4 mM and 9 mM, between 4 mM and 8 mM, between 4 mM and 7 mM, between 4 mM and 6 mM, between 4 mM and 5 mM, between 5 mM and 10 mM, between 5 mM and 9 mM, between 5 mM and 8 mM, between 5 mM and 7 mM, between 5 mM and 6 mM, between 6 mM and 10 mM, between 6 mM and 9 mM, between 6 mM and 8 mM, between 6 mM and 7 mM, between 7 mM and 10 mM, between 7 mM and 9 mM, between 7 mM and 8 mM, between 8 mM and 10 mM, between 8 mM and 9 mM, between 8 mM and 9 mM, between 0.1 mM and 5 mM, between 0.5 mM and 4.5 mM, between 1 mM and 4 mM, between 2 mM and 3 mM, between 0.5 mM and 2 mM, between 0.6 mM and 1.5 mM, between 0.7 mM and 1.3 mM, between 0.8 mM and 1.2 mM, between 0.9 mM and 1.1 mM, between 0.6 mM and 1.2 mM, between 0.6 mM and 1.2 mM, between 0.7 mM and 1.2 mM, between 0.8 mM and 1.2 mM, between 0.9 mM and 1 mM, between 0.8 mM and 1 mM, between 0.9 mM and 1.5 mM, between 0.9 mM and 1.2 mM, or between 1 mM and 2 mM.In some embodiments, the first electrophoretic medium comprises magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof, at a concentration of 1 mM. In some embodiments, the second electrophoretic medium comprises magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof, at a concentration sufficient to stabilize the aptamer-target molecule (e.g., biomolecule) complex. In some embodiments, the second electrophoretic medium comprises magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof, at a concentration of 10 mM or less (e.g., less than 10 mM, less than 9 mM, less than 8 mM, less than 7 mM, less than 6 mM, less than 5 mM, less than 4 mM, less than 3 mM, less than 2 mM, less than 1 mM, or less than 0.5 mM).In some embodiments, the first electrophoretic medium contains magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof, at a concentration between 0.1 mM and 10 mM, between 0.1 mM and 9 mM, between 0.1 mM and 8 mM, between 0.1 mM and 7 mM, between 0.1 mM and 6 mM, between 0.1 mM and 4 mM, between 0.1 mM and 3 mM, between 0.1 mM and 2 mM, between 0.1 mM and 1 mM, between 0.1 mM and 0.5 mM, between 0.5 mM and 10 mM, between 0.5 mM and 9 mM, between 0.5 mM and 8 mM, between 0.5 mM and 7 mM, between 0.5 mM and 6 mM, between 0.5 mM and 5 mM, between 0.5 mM and 4 mM, between 0.5 mM and 3 mM, between 0.5 mM and 2 mM, between 0.5 mM and 1 mM, between 1 mM and 10 mM, between 1 mM and 9 mM, between 1 mM and 8 mM, between 1 mM and 7 mM, between 1 mM and 6 mM, between 1 mM and 5 mM, between 1 mM and 3 mM, between 1 mM and 2 mM, between 2 mM and 10 mM, between 2 mM and 9 mM, between 2 mM and 8 mM, between 2 mM and 7 mM, between 2 mM and 6 mM, between 2 mM and 5 mM, between 2 mM and 4 mM, between 3 mM and 10 mM, between 3 mM and 9 mM, between 3 mM and 8 mM, between 3 mM and 7 mM, between 3 mM and 6 mM, between 3 mM and 5 mM, between 3 mM and 4 mM, between 4 mM and 10 mM, between 4 mM and 9 mM, between 4 mM and 8 mM, between 4 mM and 7 mM, between 4 mM and 6 mM, between 4 mM and 5 mM, between 5 mM and 10 mM, between 5 mM and 9 mM, between 5 mM and 8 mM, between 5 mM and 7 mM, between 5 mM and 6 mM, between 6 mM and 10 mM, between 6 mM and 9 mM, between 6 mM and 8 mM, between 6 mM and 7 mM, between 7 mM and 10 mM, between 7 mM and 9 mM, between 7 mM and 8 mM, between 8 mM and 10 mM, between 8 mM and 9 mM, between 8 mM and 9 mM, between 0.1 mM and 5 mM, between 0.5 mM and 4.5 mM, between 1 mM and 4 mM, between 2 mM and 3 mM, between 0.5 mM and 2 mM, between 0.6 mM and 1.5 mM, between 0.7 mM and 1.3 mM, between 0.8 mM and 1.2 mM, between 0.9 mM and 1.1 mM, between 0.6 mM and 1.2 mM, between 0.6 mM and 1.2 mM, between 0.7 mM and 1.2 mM, between 0.8 mM and 1.2 mM, between 0.9 mM and 1 mM, between 0.8 mM and 1 mM, between 0.9 mM and 1.5 mM, between 0.9 mM and 1.2 mM, or between 1 mM and 2 mM.In some embodiments, the second electrophoretic medium comprises magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof, at a concentration of 1 mM. Magnesium can be added to the first and / or second electrophoretic medium in the form of any known magnesium salt, such as magnesium chloride and magnesium sulfate. In some embodiments, the magnesium ions are in the form of magnesium chloride.

