Inhibition of targeted aptamer clusters

Abstract

The present disclosure relates, at least in part, to a method of sequencing low abundance aptamers in an aptamer library, the method comprising: (i) amplifying a plurality of aptamers capable of binding one or more target molecules in a sample by emulsion PCR to produce an aptamer library; (ii) carrying out sequencing on the aptamer library; and (iii) contacting antisense oligonucleotides (ASOs) targeting the abundance aptamers of the aptamer library with the aptamer library to form a mixture, wherein contacting the ASOs with the aptamer library results in inactivation of the abundance aptamers of the aptamer library.

Description

Technical Field

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

[0002] The enriched aptamers are sequenced to obtain sequencing information. However, a large amount of sequence information is lost for the low-abundance aptamers in the enriched aptamer library.

[0003] List of cited references

[0004] Patent Literature

[0005] [PTL 1] US2006 / 0160763

[0006] Non-patent literature

[0007] [NPL 1] Keefe et al., Aptamers as therapeutics. Nat. Rev. Drug Discov. 2010;9:537-550.

[0008] [NPL 2] Jayasena SD, Aptamers: An emerging class of molecules that rival antibodies in diagnostics. Clin. Chem. 1999; 45: 1628-1650.

[0009] [NPL 3] Tuerk et al., Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990; 249: 505-510.

[0010] [NPL 4] Ellington et al., In vitro selection of RNA molecules that bind specific ligands. Nature. 1990; 346: 818-822.

[0011] [NPL 5] SELEX-A (Registered Trademark) evolutionary method to generate high-affinity nucleic acid ligands. Biomolecular Engineering, 24(4): 381-403; Klug and Famulok (1994).

[0012] [NPL 6] All you wanted to know about SELEX. Molecular Biology Reports, 20:97-107; Darmostuk et al. (2015).

[0013] [NPL 7] Current approaches in SELEX: An update to aptamer selection technology. Biotechnology Advances, 33(6): 1141-1161.

[0014] [NPL 8] Schutze et al., Probing the SELEX Process with Next-Generation Sequencing. PLoS One. 2011;6(12):e29604.

[0015] [NPL 9] Hao et al., Introduction of Aptamer, SELEX, and Different SELEX Variants, Aptamers for Medical Applications, pp. 1-30.

[0016] [NPL 10] Chai et al., Principle of Emulsion PCR and Its Applications in Biotechnology, J Anim Reprod Biotechnol, 2019;34:259-266.

[0017] [NPL 11] Shao et al., Emulsion PCR: A High Efficient Way of PCR Amplification of Random DNA Libraries in Aptamer Selection. PLoS ONE, 6(9): e24910.

[0018] [NPL 12] Duffy et al., Modified nucleic acids: replication, evolution, and next-generation therapeutics, BMC Biology, vol. 18, article number: 112 (2020).

[0019] [NPL 13] Hollenstein (2015) Generation of long, fully modified, and serum-resistant oligonucleotides by rolling circle amplification. Organic Biomolecular Chemistry, 13: 9829.

[0020] [NPL 14] Kong et al. (2016). Generation of Synthetic Copolymer Libraries by Combinatorial Assembly on Nucleic Acid Templates. ACS Combinatorial Science, 18: 355-370.

[0021] [NPL 15] Abraham et al., A quick and effective in-house method of DNA purification from agarose gel, suitable for sequencing. 3Biotech. 2017 Jul;7(3):180.

[0022] [NPL 16] Leonard et al., Basic Methods in Molecular Biology, 1986, Section 5-5 - Agarose Gel Electrophoresis, pp. 58-61. Summary of the invention

[0023] Technical issues

[0024] The object of the present invention is to provide a method for sequencing low-abundance aptamers in an aptamer library.

[0025] Solution to the problem

[0026] The present disclosure relates at least in part to methods for mining sequence information of low-abundance aptamers in aptamer libraries. The aptamer library contains high-abundance aptamers (e.g., aptamers with a sequence frequency level higher than a certain percentage in the sequencing reaction) and low-abundance aptamers (e.g., aptamers with a sequence frequency level lower than a certain percentage in the sequencing reaction). In some embodiments, during the sequencing reaction, due to the low readings of the low-abundance aptamers (e.g., low readings below the detection limit), the high-abundance aptamers mask the sequence information of the low-abundance aptamers, making them unable to be effectively analyzed. In some embodiments, the present disclosure provides methods for depleting high-abundance aptamers (e.g., by antisense oligonucleotides (ASOs) targeting high-abundance aptamers), so that low-abundance aptamers can be effectively amplified (e.g., by emulsion PCR) and sequenced. In some embodiments, these methods can be described as suppression of targeted aptamer clusters (STAC).

[0027] In some aspects, the present disclosure provides a method for sequencing low-abundance aptamers in 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 produce an aptamer library; (ii) sequencing the aptamer library; and (iii) contacting a plurality of antisense oligonucleotides (ASOs) targeting high-abundance aptamers of the aptamer library with the aptamer library to form a mixture; wherein contacting the ASOs with the aptamer library results in inactivation of the high-abundance aptamers of the aptamer library.

[0028] In some embodiments, the method further comprises selecting a plurality of aptamers capable of binding to one or more target molecules in a sample in 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 one or more target molecules from the aptamer-target molecule complexes.

[0029] In some embodiments, the method further comprises repeating steps (a)-(c) and (i)-(iii), and wherein when step (a) is repeated, the mixture obtained from step (iii) comprises a plurality of candidate aptamers. 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).

[0030] In some embodiments, the ASO comprises modified nucleotides.

[0031] In some embodiments, sequencing low-abundance aptamers in the aptamer library comprises next generation sequencing (NGS).

[0032] 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, cheek 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 target molecules, which include nucleic acids, proteins, polypeptides, carbohydrates, lipids or combinations thereof. In some embodiments, the biological sample is not denatured.

[0033] In some embodiments, 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). In some embodiments, 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). In some embodiments, 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). In some embodiments, 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). In some embodiments, 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).

[0034] In some embodiments, step (b) comprises electrophoresing the composition in a first electrophoretic medium in a first direction to obtain a portion of the first electrophoretic medium containing the aptamer-target molecule complex.

[0035] In some embodiments, step (b) further comprises electrophoresing a portion of the first electrophoretic medium in a second electrophoretic medium in a second direction to obtain a portion of the second electrophoretic medium containing the aptamer-target molecule complex.

[0036] In some embodiments, the first electrophoretic medium is a first agarose gel. In some embodiments, the second electrophoretic medium is a second agarose gel.

[0037] In some embodiments, the first and second electrophoretic media comprise 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.

[0038] In some embodiments, the first and second electrophoretic 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 electrophoretic 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 electrophoretic 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.

[0039] In some embodiments, step (iii) further comprises cutting the portion of the first electrophoretic medium comprising the aptamer-target molecule complexes from the remainder of the first electrophoretic medium. In some embodiments, the portion of the first electrophoretic medium is assembled into a well in a second electrophoretic medium to perform electrophoresis in a second direction.

[0040] In some embodiments, electrophoresis in the first direction and the second direction is performed at a temperature between 10°C and 20°C.

[0041] 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).

[0042] In some embodiments, each of the plurality of candidate aptamers comprises a modified nucleotide. In some embodiments, each of the plurality of candidate aptamers comprises one or more 5-tryptamine-uracils in place of thymine.

[0043] 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.

[0044] In some embodiments, the method further comprises, before step (c), cutting a portion of the second electrophoretic medium containing the aptamer-target molecule complexes from the rest of the second electrophoretic medium, and extracting the aptamer-target molecule complexes from the portion of the second electrophoretic medium.

[0045] In some embodiments, the method further comprises denaturing and renaturing the aptamer library prior to contacting the ASO with the aptamer library.

[0046] In some embodiments, the method further comprises denaturing and renaturing the aptamer library after contacting the ASO with the aptamer library.

[0047] Beneficial effects of the present invention

[0048] The present invention provides a method for sequencing low-abundance aptamers in an aptamer library, which can perform data mining and more in-depth analysis on the aptamer library. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] [ Figure 1 ] Figure 1 The top example graph shows typical NGS data of an aptamer library, indicating that low-abundance aptamers are below the detection limit and difficult to analyze due to low read counts. The bottom example graph shows NGS data of an aptamer library in which high-abundance aptamers have been depleted, which results in an increase in read counts of low-abundance aptamers.

[0050] [ Figure 2 ] Figure 2 An exemplary strategy for sequence depletion in an aptamer library is shown. Antisense oligonucleotides (ASOs) targeting abundant aptamers induce structural changes in the targeted aptamers, resulting in the inactivation of these aptamers, thereby inhibiting the interaction of abundant aptamers with their respective target molecules.

[0051] [ Figure 3 ] Figure 3 and 4 is a graph showing that ASO depletion of high-abundance aptamers in an aptamer library resulted in an increase in the percentage of reads of low-abundance aptamers. Figure 3Shown are exemplary results of efficient and specific inactivation of high abundance aptamers by ASOs. The first 18 (1-9, 11-14, and 16-20) high abundance aptamers were specifically and effectively depleted by ASO modeling, and the remaining sequences showed an increase in the percentage of reads. In order to test the knockdown specificity, no ASO targeting aptamers #10 and #15 was designed. Therefore, after contacting the aptamer library with the ASOs, aptamers #10 and #15 showed an increase in the percentage of reads.

[0052] [ Figure 4 ] Figure 3 and 4 is a graph showing that ASO depletion of high-abundance aptamers in an aptamer library resulted in an increase in the percentage of reads of low-abundance aptamers. Figure 4 It was shown that knocking down high-abundance aptamers created more space for NGS analysis of low-abundance sequences.

[0053] [ Figure 5 ] Figure 5-7 Exemplary fold changes in sequence frequency after different PCR programs are shown. The top 100 aptamers are shown. Figure 5-6 Shown is a comparison of emulsion PCR results obtained by simultaneous replication of the same aptamer library ( Figure 5 ) and universal PCR ( Figure 6 )Exemplary data of PCR products between the methods.

[0054] [ Figure 6 ] Figure 5-7 Exemplary fold changes in sequence frequency after different PCR programs are shown. The top 100 aptamers are shown. Figure 5-6 Shown is a comparison of emulsion PCR results obtained by simultaneous replication of the same aptamer library ( Figure 5 ) and universal PCR ( Figure 6 )Exemplary data of PCR products between the methods.

[0055] [ Figure 7 ] Figure 5-7 Exemplary fold changes in sequence frequencies after different PCR programs are shown. Figure 7 Data for PAGE detection of PCR products are shown. Emulsion conditions were as follows: 1; combination of sonication and vortexing, 2; vortexing, 3; stirring by magnetic stir bar, 4; universal PCR.

[0056] [ Figure 8 ] Figure 8 and 9 An exemplary NGS analysis of an aptamer library is shown. Figure 8 An exemplary NGS analysis of an aptamer library depleted or not of a high abundance aptamer using 18 different ASOs is shown. In this experiment, sequences 10 and 15 were not targeted by any ASO to study ASO specificity.

[0057] [ Fig. 9 ] Figure 8 and 9 An exemplary NGS analysis of an aptamer library is shown. Fig. 9 An exemplary NGS analysis of an aptamer library depleted or not of a high abundance aptamer using 52 different ASOs is shown. A non-targeted sequence is shown after sequence 53 for comparison.

[0058] [ Fig.10 ] Fig.10 and 11 Shown are the changes in sequence frequency of the top 500 sequences after depletion of aptamers by addition of 52 different ASOs. Fig.10 Addition of ASOs prior to library denaturation is shown to strongly remove target sequences. Data are plotted against aptamer sequence to show changes in sequence frequency with or without ASOs. Sequences that were not targeted by the ASOs but were reduced by the addition of ASOs are shown separately as non-specific effects.

[0059] [ Fig.11 ] Fig.10 and 11 Shown are the changes in sequence frequency of the top 500 sequences after depletion of aptamers by addition of 52 different ASOs. Fig.11 Addition of ASOs after library denaturation is shown to more specifically remove target sequences. Sequences not targeted by the ASOs but reduced by the addition of ASOs are shown separately as non-specific effects.

[0060] [ Fig.12 ] Fig.12 Exemplary NGS data are shown, demonstrating the number of sequences detected due to ASO-dependent depletion of highly abundant aptamers. DETAILED DESCRIPTION

[0061] The present disclosure relates, at least in part, to methods for mining sequence information of low-abundance aptamers in aptamer libraries. The aptamer library contains high-abundance aptamers (e.g., aptamers with a sequence frequency level higher than a certain percentage in a sequencing reaction) and low-abundance aptamers (e.g., aptamers with a sequence frequency level lower than a certain percentage in a sequencing reaction). In some embodiments, during the sequencing reaction, due to the low readings of the low-abundance aptamers (e.g., low readings below the detection limit), the high-abundance aptamers mask the sequence information of the low-abundance aptamers, making them unable to be effectively analyzed. In some embodiments, the present disclosure provides methods for depleting high-abundance aptamers (e.g., by antisense oligonucleotides (ASOs) targeting high-abundance aptamers) so that low-abundance aptamers can be effectively amplified (e.g., by emulsion PCR) and sequenced.

[0062] As used herein, the term "aptamer" refers to an oligonucleotide (e.g., a single-stranded DNA (ssDNA) or 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 20 to 60 nucleotides, 25 to 55 nucleotides, 30 to 50 nucleotides, 35 to 45 nucleotides, 20 to 50 nucleotides, 20 to 40 nucleotides, 25 to 40 nucleotides, 20 to 30 nucleotides, 30 to 40 nucleotides, 30 to 60 nucleotides, 40 to 60 nucleotides or 50 to 60 nucleotides. In some embodiments, the target molecule of the aptamer includes proteins, peptides, carbohydrates, small molecules, toxins and cells (e.g., living cells). The aptamer binds to its target with high affinity, selectivity and specificity (see, e.g., non-patent literature 1 and 2). The aptamer binding is not determined by the primary sequence, but by its tertiary structure. Target recognition and binding by aptamers involve three-dimensional shape-dependent interactions as well as hydrophobic interactions, base stacking, and intercalation. Aptamers have advantages over antibodies because they can be fully engineered in vitro, are easily produced by chemical synthesis, have desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.

[0063] The concept of aptamer in vitro evolution was introduced in 1990 and is called systematic evolution of ligands by exponential enrichment or SELEX (Non-Patent Documents 3 to 7). SELEX combines combinatorial library screening rules with in vitro evolution to enrich aptamers (e.g., DNA aptamers) for various target molecules. The SELEX process involves three interrelated steps: (i) repeatedly incubating multiple candidate aptamers with target molecules to allow binding of high-affinity aptamers; (ii) separating high-affinity binders from low-affinity binders and / or non-binders; and (iii) amplifying high-affinity binders using polymerase chain reaction (PCR). Repeat the process until high-affinity aptamers are enriched in the selection pool.

[0064] In some embodiments, after each round of selection and amplification, the resulting aptamer can be further analyzed, for example, by binding assays, diversity assays or sequencing. In conventional SELEX, the terminal aptamer library after selection is cloned, and 30-100 representatives are sequenced with Sanger sequencing. In this regard, correctly identifying candidate aptamers is the key to overall selection success. The aptamer separation process retrieves a nucleic acid library rich in sequences that specifically bind to the target. In the classical method, the enriched nucleic acid pool obtained by cloning is then subjected to Sanger sequencing of 30-100 clones, with the aim of determining several aptamer candidates for further detailed characterization. Cluster analysis helps to identify aptamer candidates. Typically, the most abundant species or representatives of the largest clusters in the sequencing pool are considered potential aptamers. Many aptamers have been separated and characterized using this understandable method. In the past decade, next-generation sequencing (NGS) technology has developed into a popular method for high-throughput aptamer sequencing. In some embodiments, the aptamers generated by each round of selection are sequenced by next-generation sequencing (NGS). However, in some cases, the most abundant aptamer sequence identified by NGS may not show optimal binding to the target due to PCR bias (Non-Patent Document 8). In addition, in some embodiments, sequence information of low-level aptamers may be lost due to low reads below the detection level.

[0065] I. Inhibition of Targeted Aptamer Clusters

[0066] The present disclosure provides, at least in part, methods for suppressing targeted aptamer clusters (e.g., high-abundance aptamers with sequence frequency levels above a certain percentage in a sequencing reaction) so that the sequence frequency of low-abundance aptamers increases and can be detected. In some embodiments, the methods provided herein enable data mining and more in-depth analysis of aptamer libraries.

[0067] In some aspects, the present disclosure provides a method for sequencing low-abundance aptamers in 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 produce an aptamer library; (ii) sequencing the aptamer library; and (iii) contacting a plurality of antisense oligonucleotides (ASOs) targeting high-abundance aptamers of the aptamer library with the aptamer library to form a mixture; wherein contacting the ASOs with the aptamer library results in inactivation of the high-abundance aptamers of the aptamer library.