[0079] In some embodiments, the running buffer for electrophoresis in the first direction comprises boric acid at a concentration between 40 mM and 100 mM. In some embodiments, the running buffer for electrophoresis in the first direction comprises tris(hydroxymethyl)aminomethane at a concentration between 40 mM and 100 mM. In some embodiments, the running buffer for electrophoresis in the second direction comprises boric acid at a concentration between 40 mM and 100 mM. In some embodiments, the running buffer for electrophoresis in the second direction comprises tris(hydroxymethyl)aminomethane at a concentration between 40 mM and 100 mM.

[0080] In some embodiments, the 2D electrophoresis is performed at a temperature optimal for the movement and separation of the molecules in the composition (e.g., aptamer-biological molecule complexes and unbound aptamers). In some embodiments, the electrophoresis in the first direction is performed at a temperature between 8°C and 22°C, between 9°C and 21°C, or between 10°C and 20°C, between 11°C and 19°C, between 12°C and 18°C, between 13°C and 17°C, between 14°C and 16°C, between 10°C and 15°C, between 11°C and 14°C, between 12°C and 13°C. In some embodiments, the electrophoresis in the first direction is performed at 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, or 22°C. In some embodiments, the electrophoresis in the second direction is performed at a temperature between 8°C and 22°C, between 9°C and 21°C, or between 10°C and 20°C, between 11°C and 19°C, between 12°C and 18°C, between 13°C and 17°C, between 14°C and 16°C, between 10°C and 15°C, between 11°C and 14°C, between 12°C and 13°C. In some embodiments, the electrophoresis in the second direction is performed at 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C. In some embodiments, the amount of salt (e.g., sodium chloride and / or magnesium chloride) in the first and second electrophoresis media raises the temperature of the electrophoresis media to a temperature that can interfere with the stability of the aptamer-target molecule complex. In some embodiments, to maintain the temperature within the optimal temperature range described herein, the electrophoresis unit is placed in a cold water bath filled with ice.

[0081] As described herein, in some embodiments, the method further comprises extracting an aptamer capable of binding to one or more target molecules (e.g., biomolecules) from a portion of a second electrophoretic medium containing an aptamer-target molecule complex. In some embodiments, the method further comprises amplifying a plurality of aptamers extracted from an aptamer-target molecule (e.g., biomolecule) complex to form an aptamer library. In some embodiments, the amplification of an aptamer capable of binding to one or more target molecules (e.g., biomolecules) is performed by polymerase chain reaction (PCR). Polymerase chain reaction (PCR) is an experimental technique used to amplify a DNA sequence by using short DNA sequences called primers. The temperature of the sample is repeatedly raised and lowered to facilitate the DNA replication enzyme to copy the target DNA sequence. Non-limiting examples of PCR include emulsion PCR, asymmetric PCR, convection PCR, dial-out PCR, digital PCR, helicase-dependent amplification, hot start PCR, in silico PCR, inverse PCR, PCR utilizing ligation, mini-primers PCR, multiplex ligation-dependent probe amplification, multiplex PCR, nanoparticle-assisted PCR, nested PCR, overlap extension PCR, quantitative PCR, reverse complement PCR, single specific primer-PCR, and solid-phase PCR. In some embodiments, the aptamer is amplified by emulsion PCR.