[0068] In some embodiments, the plurality of aptamers capable of binding to one or more target molecules in a sample may be a plurality of aptamers enriched for target molecules 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).

[0069] In some embodiments, multiple aptamers capable of binding one or more target molecules in a sample are amplified to form an aptamer library before sequencing. In some embodiments, multiple aptamers include aptamers of different abundances (i.e., sequence order information). In some embodiments, aptamers that bind target molecules with higher affinity are present in multiple aptamers capable of binding target molecules with higher abundance than aptamers that bind target molecules with lower affinity. In some embodiments, it is crucial to retain sequence order information during amplification so that the sequencing step can correctly identify high-abundance aptamers in the aptamer library. In some embodiments, the methods described herein can use any amplification method, as long as the amplification method (e.g., PCR) can retain sequence order information in multiple aptamers capable of binding one or more target molecules (e.g., biomolecules) after forming an aptamer library by amplification.

[0070] In some embodiments, the present disclosure is based at least in part on the following discovery: multiple aptamers capable of binding one or more target molecules in a sample are amplified by emulsion PCR to retain correct sequence order information. As used herein, the term "emulsion PCR" refers to a PCR reaction performed on an aqueous droplet emulsified in the oil phase of an oil-in-water emulsion. In some embodiments, the aqueous droplet acts as a miniaturized "reactor" for each PCR reaction and is physically separated from each other without exchanging macromolecules, especially PCR products. Individual DNA molecules are compartmentalized into these different reaction droplets, thereby allowing them to be amplified independently of each other. By emulsion PCR, the formation of non-productive chimeras and other by-products is avoided, and the overall amplification deviation is reduced. (See, e.g., non-patent literature 10 and 11). In some embodiments, the emulsion PCR reaction is prepared by mixing a PCR solution with an emulsified oil. In some embodiments, the emulsified oil comprises oil (e.g., mineral oil). In some embodiments, the emulsified oil comprises 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 emulsified oil comprises 95.05% oil (e.g., mineral oil). In some embodiments, the emulsified PCR reaction is vigorously shaken to form water-in-oil emulsion droplets. In some embodiments, a pre-run of the emulsion PCR is performed with a fixed cycle prior to performing the amplification step, and the PCR products are analyzed to select an appropriate PCR cycle that provides a single PCR product population (e.g., displayed as a clear single band on DNA electrophoresis). In some embodiments, 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 multiple aptamers.

[0071] In some embodiments, the method further comprises sequencing the aptamers in the aptamer library generated from the emulsion PCR amplification. As used herein, the term "sequencing" with respect to nucleic acids refers to the process of determining the nucleotide sequence of a given nucleic acid fragment. Nucleic acid (e.g., aptamer) sequencing methods are known in the art, including but 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, pyrophosphate sequencing, sequencing by synthesis (e.g., MiSeq), combined 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 tag sequencing (MPSS), polymerase clone sequencing, 454 pyrophosphate sequencing, Illumina (Solexa) sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, microfluidic system). In some embodiments, the aptamer is sequenced by next generation sequencing (e.g., MiSeq).

[0072] In some embodiments, the sequencing data (e.g., NGS data) of the aptamer library comprises high-frequency regions corresponding to high-abundance aptamers in the aptamer library and low-frequency regions corresponding to low-abundance aptamers in the aptamer library. In some embodiments, the high-abundance aptamer is 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%, higher than 0.51%, higher than 0.52%, higher than 0.53%, higher than 0.54%, higher than 0.55%, higher than 0.56%, higher than 0. In some embodiments, the aptamers with low abundance are 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 more. In some embodiments, the sequence information of low abundance aptamers is below the detection limit, making it impossible to obtain the sequence information of these aptamers. In some embodiments, high abundance aptamers mask low abundance aptamers, making them unable to be effectively sequenced and analyzed.

[0073] The present disclosure is based at least in part on the discovery that by removing high-abundance aptamers from an aptamer library, sequence information of low-abundance aptamers can be obtained in the next round of selection. In some aspects, the methods provided herein further include contacting a plurality of antisense oligonucleotides (ASOs) targeting high-abundance aptamers of an aptamer library with the aptamer library to form a mixture. In some embodiments, the ASO targeting high-abundance aptamers of an aptamer library is designed based on the sequence information of the high-abundance aptamers obtained from the sequencing reaction.

[0074] 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 it hybridizes, wherein such hybridization results in at least one antisense activity (eg, inactivation of the aptamer).

[0075] In some embodiments, the ASO targeting an aptamer is designed to cause a conformational change in the aptamer so that it no longer binds to its target molecule. In some embodiments, the ASO targeting an aptamer comprises a region complementary 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 an aptamer comprises a region complementary to any one of the high abundance aptamers in the aptamer library. In some embodiments, the aptamer-targeting ASO comprises a region of complementarity 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 contiguous nucleotides of the nucleotide sequence of any one of the highly abundant aptamers in the aptamer library. In some embodiments, the ASO targeting an aptamer comprises a region of complementarity 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 contiguous nucleotides, in addition to at least 1, at least 2, at least 3, at least 4, or at least 5 mismatches of the aptamer.

[0076] ASOs can have various 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, 8 to 50 nucleotides, 8 to 40 nucleotides, 8 to 30 nucleotides, 10 to 15 nucleotides, 10 to 20 nucleotides, 15 to 25 nucleotides, or 21 to 23 nucleotides in length.

[0077] In some embodiments, for the purposes of the present disclosure, an ASO specifically hybridizes (e.g., has complementarity) to an aptamer, where binding of the ASO to the aptamer mRNA interferes with the normal function of the aptamer to cause a loss of activity (e.g., inhibits binding to the target molecule of the aptamer), and where it is desired to avoid nonspecific binding, such as under conditions where the assay is performed under suitable stringent conditions, there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense oligonucleotide sequence to a non-target aptamer. Thus, in some embodiments, the ASO may 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 does not need to be 100% complementary to a consecutive region of the aptamer to specifically hybridize or be specific to the aptamer.

[0078] In some embodiments, one or more thymine bases (T) in any one ASO may optionally be a uracil base (U), and / or one or more U may optionally be a T.

[0079] The antisense oligonucleotides described herein can be modified, e.g., to comprise modified sugar moieties, modified internucleotide linkages, modified nucleobases, modified nucleotides, and / or (e.g., and) combinations thereof.

[0080] In some embodiments, the antisense oligonucleotides described herein comprise at least one nucleoside modified at the 2' position of the sugar. In some embodiments, the oligonucleotide comprises at least one 2'-modified nucleoside. In some embodiments, all nucleosides in the oligonucleotide are 2'-modified nucleosides.

[0081] In some embodiments, the antisense oligonucleotides described herein comprise 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA) modified nucleosides. In some embodiments, the antisense oligonucleotides comprise one or more 2'-O-methoxyethyl (2'-MOE) modified nucleosides. In some embodiments, each nucleoside of the antisense oligonucleotide is a 2'-O-methoxyethyl (2'-MOE) modified nucleoside. In some embodiments, the antisense oligonucleotides described herein comprise one or more 2'-4' bicyclic nucleosides, wherein the ribose ring comprises a bridge portion connecting two atoms in the ring, for example, connecting the 2'-O atom to the 4'-C atom via a methylene (LNA) bridge, an ethylene (ENA) bridge, or an (S)-constrained ethyl (cEt) bridge. Examples of LNAs are described in International Patent Application Publication No. WO 2008 / 043753, published on April 17, 2008 and entitled "RNA Antagonist Compounds for Modulating PCSK9," the contents of which are incorporated herein by reference in their entirety. Examples of ENAs are provided in International Patent Publication No. WO 2005 / 042777, published May 12, 2005, and 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, Oxford, 49:171-172, 2005; the disclosures of which are incorporated herein by reference in their entirety. Examples of cEt are provided in US Patents 7,101,993; 7,399,845; and 7,569,686, each of which is incorporated herein by reference in its entirety.

[0082] In some embodiments, the antisense oligonucleotide comprises a modified nucleoside disclosed in one of the following U.S. patents or patent application publications: U.S. Patent No. 7,399,845, issued on July 15, 2008, and entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Patent No. 7,741,457, issued on June 22, 2010, and entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Patent No. 8,022,193, issued on September 20, 2011, and entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Patent No. 7,569,684, issued on August 4, 2009, and entitled “Compounds and Methods for Synthesizing Bicyclic Nucleic Acid Analogs”; 6; U.S. Patent 7,335,765, issued on February 26, 2008, and entitled “Novel Nucleoside and Oligonucleotide Analogs”; U.S. Patent 7,314,923, issued on January 1, 2008, and entitled “Novel Nucleoside and Oligonucleotide Analogs”; U.S. Patent 7,816,333, issued on October 19, 2010, and entitled “Oligonucleotide Analogs and Methods of Using the Same” and U.S. Publication No. 2011 / 0009471, issued on February 17, 2015, and entitled “Oligonucleotide Analogs and Methods of Using the Same,” now U.S. Patent 8,957,201, the entire contents of each of which are incorporated herein by reference.