[0082] In some embodiments, the plurality of aptamers capable of binding to one or more target molecules (e.g., biomolecules) extracted from a portion of the second electrophoretic medium containing the aptamer-target molecule complex contain different amounts of aptamers (i.e., sequence of the sequences), for example, aptamers that bind to the target molecule with high affinity are present at high levels, while aptamers that bind to the target molecule with low affinity are present at low levels. In some aspects, the present disclosure attempts to retain the sequence information of the sequences in the plurality of aptamers capable of binding to one or more target molecules (e.g., biomolecules) after generating an aptamer library by amplification. In some embodiments, the present disclosure is based on the discovery that emulsion PCR can preserve the sequence information of the sequences in the plurality of aptamers capable of binding to one or more target molecules (e.g., biomolecules) after generating an aptamer library by amplification.

[0083] In some embodiments, sequencing of low-abundance aptamers in an aptamer library is performed through multiple rounds consisting of the steps of selecting aptamers that can bind to one or more target molecules (e.g., biomolecules) in a sample (e.g., a biological sample), amplifying the aptamers that can bind to the target molecules by emulsion PCR to form an aptamer library, sequencing the aptamer library, and knocking down high-abundance aptamers using ASO. In some embodiments, the steps of the methods described herein are repeated at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least eleven times, at least twelve times, at least thirteen times, at least fourteen times, at least fifteen times, or more than that. In some embodiments, the steps of the methods described herein are repeated four times. In some embodiments, the steps of the methods described herein are repeated nine times. In some embodiments, the aptamer library obtained after amplification (e.g., emulsion PCR) is used as a plurality of starting candidate aptamers for contacting the target molecules (e.g., biomolecules) in the sample (e.g., a biological sample) in the next round.

[0084] Other embodiments All of the features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification may be replaced by alternative features that serve the same, equivalent, or similar purpose. Thus, unless specifically stated otherwise, each feature disclosed is only an example of a general series of equivalent or similar features.

[0085] From the above description, those skilled in the art can easily identify the essential features of the present disclosure and make various changes and modifications to the present invention so that the present invention can be adapted to various uses and conditions without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are also included within the scope of the claims.

[0086] Equivalents Although several embodiments of the present invention have been described and explained herein, those skilled in the art will readily conceive of various other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each such variation and / or modification is considered to be within the scope of the embodiments of the invention described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and spatial arrangements described herein are typical, and that actual parameters, dimensions, materials, and / or spatial arrangements will depend on one or more specific applications in which one or more teachings of the present invention are used. Those skilled in the art will recognize or be able to confirm many equivalents of the specific embodiments of the present invention described herein using only ordinary experimentation. Accordingly, it is understood that the foregoing embodiments are described for purposes of illustration only, and that embodiments of the present invention may be practiced in other ways within the scope of the appended claims and their equivalents, other than as specifically described and claimed herein. Embodiments of the invention disclosed herein are directed to each individual feature, system, article, material, kit, and / or method described herein. Additionally, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the invention disclosed herein if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0087] It is understood that all definitions defined and used herein shall prevail over dictionary definitions, definitions in incorporated by reference documents, and / or ordinary meanings of defined terms.

[0088] All references, patents, and patent applications disclosed herein are incorporated by reference for the subject matter for which each is cited, which in some cases may include the entire document.

[0089] The indefinite articles "a" and "an" should be understood to mean "at least one" as used herein in the specification and claims, unless clearly indicated otherwise.

[0090] As used herein in the specification and claims, the expression "and / or" should be understood to mean "either or both" of the elements so combined, i.e., elements that are present conjunctively in some cases and disjunctively in other cases. Multiple elements listed using "and / or" should be construed in the same manner, i.e., "one or more" of the elements so combined. Other elements other than those specifically identified by the phrase "and / or" may optionally be present, whether or not they are related to those specifically identified elements. Thus, by way of non-limiting example, a reference to "A and / or B", when used in combination with an open-ended term such as "comprising", may refer to, for example, only A in one embodiment (optionally including elements other than B), only B in another embodiment (optionally including elements other than A), or both A and B in yet another embodiment (optionally including other elements).

[0091] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be construed as inclusive, i.e., including not only at least one of many elements or list of elements, but also two or more, and optionally additional unlisted items. Only terms such as "only one" or "exactly one" that clearly indicate the contrary to the foregoing, or "consisting of" when used in the claims, refer to including exactly one element of many elements or list of elements. Ordinarily, the term "or" as used herein should be construed to indicate only exclusive alternatives (i.e., "either one or the other, but not both") when preceded by exclusive terms such as "any one", "one", "only one", or "exactly one". "Consisting essentially of", when used in the claims, has its ordinary meaning as used in the field of patent law.

[0092] As used herein in the specification and claims, the expression "at least one" in reference to a listing of one or more elements means at least one element selected from any one or more of the elements in the listing of elements, but does not necessarily include at least one of each and every element specifically listed in the listing of elements, and is understood not to exclude any combinations of elements in the listing of elements. This definition also allows for the optional presence of elements other than those specifically identified within the listing of elements to which the expression "at least one" refers, whether or not such other elements are related to those specifically identified elements. Thus, by way of non-limiting example, "at least one of A and B" (or equivalently "at least one of A or B", or equivalently "at least one of A and / or B") can, in one embodiment, refer to, for example, at least one A, including two or more A's optionally, where B is absent (and optionally including elements other than B); in another embodiment, it can refer to at least one B, including two or more B's optionally, where A is absent (and optionally including elements other than A); and in yet another embodiment, it can refer to at least one A including two or more A's optionally and at least one B including two or more B's optionally (and optionally including other elements).

[0093] Also, it should be understood that, unless otherwise clearly indicated, in any method recited in the claims herein that includes two or more steps or acts, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Examples

[0094] Example 1: Materials and Methods Design of Antisense Oligonucleotides Antisense oligonucleotides (ASOs) were systematically designed based on aptamer sequence information obtained by next-generation sequencing analysis (NGS) after SELEX. The length of the ASOs was fixed at 25 - 30 nucleotides for all ASOs. The target domain started from positions 7 to 10 at the 5'-end of the random region. The target sequences knocked down by the ASOs were determined based on the sequence frequency level exceeding 0.1 - 0.2% so that the available sequencing reads of the remaining sequences after ASO-based reduction were maximized. All ASOs were composed of DNA.

[0095] Interaction between antisense oligonucleotides and aptamer library The ASO was dissolved in deionized water before use. The working concentration of the ASO was set 100 - 1000 times higher than the target sequence concentration that could be calculated from the NGS data of the aptamer library. A 50× ASO solution was prepared by mixing the ASO in a tube. The targeted core library prepared by the 2D electrophoresis-based aptamer library preparation method was diluted into a selection buffer of the working concentration (PBS with 0.005% Nonidet-P40 and 1 mM magnesium chloride) for selection. The 50× ASO solution was added to the aptamer library solution, and then the library was denatured to cause a strong knockdown efficiency. The solution was denatured at 95 °C for 3 minutes and then slowly cooled to room temperature over 30 minutes to form a stable ssDNA structure. At this step, the aptamers targeted by the ASO are inactivated due to the inhibition of the formation of functional structures. If target specificity is more important than knockdown efficiency, the 50× ASO solution was added after the reconstitution step. The reconstituted library solution was mixed with an appropriately diluted biological fluid sample solution with a reaction volume of 10 μL. The biological fluid sample was mixed with competitor substances such as salmon sperm DNA and any irrelevant oligo DNA, and then mixed with the ssDNA library solution. Dextran sulfate in the concentration range of 0.001% - 1% was also used to reduce the charge-dependent non-specific interaction between ssDNA and biomolecules. The mixture was incubated at room temperature for 10 minutes, then 2 μL of 6× loading buffer containing 36% glycerol and 6 mM magnesium chloride was added to the reaction mixture, and immediately after that, the sample was loaded onto an agarose gel.