[0083] In certain embodiments, antisense oligonucleotides can contain phosphorothioate or other modified internucleotide connections. In certain embodiments, antisense oligonucleotides include phosphorothioate internucleotide connections. In certain embodiments, antisense oligonucleotides are included in phosphorothioate internucleotide connections between at least two nucleotides. In certain embodiments, antisense oligonucleotides are included in phosphorothioate internucleotide connections between all nucleotides. For example, in certain embodiments, antisense oligonucleotides are included in the first, second and / or (for example, and) third internucleotide connections of the 5' or 3' ends of the nucleotide sequence.

[0084] Phosphorus-containing linkages that can be used include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3'-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thiocarbonylphosphoramidates, thiocarbonylalkylphosphonates, thiocarbonylalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linkage analogs of these as well as those with reversed polarity, wherein adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see U.S. Pat. No. 3,687,808; and 5,625,050.

[0085] In some embodiments, the method includes designing multiple ASOs targeting multiple high-abundance aptamers (e.g., aptamers with a sequence frequency level greater than 0.1%). In some embodiments, contacting multiple ASOs targeting multiple high-abundance aptamers results in the inactivation of high-abundance aptamers, so that they are no longer able to bind to target molecules. In some embodiments, contacting multiple ASOs targeting multiple high-abundance aptamers results in the inactivation of high-abundance aptamers, so that they are no longer able to bind to target molecules. In some embodiments, contacting multiple ASOs targeting multiple high-abundance aptamers results in depletion or knockdown of high-abundance aptamers in an aptamer library. In some embodiments, contacting multiple ASOs targeting multiple high-abundance aptamers results in depletion or knockdown of high-abundance aptamers in an aptamer library by 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%.

[0086] In some embodiments, the aptamer library is denatured and renatured to promote the formation of the 3D conformation of the aptamer. In some embodiments, the aptamer library is denatured including heating the aptamer library for a period of time at a temperature sufficient to destroy the 3D structure of the aptamer. In some embodiments, the aptamer library is denatured including heating the aptamer library at 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, the aptamer library is denatured including heating the aptamer library at 95°C for 3 minutes. In some embodiments, the denatured aptamer is renatured to form the correct 3D conformation. In some embodiments, the aptamer in the aptamer library is renatured including cooling the aptamer library at a certain temperature for a period of time to a temperature at which the 3D structure of the aptamer is re-formed. In some embodiments, the methods described herein include contacting the ASO with the aptamer library prior to denaturation of the aptamer library. Contacting the ASO with the aptamer library prior to denaturation results in a stronger knockdown efficiency, but a higher non-specific knockdown. In some embodiments, the methods described herein include contacting the ASO with the aptamer library after renaturation of the aptamer library. Contacting the ASO with the aptamer library prior to denaturation results in a higher specific knockdown.

[0087] In some embodiments, the methods described herein further include enriching a plurality of aptamers capable of binding to one or more target molecules in a sample in the following steps: 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; purifying the plurality of aptamer-target molecule complexes; and extracting a plurality of aptamers capable of binding to one or more target molecules from the aptamer-target molecule complexes.

[0088] As used herein, the term "aptamer-target molecule complex" refers to a molecular complex formed between an aptamer and its 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 to a binding partner (e.g., a target molecule) with a degree of affinity or avidity that enables the molecule to be used to distinguish the binding partner from an appropriate control in a binding assay or other binding context. With respect to aptamers, the term "specific binding" refers to the ability of an aptamer to bind to a target molecule with a degree of affinity or avidity compared to one or more appropriate reference target molecules, which enables the aptamer to be used to distinguish a specific target molecule from other target molecules. In some embodiments, if the K of the aptamer binding target molecule D is at least about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10M, 10 -11 M, 10 -12 M, 10 -13 M or lower, the aptamer specifically binds to the target molecule.

[0089] As used herein, the term "candidate aptamer" refers to a pool of random aptamers or random oligonucleotides, which can be aptamers to be subjected to the methods described herein for enriching aptamers capable of binding to one or more target molecules (e.g., biomolecules) in a sample (e.g., a biological sample).

[0090] The initial pool of candidate aptamer sequences comprises a random core sequence of 20-60-nt long species. In some embodiments, the core sequence is flanked by regions (e.g., primer binding sites) for library re-amplification. In some embodiments, it is important to have a large diversity in the initial candidate aptamer pool so that target binding aptamers may be present in the initial candidate aptamers.

[0091] In some embodiments, when generating the initial plurality of candidate aptamers, the ratio of nucleotides is optimized, for example, A:C:G:T is 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 comprises 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 One or more candidate aptamers.

[0092] In some embodiments, the candidate aptamer is an initial pool of random aptamers or random oligonucleotides, which may be an aptamer that has not yet been selected. In some embodiments, the candidate aptamer is a plurality of aptamers obtained from the last round of enrichment using the methods described herein that can bind to one or more target molecules (e.g., biomolecules) in a sample (e.g., a biological sample). In some embodiments, a plurality of candidate aptamers are DNA or RNA. In some embodiments, the candidate aptamer is a single-stranded DNA (ssDNA). In some embodiments, a plurality of candidate aptamers are folded with their appropriate tertiary structure to bind to a target molecule. In some embodiments, a plurality of candidate aptamers comprise modified nucleotides. Modified nucleotides have been previously described, see, e.g., non-patent document 12. In some embodiments, each of a plurality of candidate aptamers comprises at least one modified nucleotide. In some embodiments, each of a plurality of candidate aptamers comprises a modified nucleotide (e.g., a modified nucleotide that can be used as a DNA polymerase substrate). Modified nucleotides are well known in the art (see, e.g., patent document 1 and non-patent document 13 and 14;). In some embodiments, each of the plurality of candidate aptamers includes, but is not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylquenoside, 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, β-D-mannosyl braided glycoside, 5'-methoxycarboxymethyl uracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, braided glycoside, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, 2,6-diaminopurine or 5-tryptamine-uracil. In some embodiments, the modified nucleotide is a 2'-modified nucleotide. For example, 2'-modified nucleotides can be 2'-deoxy, 2'-fluoro, 2'-O-methoxyethyl, 2'-amino, and 2'-aminoalkoxy modified nucleotides. In some embodiments, each of the plurality of candidate aptamers comprises, but is not limited to, one or more 5-tryptamine-uracils in place of thymine.In some embodiments, at least one thymine in each of the plurality of candidate aptamers is replaced with 5-tryptamine-uracil. In some embodiments, all thymines in each of the plurality of candidate aptamers are replaced with 5-tryptamine-uracil.

[0093] In some embodiments, each of a plurality of candidate aptamers comprises a detectable label. A detectable label can promote the detection of an aptamer-target molecule (e.g., biomolecule) complex. In some embodiments, a detectable label is a protein capable of producing a colorimetric change. In some embodiments, the protein capable of producing a colorimetric change is alkaline phosphatase, horseradish peroxidase, or luciferase. In some embodiments, a detectable label is a fluorescent molecule. In some embodiments, fluorescent molecules include but are not limited to fluorescent dyes TYE665, fluorescent 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 a plurality of candidate aptamers is labeled at a 5' terminal phosphate group. In some embodiments, each of a plurality of candidate aptamers is labeled at its 3' terminal hydroxyl group. In some embodiments, each of a plurality of candidate aptamers comprises one or more than one detectable label. In some embodiments, each of the plurality of candidate aptamers comprises a TYE665 fluorophore at the 5' end.

[0094] In some embodiments, a plurality of candidate aptamers are prepared in a selection buffer prior to contacting a target molecule (e.g., a biomolecule) in a sample (e.g., a biological sample). In some embodiments, the selection buffer comprises nonidet-P40. In some embodiments, the selection buffer comprises nonidet-P40 at a concentration between 0.001% and 0.01%. In some embodiments, the selection buffer comprises nonidet-P40 at a concentration of 0.005%. In some embodiments, the selection buffer comprises a salt (e.g., a magnesium ion) capable of stabilizing the tertiary structure of the aptamer. In some embodiments, the selection buffer comprises magnesium ions at a concentration of 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 comprises magnesium at a concentration of 1 mM. In some embodiments, the magnesium ions are in the form of magnesium chloride. In some embodiments, a plurality of candidate aptamers are expressed in a selection buffer at a concentration of 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 10 ... The invention can be prepared with a concentration of between 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.