[0096] Preparation of agarose gels for 1D and 2D electrophoresis Agarose gels for 1D electrophoresis and 2D electrophoresis were prepared as follows. Agarose powder was mixed at a concentration of 1.0 - 1.2 wt% in 1×TB buffer (80 mM Tris base, 80 mM boric acid) containing a NaCl concentration of 100 - 200 mM according to the selected conditions. The mixture was heated by microwave to completely dissolve the agarose powder. Then, after cooling the agarose solution to approximately 60 °C, 1 M MgCl2 solution was added to a MgCl2 concentration of 1 mM and mixed thoroughly. Next, 11 mL of the solution was poured into a gel tray (55 mm (W) × 60 mm (L)) with wells at predetermined positions. The number of wells was 3 - 4 wells (10 mm (W) × 1 mm (L)) for 1D electrophoresis and 1 well (45 mm (W) × 2 mm (L)) for 2D electrophoresis. To reduce the heat generated from the high-salt agarose gel during electrophoresis, the thickness of the agarose gel was adjusted to be relatively thin. Before use, the gel was placed at room temperature for at least 3 hours but less than 6 hours.

[0097] Removal of Targeted Aptamer Clusters from Libraries by 2D Electrophoresis An electrophoresis unit such as Mupid2 was filled with 1×TB buffer. The gel for 1D electrophoresis prepared above was placed in a gel box. Since a high-salt agarose gel was used, the electrophoresis unit was placed in a cold water bath filled with crushed ice to maintain the buffer temperature between 10 - 20 °C during electrophoresis. The gel was prerun at 100 V for 10 minutes, and then the samples were run. The sample mixture was carefully loaded into the wells of the gel. One-dimensional electrophoresis was performed in the dark at 100 V for 55 - 60 minutes. After the run, the gel was taken out of the gel box and visualized by a ChemiDoc MP Imaging System (Bio-Rad). The entire region of the aptamer-biological molecule complex that was present overall on the free aptamer region was cut out for 2D electrophoresis. The size of the cut-out gel was adjusted to fit the well of the 2D electrophoresis gel.

[0098] Similar to 1D electrophoresis, 2D electrophoresis was performed. After running, the gel was taken out of the gel box for visualization by a ChemiDoc MP Imaging System (Bio-Rad), and only the diagonal regions formed by the aptamer-biological molecule complexes were excised. The excised gel pieces were placed in microtubes.

[0099] Extraction of DNA from agarose gel The excised gel pieces were melted at 95 °C for 3 minutes. The melted agarose solution was dispensed into several tubes with 110 μL aliquots. The tubes were incubated at 55 °C for 1 minute. Heat-resistant β-agarase was added to each tube in 2 μL aliquots, and the tubes were incubated at 55 °C for 15 minutes to enzymatically digest the agarose. The ssDNA library was recovered by Oligo Clean and Concentrator Kits (Zymo Research) according to the manufacturer's instructions. The recovered ssDNA was eluted with 30 μL of deionized water.

[0100] Emulsion PCR amplification of the recovered ssDNA library The recovered ssDNA was subjected to emulsion PCR amplification in a two-step process. Note that all PCR reactions should be performed by emulsion PCR in order to maintain the sequence information of the sequences in the library after removing frequently occurring sequences. The emulsion PCR solution was prepared as follows: 100 μL of PCR solution was mixed with 250 μL of emulsion oil (4.5% Span80, 0.4% Tween80, 0.05% Triton-X100, and 95.05% mineral oil), and this solution was vigorously mixed with a magnetic stir bar until thoroughly mixed. The emulsion PCR reaction was tested by different numbers of consecutive PCR cycles in small volumes. The PCR products at the specified cycles were recovered by chloroform extraction and analyzed by gel electrophoresis (6% polyacrylamide with 0.5×TBE buffer) to determine the appropriate PCR cycle that could provide a distinct single band for DNA products without any concatemers or truncations. The remaining PCR samples were amplified using the determined PCR cycle. The PCR products were recovered by chloroform extraction and subjected to purification with Oligo Clean and Concentrator Kits. The amplified DNA was eluted with 10 μL of deionized water and quantified by a Qubit 4 fluorometer (Invitrogen). This PCR amplification step was repeated until a sufficient amount of DNA was obtained.

[0101] Generation of the aptamer library by primer extension In some cases, 2 - 3 cycles are required to sufficiently remove the target sequences from the library. The amplified DNA was diluted to a concentration of 1 ng / μL to prepare a template DNA sample for the production of the aptamer library by primer extension. Then, 2 μL of the solution was used for each 100 μL of PCR amplification. In this PCR amplification step, an antisense strand primer biotinylated at the 5’ end was used, and the number of PCR cycles was fixed at 8 cycles. The PCR products were recovered by chloroform extraction and then purified using Oligo Clean and Concentrator Kits. The purified DNA was subjected to primer extension using a 5’-TYE665 fluorophore-labeled primer. The product was directly immobilized on streptavidin agarose beads filled with PBSN buffer (PBS with 0.005% Nonidet-P40). The beads were incubated at 16 °C and 1500 rpm for 15 minutes every 2 minutes with shaking. The beads were washed 3 times with PBS, filled with 40 μL of 20 mM NaOH solution to denature the product DNA, and dissociate the 5’-TYE665-labeled aptamer. The bead solution was incubated at 37 °C and 1500 rpm for 1 minute with shaking. The solution was centrifuged for 30 seconds using a benchtop mini centrifuge. The supernatant was transferred to a new tube. The denaturation step was performed one more time, and the supernatants were combined in the same tube. The recovered supernatant was quenched with 80 mM HCl to adjust the pH of the solution to approximately 7.0 - 8.0 and purified using Oligo Clean and Concentrator Kits. The aptamer library was eluted with 10 μL of deionized water and quantified using NanoDrop. The aptamer library was used for the next selection cycle.

[0102] Sequencing analysis of the aptamer library The PCR products obtained after 2D electrophoresis were amplified by emulsion PCR in a two-step process and prepared for next-generation sequencing (NGS) analysis by MiSeq (Illumina). An adapter sequence primer set was used in the first step, and then an index sequence primer set was used in the second step. Thermal cycling was performed as per the manufacturer's instructions. The PCR products were purified by NucleoSpin Gel and PCR Clean-up (Macherey Nagel) and quantified by a Qubit 4 fluorometer in the first step and by a Bioanalyzer in the second step. The products were analyzed by MiSeq according to the manufacturer's instructions. All sequencing data for each round were created as FASTAQ files. After extracting the aptamer domain by trimming the 5'- and 3'-primer regions, all sequences were used for frequency analysis.

[0103] Example 2: Reduction of highly abundant aptamers in an aptamer library by ASO This example describes a method for knocking down highly abundant aptamers in a library of aptamers. In an aptamer library, highly abundant aptamers (e.g., aptamers with high affinity for a target molecule) contain a larger number of NGS reads than less abundant aptamers (e.g., aptamers with low affinity for a target molecule). Similarly, less abundant aptamers exhibit a lower detection ability by NGS analysis, i.e., aptamers in low-frequency regions are difficult to analyze due to fewer reads, but more sequences exist below the detection limit region. When the highly frequent aptamers are reduced from the aptamer library, the detection ability of less abundant aptamers that can bind to the target biomolecule increases, i.e., the reduction of highly frequent sequences increases the less frequent sequences (Figure 1).

[0104] This method further describes reducing high-abundance aptamers from an aptamer library by ASO. The ASO is designed to target high-abundance aptamers (e.g., aptamers whose percentage of reads exceeds a certain threshold). Hybridization of the ASO to high-abundance aptamers induced a structural change that inactivated the binding ability of the high-abundance aptamers to the target molecule (Figure 2).

[0105] Example 3: Manipulation of an Aptamer Library for Mouse Serum The method according to the present disclosure was applied to the manipulation of an aptamer library prepared for mouse serum.

[0106] The aptamer library was prepared and sequenced based on the method for preparing the aptamer library using separately prepared unpurified biological samples.

[0107] According to the present disclosure, emulsion PCR was used to maintain the sequence information of the sequences after reducing the targeted sequences. Figures 5 and 6 show typical data comparing PCR products between the emulsion PCR method and the general PCR method obtained by simultaneously replicating the same aptamer library. Figure 7 shows data for the confirmation of PCR products by PAGE. The emulsion conditions are as follows: 1: combination of sonication and vortexing, 2: vortexing, 3: stirring with a magnetic stirrer bar, 4: general PCR.