[0095] In some embodiments, enriching a plurality of aptamers is also described as selecting a plurality of aptamers. The terms "enriching," "enrichment," or "enrichment process" are used interchangeably with selecting, selecting, or selecting process, respectively. In some embodiments, enriching a plurality of aptamers comprises 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 (e.g., biomolecules). In some embodiments, enriching a plurality of aptamers comprises selecting aptamers that have a higher affinity for one or more target molecules (e.g., biomolecules) relative to aptamers that have a lower affinity for one or more target molecules (e.g., biomolecules).

[0096] In some embodiments, the methods described herein include contacting a plurality of candidate aptamers with a sample. In some embodiments, the sample comprises one or more target molecules (e.g., proteins, nucleic acids, toxins, and / or small molecules). In some embodiments, the sample comprises one target molecule. In some embodiments, the sample comprises more than one target molecule.

[0097] In some embodiments, the methods described herein include contacting a plurality of candidate aptamers with a composite sample (e.g., a biological sample) comprising a plurality of different target molecules. In some embodiments, the composite sample comprises a plurality of different target molecules of the same type (e.g., the composite sample contains a plurality of different proteins). In some embodiments, the composite sample contains a plurality of different types of target molecules (e.g., the composite sample contains proteins, nucleic acids, small molecules, toxins, etc.). In some embodiments, the composite sample contains different types of target molecules, and each type of target molecule further contains different single target molecules (e.g., the composite 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 composite 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, atmosphere, external space, etc.), manufacturing samples (e.g., samples obtained from bioreactors, samples obtained from HPLC flow-throughs, samples containing small molecule drug intermediates, etc.). In some embodiments, the composite sample is a biological sample. As used herein, the term "biological sample" refers to a sample obtained from a biological subject.

[0098] 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, cheek swab, feces, gastrointestinal fluid, liposuction samples, saliva, milk, nasal swab, peritoneal fluid, semen, sputum, synovial fluid, tears, vaginal fluid, tissue biopsy, autopsy samples, cells or cell lysates, cultured cells, tissue samples (e.g., tissue samples from humans, 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), a protein, a peptide, a lipid, a polysaccharide, a proteoglycan, and a 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 having, suspected of having, or at risk of developing a disease.

[0099] In some embodiments, the target molecule (e.g., biomolecule) in the sample (e.g., biological sample) is denatured before contacting with a plurality of candidate aptamers. In some embodiments, the target molecule (e.g., biomolecule) in the sample (e.g., biological sample) is not denatured before contacting with a plurality of candidate aptamers. As used herein, the term "denature, denaturation, or denatured" refers to conditions that subject a sample (e.g., biological sample) to destruction of connections (e.g., disulfide connections), 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, which are responsible for the highly ordered structure of the target molecule (e.g., biomolecules such as proteins) in its natural (native) state. In some embodiments, the denatured sample comprises a sample in which the biomolecules (e.g., proteins or nucleic acids) in the sample have lost their quaternary, tertiary, and secondary structures, thereby retaining only their primary structure (e.g., linear amino acid sequence or nucleic acid sequence), such that the biomolecules do not retain their respective structures and / or specified functions. In some embodiments, the denatured sample comprises the following sample, wherein the biomolecule (e.g., protein or nucleic acid) has been degraded into fragments so that the biomolecule no longer retains its structure and / or specified function. Therefore, the denatured sample comprises biomolecules (e.g., proteins or nucleic acids) that do not retain their specified functions (e.g., binding capacity, biological activity, etc.). On the contrary, the non-denatured sample is a sample containing target molecules (e.g., biomolecules) that retain their natural conformation and their respective specified functions. Methods for denaturing the target molecules in the sample are known in the art, such as by heating, by treating with alkali, acid, urea or detergent, or by vigorous shaking. In some embodiments, the target molecule (e.g., biomolecule) is in its natural conformation. In some embodiments, it is advantageous to contact a plurality of candidate aptamers with target molecules (e.g., biomolecules) in their natural conformations, because the selected aptamers can bind to their target molecules (e.g., biomolecules) under other conditions (e.g., in a subject) except the conditions for selecting the aptamer. Those of ordinary skill in the art will understand that denaturation includes conditions (e.g., heating, sonication, incubation in the presence of a detergent, etc.) that subject a target molecule (e.g., a biomolecule) in a sample (e.g., a biological sample) to any 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) sufficient to render it unable to perform its designated function. As used herein, the term "undenatured" refers to maintaining a structure of a target molecule (e.g., a biomolecule) in a sample (e.g., a biological sample) sufficient to perform its designated function. In some embodiments, the candidate aptamers of the present disclosure can be contacted with an undenatured biological sample.In some embodiments, 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 and their respective designated functions.

[0100] The present disclosure recognizes the difficulty of enriching aptamers that can bind to target molecules in their native conformations in complex samples (e.g., biological samples). The present disclosure also identifies the disadvantages of using one-dimensional electrophoresis (1D electrophoresis) and / or running electrophoresis under mild conditions (e.g., low salt conditions) to enrich aptamer-target molecule (e.g., biological molecule) complexes. For example, and without wishing to be bound by any particular theory, 1D electrophoresis may not be able to separate aptamer-target molecule complexes from aptamers that bind to target molecules due to nonspecific binding. With respect to aptamers, as used herein, the term "nonspecific binding" refers to the ability of an aptamer to bind to a molecule with a certain degree of affinity or avidity compared to one or more appropriate reference target molecules, which makes the aptamer unable to be used to distinguish the molecule from other molecules. In some embodiments, if the K of the aptamer binding molecule is D For 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M, 10 -1 M or higher, the aptamer binds nonspecifically to the molecule.

[0101] Additionally, the conditions of 1D electrophoresis may not be sufficient to disrupt the nonspecific binding between certain aptamers and target molecules. Therefore, the present disclosure attempts to enrich multiple aptamers that can bind to one or more biomolecules in a biological sample by enriching the aptamer-target molecule complex by 2D electrophoresis. 2D electrophoresis can be performed under salt conditions that are sufficient to disrupt the nonspecific interactions between the aptamer and the target molecule but not sufficient to disrupt the specific binding between the aptamer and its target molecule. Therefore, the present disclosure enables the simultaneous enrichment of multiple aptamers that can bind to multiple target molecules (e.g., biomolecules) in a complex sample (e.g., a biological sample).

[0102] In some embodiments, the sample (e.g., biological sample) is not diluted prior to contacting with the plurality of candidate aptamers. In some embodiments, the sample (e.g., biological sample) is diluted prior to contacting with the plurality of candidate aptamers. In some embodiments, prior to contacting the biological sample with the plurality of candidate aptamers, the sample is diluted at a concentration of 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. The biological sample is diluted in a ratio of between 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. In some embodiments, the biological sample is serum, and the serum is diluted at a ratio of 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 1:100, 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 1:200. In some embodiments, the serum is diluted at a ratio of 1:200 prior to contacting 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. In some embodiments, the biological sample is diluted in any suitable dilution buffer prior to contact with a plurality of candidate aptamers. Non-limiting examples of dilution buffers include phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline (DPBS), Hanks balanced salt solution (HBSS), Dulbecco's modified Eagle medium (DMEM). One of ordinary skill can select a suitable dilution buffer according to the biological sample used.

[0103] In some embodiments, the method further includes contacting the sample (e.g., biological sample) with a plurality of competing nucleic acids prior to contacting the sample (e.g., biological sample) with a plurality of candidate aptamers. The competing nucleic acids are used to block non-specific binding between the aptamers and the target molecule. In some embodiments, the competing nucleic acids are a set of random unrelated nucleic acids. In some embodiments, the competing nucleic acids are salmon sperm DNA.

[0104] In some embodiments, contacting a plurality of candidate aptamers with a sample (e.g., a biological sample) results in the binding of the aptamers to their target molecules (e.g., biomolecules) to produce a composition. In some embodiments, the composition comprises an aptamer-target molecule (e.g., aptamer-biomolecule) complex. In some embodiments, the composition comprises an unbound aptamer. In some embodiments, the composition comprises an unbound target molecule (e.g., unbound biomolecule). In some embodiments, the composition comprises an aptamer-target molecule (e.g., aptamer-biomolecule) complex, an unbound aptamer, and / or an unbound target molecule (e.g., unbound biomolecule). In some embodiments, the composition further comprises an aptamer that binds to a biomolecule due to nonspecific binding.