[0108] In the verification experiment, the target aptamer sequence was selected from the most frequently occurring sequences found in the next-generation sequencing analysis of the aptamer library. Two sequences were taken out from the target list to examine the specificity of the present invention. The antisense oligonucleotide library was systematically designed based only on the sequence information obtained from next-generation sequencing. The length was fixed at 30 nucleotides, and the target sequence was set from the 7th position of the aptamer domain. DNA oligonucleotides were chemically synthesized without any specific chemical modification. The results of the simultaneous inactivation of 18 aptamers by 18 different ASOs are shown in FIGS. 3 and 8. The 10th and 15th aptamers were not targeted by any ASO in this experiment to examine the specificity of the ASO. The results of the simultaneous inactivation of 52 aptamers by 52 different ASOs are shown in FIG. 9. The change in the sequence frequency after the addition of the ASO was examined. FIG. 10 shows the change in the sequence frequency in the aptamer library in which the ASO was added at the time of aptamer denaturation to hybridize the ASO with the target aptamer. The sequence comparison analysis of the non-specific inactivation sequences by the added ASO revealed that non-specific inactivation occurred in sequences with relatively high homology to the ASO. FIG. 11 shows the change in the sequence frequency in the aptamer library in which the ASO was added after the formation of the aptamer structure to reduce the unexpected hybridization between the aptamer and the ASO caused by the highly homologous sequence. The analysis of the number of available sequences after the reduction of the target is shown in FIG. 12. It was revealed that approximately 30% more sequences were found at a sufficient sequence frequency for sequencing analysis (FIG. 4).

Claims

**Claim 1** A method for sequencing low-abundance aptamers from an aptamer library, comprising: (i) amplifying a plurality of aptamers capable of binding to one or more target molecules in a sample by emulsion PCR to produce an aptamer library; (ii) sequencing said aptamer library; and (iii) contacting a plurality of antisense oligonucleotides (ASOs) targeting high-abundance aptamers in said aptamer library with said aptamer library to form a mixture, wherein contacting said ASOs with said aptamer library results in inactivation of said high-abundance aptamers in said aptamer library. A method comprising the above steps. **Claim 2** Steps (a) to (c): (a) contacting a plurality of candidate aptamers with a sample containing one or more target molecules to form a composition comprising a plurality of aptamer-target molecule complexes; (b) purifying said plurality of aptamer-target molecule complexes; and (c) extracting a plurality of aptamers capable of binding to one or more target molecules from said aptamer-target molecule complexes. The method according to claim 1, further comprising selecting a plurality of aptamers capable of binding to one or more target molecules in the sample. **Claim 3** The method according to claim 2, further comprising repeating steps (a) to (c) and (i) to (iii), wherein the mixture obtained from step (iii) comprises said plurality of candidate aptamers when repeating step (a). **Claim 4** The method according to claim 3, wherein the repetition is performed at least three times. **Claim 5** The method according to any one of claims 1 to 4, further comprising sequencing the aptamer library obtained from step (iii). **Claim 6** The method according to any one of claims 1 to 5, wherein the ASO comprises a modified nucleotide. **Claim 7** The method according to any one of claims 1 to 6, wherein sequencing of low-abundance aptamers from the aptamer library comprises next-generation sequencing (NGS). **Claim 8** The method according to any one of claims 1 to 7, wherein the sample is a biological sample. **Claim 9** The method according to claim 8, wherein the biological sample is serum, plasma, cerebrospinal fluid (CSF), urine, amniotic fluid, bone marrow, bronchoalveolar lavage fluid, oral swab, feces, gastrointestinal fluid, liposuction sample, saliva, milk, nasal swab, peritoneal fluid, semen, sputum, synovial fluid, tears, vaginal fluid, tissue biopsy, cell lysate, cultured cells, tissue sample, or in vivo endothelial cells.

10. The method according to claim 8 or 9, wherein the biological sample comprises a target molecule comprising nucleic acid, protein, polypeptide, carbohydrate, lipid, or a combination thereof.

11. The method according to any one of claims 8 to 10, wherein the biological sample is not denatured.

12. The method according to any one of claims 1 to 11, wherein the high-abundance aptamer in the aptamer library is an aptamer having a sequence frequency level higher than 0.05% in the sequencing reaction of step (ii).

13. The method according to any one of claims 1 to 12, wherein the high-abundance aptamer in the aptamer library is an aptamer having a sequence frequency level higher than 0.1% in the sequencing reaction of step (ii).

14. The method according to any one of claims 1 to 13, wherein the high-abundance aptamer in the aptamer library is an aptamer having a sequence frequency level higher than 0.15% in the sequencing reaction of step (ii).

15. The method according to any one of claims 1 to 14, wherein the high-abundance aptamer in the aptamer library is an aptamer having a sequence frequency level higher than 0.2% in the sequencing reaction of step (ii).

16. The method according to any one of claims 1 to 15, wherein the high-abundance aptamer in the aptamer library is an aptamer having a sequence frequency level higher than 0.5% in the sequencing reaction of step (ii).

17. The method according to any one of claims 2 to 16, wherein step (b) comprises subjecting the composition to electrophoresis in a first electrophoresis medium in a first direction to obtain a portion of the first electrophoresis medium containing the aptamer-target molecule complex.

18. The method according to claim 17, wherein step (b) further comprises subjecting the portion of the first electrophoresis medium to electrophoresis in a second electrophoresis medium in a second direction to obtain a portion of the second electrophoresis medium containing the aptamer-target molecule complex.

19. The method according to claim 17 or 18, wherein the first electrophoresis medium is a first agarose gel.

20. The method according to claim 18 or 19, wherein the second electrophoresis medium is a second agarose gel.

21. The method according to claim 19 or 20, wherein the first and second electrophoresis media contain sodium ions, potassium ions, lithium ions, ammonium ions, or any combination thereof, at a concentration between 100 mM and 200 mM.

22. The method according to claim 21, wherein the sodium ions are in the form of sodium chloride.

23. The method according to any one of claims 19 to 22, wherein the first and second electrophoresis media contain magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof, at a concentration of 10 mM or less.

24. The method according to any one of claims 19 to 23, wherein the first and second electrophoresis media contain magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof, at a concentration between 0.5 mM and 2 mM.

25. The method according to any one of claims 19 to 24, wherein the first and second electrophoresis media contain magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof, at a concentration of 1 mM.

26. The method according to any one of claims 23 to 25, wherein the magnesium ions are in the form of magnesium chloride.

27. The method according to any one of claims 19 to 26, wherein step (b) further comprises cutting out a portion of the first electrophoresis medium containing the aptamer-target molecule complex from the remaining portion of the first electrophoresis medium.

28. The method according to claim 27, wherein a portion of the first electrophoresis medium is adapted to a well in the second electrophoresis medium for performing electrophoresis in a second direction.

29. The method according to any one of claims 17 to 28, wherein the electrophoresis in the first direction and the second direction is performed at a temperature between 10 °C and 20 °C.

30. The method according to any one of claims 1 to 29, wherein the plurality of candidate aptamers are single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA, or peptides.

31. The method according to claim 30, wherein the plurality of candidate aptamers are single-stranded DNA (ssDNA).

32. The method according to claim 31, wherein each of the plurality of candidate aptamers comprises a modified nucleotide.

33. The method according to any one of claims 1 to 32, wherein each of the plurality of candidate aptamers is labeled.

34. The method according to claim 33, wherein each of the plurality of candidate aptamers is fluorescently labeled.

35. Before step (c), the method according to any one of claims 1 to 34, further comprising the step of cutting out a portion of the second electrophoretic medium containing the aptamer-target molecule complex from the remaining portion of the second electrophoretic medium, and the step of extracting the aptamer-target molecule complex from the portion of the second electrophoretic medium.

36. The method according to any one of claims 1 to 35, further comprising the step of denaturing and reconstituting the aptamer library before the step of contacting the ASO with the aptamer library.

37. The method according to any one of claims 1 to 36, further comprising the step of denaturing and reconstituting the aptamer library after the step of contacting the ASO with the aptamer library.

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