[0105] In some embodiments, the methods described herein include separating an aptamer-target molecule (e.g., aptamer-biomolecule) complex from an unbound aptamer. In some embodiments, the methods described herein include separating an aptamer-target molecule (e.g., aptamer-biomolecule) complex from an unbound aptamer. In some embodiments, the methods described herein include separating an aptamer-target molecule (e.g., aptamer-biomolecule) complex from an aptamer that binds to a biomolecule due to non-specific binding. In some embodiments, the methods of the invention include enriching an aptamer-target molecule (e.g., aptamer-biomolecule complex) from a composition that also includes an unbound aptamer and / or an aptamer that binds to a target molecule due to non-specific binding.

[0106] In some embodiments, enriching aptamer-target molecules (e.g., aptamer-biomolecule complexes) from a composition that also includes unbound aptamers and / or aptamers that bind to target molecules due to non-specificity includes electrophoresis of the composition. As used herein, the term "electrophoresis" refers to a technique for separating molecules (e.g., DNA, RNA, proteins, aptamer-target molecule complexes) based on the size and charge of the separated molecules. The electrophoresis system includes two oppositely charged electrodes (anode, cathode) connected by a conductive electrophoretic medium. The current is used to move the molecules to be separated through the electrophoretic medium. In some embodiments, a negative charge is applied to move the molecules toward the positive charge. In addition, the electrophoretic medium typically includes pores that allow smaller molecules to move faster than larger molecules. In some embodiments, the present disclosure is based on the following theory: the size of multiple aptamer-target molecule (e.g., biomolecule) complexes is different from that of unbound aptamers (e.g., biomolecules). In some embodiments, the size of the unbound aptamer is smaller than that of the aptamer-target molecule (e.g., biomolecule) and therefore migrates faster during electrophoresis. In some embodiments, the sizes of individual aptamers-target molecules (e.g., biomolecules) in a plurality of aptamer-target molecule (e.g., biomolecule) complexes are different from each other. In some embodiments, individual aptamers-target molecules (e.g., biomolecules) in a plurality of aptamer-target molecule (e.g., biomolecule) complexes migrate at different speeds during electrophoresis. In some embodiments, the methods described herein include subjecting the composition to electrophoresis in a first direction in a first electrophoretic medium. In some embodiments, the size difference between the aptamer-target molecule (e.g., biomolecule) complex and the aptamer that binds to the target molecule (e.g., biomolecule) due to non-specific binding is insufficient to separate the aptamer-target molecule (e.g., biomolecule) complex from the aptamer that binds to the target molecule (e.g., biomolecule) due to non-specific binding. In some embodiments, after electrophoresis in the first direction, a portion of the first electrophoretic medium contains the aptamer-target molecule complex, and the same portion may also contain the aptamer that binds to the target molecule (e.g., biomolecule) due to non-specific binding.

[0107] Thus, in some embodiments, the present disclosure attempts to enrich a plurality of aptamers capable of binding to one or more biomolecules in a biological sample by enriching aptamer-target molecule (e.g., biomolecule) complexes by 2D electrophoresis. In some embodiments, the method includes subjecting a portion of a first electrophoretic medium containing an aptamer-target molecule complex to electrophoresis in a second direction in a second electrophoretic medium, the portion also containing an aptamer that binds to a target molecule (e.g., biomolecule) due to non-specific binding. The term "second direction" as used herein refers to a direction different from the first direction. In some embodiments, the second direction is orthogonal to the first direction. In some embodiments, the method further includes cutting out a portion of the first electrophoretic medium containing an aptamer-target molecule complex (which may also contain an aptamer that binds to a target molecule (e.g., biomolecule) due to non-specific binding) from the remainder of the first electrophoretic medium. In some embodiments, the method further includes assembling the cut portion of the first electrophoretic medium containing an aptamer-target molecule complex (which may also contain an aptamer that binds to a target molecule (e.g., biomolecule) due to non-specific binding) in a hole in the second electrophoretic medium. Electrophoresis of the composition in a second dimension disrupts weak non-specific binding between the aptamer and the target molecule (e.g., biomolecule), such that the 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 a diagonal region of the second electrophoretic medium after electrophoresis in the second direction. In some embodiments, the method further comprises cutting a portion of the second electrophoretic medium containing the aptamer-target molecule complex from the remainder of the second electrophoretic medium.

[0108] In some embodiments, the method further comprises extracting an aptamer capable of binding one or more target molecules (e.g., a biomolecule) from a second electrophoretic medium portion containing the aptamer-target molecule complex. In some embodiments, the aptamer is separated from the aptamer-target molecule complex during this step. Methods for extracting aptamers from a portion of an electrophoretic medium have been previously described, such as by commercially available kits, such as Oligo Clean & Concentrator Kits from Zymo Research, QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany), Wizard SV Gel and PCR Cleanup System (Promega, Madison, Wisconsin, USA), and GENECLEAN (registered trademark) II Kit (MP BIOMEDICALS, Solon, Ohio, USA), or by the methods described in Non-Patent Literature 15.

[0109] In some embodiments, the electrophoretic medium comprises a hole that allows the molecules subjected to electrophoresis to migrate. Non-limiting examples of electrophoretic media include agarose, polyacrylamide, a silicon dioxide matrix or starch. In some embodiments, the first electrophoretic medium is agarose. In some embodiments, the second electrophoretic medium is agarose. In some embodiments, the first electrophoretic medium is agarose, and the second electrophoretic medium is agarose. The method for preparing agarose gel for electrophoresis is known in the art, for example, as described in non-patent literature 16. In some embodiments, agarose gel is prepared from dry agarose powder (for example, agarose powder is dissolved in a suitable buffer such as Tris buffer by heating, and agarose is solidified by cooling to room temperature). In some embodiments, agarose gel is a precast gel purchased from a supplier. In some embodiments, the first electrophoretic medium is an agarose gel and comprises agarose at any concentration between 0.5% and 3% (e.g., 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%), 1%-2%, 0.5%-1%, 1.5%-2%, or 2%-2.5%.

[0110] In some aspects, the disclosure also contemplates optimal conditions (e.g., salt, temperature) for performing electrophoresis. In some embodiments, 2D electrophoresis is performed under salt conditions (e.g., the electrophoresis medium comprises 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., the electrophoresis medium comprises a certain concentration of ions) that are sufficient to destroy the nonspecific interactions between the aptamer and the target molecule but not sufficient to destroy the specific binding between the aptamer and its target molecule. In some embodiments, the first electrophoresis medium comprises sodium ions (e.g., sodium chloride), potassium ions, lithium ions, ammonium ions, or any combination thereof, at a concentration sufficient to destroy the nonspecific 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). As used herein, the term "a concentration of any combination thereof" refers to the total concentration of one or more ions in a combination (e.g., any combination described herein). For example, a combination of two ions at concentrations between X and Y means that the total concentration of the two ions is within the range of X and Y; a combination of three ions at concentrations between X and Y means that the total concentration of the three ions is within the range of X and Y, and so on. Any unit known in the art can be used to describe concentration, for example, 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, when the electrophoretic medium contains a combination of ions (e.g., sodium ions, potassium ions, lithium ions, and / or ammonium ions) with a total concentration between 100 mM and 200 mM, this means that the total concentration of its ions is between 100 mM and 200 mM. It is within the skill of one of ordinary skill in the art to select a concentration for each ion in the combination to achieve a total concentration within a specified range.

[0111] 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 120mM and 150mM, between 120mM and 160mM, between 120mM and 170mM, between 120mM and 130mM, between 150mM and 160mM, between 150mM and 170mM, between 150mM and 180mM, between 150mM and 190mM, between 100mM and 120mM, between 120mM and 130mM, between 130mM and 140mM, between 140mM and 150mM, between 150mM and 160mM, between 160mM and 170mM, between 170mM and 180mM, between 180mM and 190mM, or between 190mM and 200mM. In some embodiments, the first electrophoretic medium comprises sodium ions (eg, sodium chloride), potassium ions, lithium ions, ammonium ions, or any combination thereof, at a concentration between 100 mM and 200 mM (eg, any concentration between 100 mM and 200 mM).In some embodiments, the second electrophoretic medium comprises sodium chloride at a concentration between 50mM and 500mM, between 80mM and 450mM, between 100mM and 400mM, between 150mM and 350mM, between 200mM and 300mM, between 100mM and 400mM, between 100mM and 300mM, between 100mM and 200mM, between 100mM and 150mM, between 150mM and 200mM, between 110mM and 190mM, between 120mM and 180mM, between 130mM and 170mM, between 140mM and 160mM, between 120mM and 150mM or between 100mM and 120mM, between 120mM and 130mM, between 130mM and 140mM, between 140mM and 150mM, between 150mM and 160mM, between 120mM and 170mM, between 120mM and 130mM, between 150mM and 160mM, between 150mM and 170mM, between 150mM and 180mM, between 150mM and 190mM, between 100mM and 120mM, between 120mM and 130mM, between 130mM and 140mM, between 140mM and 150mM, between 150mM and 160mM, between 160mM and 170mM, between 170mM and 180mM, between 180mM and 190mM, or between 190mM and 200mM. In some embodiments, the second electrophoretic medium comprises sodium ions (eg, sodium chloride), potassium ions, lithium ions, ammonium ions, or any combination thereof, at a concentration between 100 mM and 200 mM (eg, any concentration between 100 mM and 200 mM).

[0112] Additionally, and in some embodiments, 2D electrophoresis is performed under salt conditions (e.g., a concentration of divalent ions) sufficient to stabilize the structure of the aptamer and / or aptamer-target molecule (e.g., biomolecule) complex. Non-limiting examples of divalent ions capable of stabilizing the structure of the aptamer and / or 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 electrophoretic medium comprises magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof, at a concentration sufficient to stabilize the aptamer structure and / or aptamer-target molecule (e.g., biomolecule) complex. In some embodiments, the first 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 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 ...2 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 2 mM, between 0.1 mM and 2 mM, between 0.1 mM and 2 mM, between 0.1 mM and 2 mM, between 0.1 mM and 2 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 Between 0.1mM and 1mM, between 0.1mM and 0.5mM, between 0.5mM and 10mM, between 0.5mM and 9mM, between 0.5mM and 8mM, between 0.5mM and 7mM, between 0.5mM and 6mM, between 0.5mM and 5mM, between 0.5mM and 4mM, between 0.5mM and 3mM, between 0.5mM and 2mM, between 0.Between 5mM and 1mM, between 1mM and 10mM, between 1mM and 9mM, between 1mM and 8mM, between 1mM and 7mM, between 1mM and 6mM, between 1mM and 5mM, between 1mM and 3mM, between 1mM and 2mM, between 2mM and 10mM, between 2mM and 9mM, between 2mM and 8mM, between 2mM and 7mM, between 2mM and 6mM, between 2mM and 5mM, between 2mM and 4mM, between 3mM and 10m M, between 3mM and 9mM, between 3mM and 8mM, between 3mM and 7mM, between 3mM and 6mM, between 3mM and 5mM, between 3mM and 4mM, between 4mM and 10mM, between 4mM and 9mM, between 4mM and 8mM, between 4mM and 7mM, between 4mM and 6mM, between 4mM and 5mM, between 5mM and 10mM, between 5mM and 9mM, between 5mM and 8mM, between 5mM and 7mM, between 5m Between M and 6mM, between 6mM and 10mM, between 6mM and 9mM, between 6mM and 8mM, between 6mM and 7mM, between 7mM and 10mM, between 7mM and 9mM, between 7mM and 8mM, between 8mM and 10mM, between 8mM and 9mM, between 8mM and 9mM, between 0.1mM and 5mM, between 0.5mM and 4.5mM, between 1mM and 4mM, between 2mM and 3mM, between 0.5mM and 2mM, between 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.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 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 Between 1mM and 5mM, between 0.5mM and 4mM, between 0.5mM and 3mM, between 0.5mM and 2mM, between 0.5mM and 1mM, between 1mM and 10mM, between 1mM and 9mM, between 1mM and 8mM, between 1mM and 7mM, between 1mM and 6mM, between 1mM and 5mM, between 1mM and 3mM, between 1mM and 2mM, between 2mM and 10mM, between 2mM and 9mM, between 2mM and 8mM, between 2mM and 7mM, between 2mM and 6mM, between 2mM and 5mM, between 2mM and 4mM, between 3mM and 10mM, between 3mM and 9mM , between 3mM and 8mM, between 3mM and 7mM, between 3mM and 6mM, between 3mM and 5mM, between 3mM and 4mM, between 4mM and 10mM, between 4mM and 9mM, between 4mM and 8mM, between 4mM and 7mM, between 4mM and 6mM, between 4mM and 5mM, between 5mM and 10mM, between 5mM and 9mM, between 5mM and 8mM, between 5mM and 7mM, between 5mM and 6mM, between 6mM and 10mM, between 6mM and 9mM, between 6mM and 8mM, between 6mM and 7mM, between 7mM and 10mM, between 7mM and 9mM Between 7mM and 8mM, between 8mM and 10mM, between 8mM and 9mM, between 8mM and 9mM, between 0.1mM and 5mM, between 0.5mM and 4.5mM, between 1mM and 4mM, between 2mM and 3mM, between 0.5mM and 2mM, between 0.6mM and 1.5mM, between 0.7mM and 1.3mM, between 0.8mM and 1.2mM, between 0.9mM and 1.1mM, between 0.6mM and 1.2mM, between 0.6mM and 1.2mM, between 0.7mM and 1.2mM, between 0.8mM and 1.2mM, between 0.9mM and 1mM, between 0.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 may be added to the first and / or second electrophoretic medium as any known magnesium salt (from, for example, magnesium chloride and magnesium sulfate). In some embodiments, the magnesium ions are in the form of magnesium chloride.

[0113] In some embodiments, the running buffer for electrophoresis in the first direction comprises boric acid at a concentration between 40mM and 100mM. In some embodiments, the running buffer for electrophoresis in the first direction comprises tris(hydroxymethyl)aminomethane at a concentration between 40mM and 100mM. In some embodiments, the running buffer for electrophoresis in the second direction comprises boric acid at a concentration between 40mM and 100mM. In some embodiments, the running buffer for electrophoresis in the second direction comprises tris(hydroxymethyl)aminomethane at a concentration between 40mM and 100mM.

[0114] In some embodiments, 2D electrophoresis is performed at a temperature that is optimal for the migration and separation of molecules in the composition (e.g., aptamer-biomolecule complexes and unbound aptamers). In some embodiments, the first direction electrophoresis 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 first direction electrophoresis 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 second direction electrophoresis 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 second direction electrophoresis 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 electrophoretic media increases the temperature of the electrophoretic media to a temperature that may interfere with the stability of the aptamer-target molecule complex. In some embodiments, in order to maintain the temperature in the optimal temperature range described herein, the electrophoresis unit is placed in a cold water bath filled with ice.

[0115] As described herein, in some embodiments, the method further comprises extracting an aptamer capable of binding one or more target molecules (e.g., biomolecules) from a second electrophoretic medium portion 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 aptamer capable of binding one or more target molecules (e.g., biomolecules) is amplified by polymerase chain reaction (PCR). Polymerase chain reaction (PCR) is a laboratory technique for amplifying DNA sequences by using short DNA sequences called primers. Repeatedly raising and lowering the temperature of the sample to help the DNA replicase to replicate 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, computer simulation PCR, inverse PCR, ligation-mediated PCR, mini-primer PCR, multiple ligation-dependent probe amplification, multiplex PCR, nanoparticle-assisted PCR, nested PCR, overlap extension PCR, quantitative PCR, reverse complementary PCR, single-specific primer PCR, and solid phase PCR. In some embodiments, aptamers are amplified by emulsion PCR.

[0116] In some embodiments, the plurality of aptamers capable of binding one or more target molecules (e.g., biomolecules) extracted from the second electrophoretic medium portion containing the aptamer-target molecule complex comprises aptamers of different amounts (i.e., sequence order), for example, aptamers that bind to target molecules with higher affinity are present at higher levels, while aptamers that bind to target molecules with lower affinity are present at lower levels. In some aspects, the present disclosure attempts to retain sequence order information in a plurality of aptamers capable of binding to one or more target molecules (e.g., biomolecules) after forming an aptamer library by amplification. In some embodiments, the present disclosure is based on the discovery that emulsion PCR is capable of retaining sequence order information in a plurality of aptamers capable of binding to one or more target molecules (e.g., biomolecules) after forming an aptamer library by amplification.

[0117] In some embodiments, an aptamer that can bind to one or more target molecules (e.g., biomolecules) in a sample (e.g., a biological sample) is selected by multiple rounds, an aptamer that can bind to a target molecule is amplified by emulsion PCR to form an aptamer library, and the aptamer library is sequenced; and the low-abundance aptamer in the aptamer library is sequenced by knocking down the high-abundance aptamer using ASO. In some embodiments, the steps of the method 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. In some embodiments, the steps of the method described herein are repeated four times. In some embodiments, the steps of the method described herein are repeated nine times. In some embodiments, the aptamer library obtained after amplification (e.g., emulsion PCR) is used as a starting multiple candidate aptamers for the next round of contact with the target molecule (e.g., biomolecule) in the sample (e.g., biological sample).

[0118] Other embodiments

[0119] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature for the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is only an example of a general series of equivalent or similar features.

[0120] From the above description, those skilled in the art can easily determine the essential features of the present disclosure, and without departing from its spirit and scope, various changes and modifications may be made to the present invention to adapt it to various usages and conditions. Therefore, other embodiments are also within the scope of the claims.

[0121] Equivalent

[0122] Although several embodiments of the present invention have been described and illustrated herein, it will be readily apparent to those of ordinary skill in the art that various other means and / or structures for performing functions and / or obtaining results and / or one or more advantages described herein will be contemplated, and each of such changes and / or modifications is considered to be within the scope of the embodiments of the present invention described herein. More generally, it will be readily understood by those skilled in the art that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications for which the present invention is taught. Those skilled in the art will recognize or be able to determine the equivalents of the specific embodiments of the invention described herein using experimental methods that do not exceed the routine. Therefore, it should be understood that the above embodiments are presented only by way of example, and within the scope of the appended claims and their equivalents, embodiments of the present invention may be practiced in a manner different from that specifically described and required. The embodiments of the present invention disclosed herein relate to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods (if such features, systems, articles, materials, kits, and / or methods are not mutually contradictory) is included within the scope of the invention disclosed herein.

[0123] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0124] All references, patents, and patent applications disclosed herein are incorporated by reference, in some cases in their entirety, to the extent that their respective cited subject matter is incorporated herein by reference.

[0125] Unless explicitly indicated to the contrary, the indefinite articles "a" and "an" as used herein in the specification and in the claims should be understood to mean "at least one".

[0126] In the specification and in the claims, the phrase "and / or" as used herein should be understood to mean "any one or both" of the elements so combined, i.e., the elements are present jointly in some cases and separately in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so combined. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with an open language such as "comprises / includes", may refer to only A (optionally including elements other than B) in some embodiments; to only B (optionally including elements other than A) in another embodiment; to both A and B (optionally including other elements) in yet another embodiment; and so on.

[0127] In the specification and in the claims, "or" as used herein should be understood to have the same meaning as "and / or" as defined above. For example, when separating the items in a list, "or" or "and / or" should be interpreted as inclusive, that is, including at least one of a plurality of elements or a list of elements, but also including more than one element, and optionally including additional unlisted items. Only terms that clearly indicate the opposite, such as "only one of" or "exactly one of", or when used in the claims, "consisting of", will refer to including exactly one element in a plurality of elements or a list of elements. Usually, when preceded by an exclusive term, such as "any", "one of", "only one of" or "exactly one of", the term "or" as used herein should only be interpreted as indicating an exclusive alternative (i.e. "one or the other, not both"). When used in the claims, "consisting essentially of" should have its ordinary meaning when used in the field of patent law.

[0128] In the specification and in the claims, as used herein, the phrase "at least one" with respect to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, and does not exclude any combination of multiple elements in the list of elements. This definition also allows for the optional presence of elements other than the elements specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a 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") may refer to at least one (optionally including more than one) A without B (and optionally including elements other than B) in one embodiment; to at least one (optionally including more than one) B without A (and optionally including elements other than A) in another embodiment; to at least one (optionally including more than one) A and at least one (optionally including more than one) B (and optionally including other elements) in yet another embodiment; and so on.

[0129] It should also be understood that in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are recited unless explicitly indicated to the contrary.

[0130] Examples

[0131] Example 1: Materials and Methods

[0132] Antisense oligonucleotide design

[0133] Antisense oligonucleotides (ASOs) were systematically designed based on the aptamer sequence information obtained by next generation sequencing analysis (NGS) after SELEX. For all ASOs, the length of the ASO was fixed at 25-30 nucleotides. The target domain started from position 7-10 at the 5'-end of the random region. The target sequence knocked down by ASO was determined based on a sequence frequency level exceeding 0.1%-0.2% to maximize the available sequencing reads of the remaining sequence after ASO-based depletion. All ASOs are composed of DNA.

[0134] Interaction between antisense oligonucleotides and aptamer library

[0135] ASO was dissolved in deionized water before use. The working concentration of ASO was set to 100-1000 times higher than the target sequence concentration, which can be calculated based on the NGS data of the aptamer library. A 50x ASO solution was prepared by mixing ASO in a tube. The targeted nucleic acid library prepared by the 2D electrophoresis-based aptamer library generation method was diluted at a working concentration in the selection buffer (PBS containing 0.005% nonidet-P40 and 1mM magnesium chloride) for selection. Before the library was denatured, 50X ASO solution was added to the aptamer library solution to cause strong knockdown efficiency. The solution was denatured at 95°C for 3min and then slowly cooled to room temperature within 30min to form a stable ssDNA structure. In this step, the aptamer targeted by ASO is inactivated due to the inhibition of functional structure formation. If target specificity is more important than knockdown efficiency, 50x ASO solution is added after the renaturation step. The renatured library solution was mixed with an appropriately diluted biological fluid sample solution at a reaction volume of 10 μL. Before mixing with the ssDNA library solution, the biofluid sample is mixed with competitors (such as salmon sperm DNA and any unrelated oligonucleotide DNA). Dextran sulfate is also used at a concentration of 0.001% to 1% to reduce the charge-dependent nonspecific interactions of ssDNA with biomolecules. The mixture is incubated at room temperature for 10 minutes, and then 2 μL of 6x loading buffer containing 36% glycerol and 6mM magnesium chloride is quickly added to the reaction mixture, and the sample is then loaded on an agarose gel.

[0136] Preparation of agarose gels for 1D and 2D electrophoresis

[0137] Agarose gels for 1D and 2D electrophoresis were prepared as follows. Agarose powder was mixed in 1x TB buffer (80mM tris base, 80mM boric acid) at a concentration of 1.0-1.2wt%, which included 100-200mM NaCl concentration, depending on the selected conditions. The mixture was heated by microwave to completely dissolve the agarose powder. Then, after the agarose solution was cooled to about 60°C, 1M MgCl2 solution was added to a concentration of 1mM MgCl2 and mixed thoroughly. Then, 11mL of the solution was poured into a gel tray (55mm(W)x 60mm(L)) with a hole comb. The number of holes for 1D electrophoresis was 3-4 holes (10mm(W)x 1mm(L)), and 2D electrophoresis was 1 hole (45mm(W)x 2mm(L)). The thickness of the agarose gel was adjusted to be relatively thin to reduce the heat generated by the high-salt agarose gel during electrophoresis. Allow the gel to stand at room temperature for at least 3 hours but less than 6 hours before use.

[0138] Removal of targeted aptamer clusters from the library by 2D electrophoresis

[0139] Fill the electrophoresis unit, such as Mupid2, with 1X TB buffer. The 1D electrophoresis gel prepared above is placed in a gel box, and the electrophoresis unit is placed in a cold water bath filled with crushed ice. Due to the use of high-salt agarose gel, the buffer temperature is maintained between 10°C-20°C during electrophoresis. Before the sample is run, the gel is pre-run at 100V for 10min. The sample mixture is carefully loaded onto the wells of the gel. The first dimension electrophoresis is performed at 100V in the dark for 55-60min. After the run, the gel is removed from the gel box and visualized by the ChemiDoc MP imaging system (Bio-Rad). The entire area of ​​the aptamer-biomolecule complex that is usually present above the free aptamer area is excised for 2D electrophoresis. The size of the excised gel is adjusted to the wells of the 2D electrophoresis gel.

[0140] 2D electrophoresis was performed the same as 1D electrophoresis. After the run, the gel was removed from the gel box, visualized by ChemiDoc MP imaging system (Bio-Rad), and only the diagonal area formed by the aptamer-biomolecule complex was excised. The excised gel slice was placed in a microtube.

[0141] Extraction of DNA from agarose gel

[0142] The excised gel slices were melted at 95°C for 3 min. The molten agarose solution was distributed into several tubes in 110 μL aliquots. The tubes were incubated at 55°C for 1 min. 2 μL of thermostable β-agarase was added to each tube, and the tubes were incubated at 55°C for 15 min to enzymatically digest the agarose. The ssDNA library was recovered by Oligo Clean & Concentrator kit (Zuomo Research) according to the manufacturer's instructions. The recovered ssDNA was eluted with 30 μL of deionized water.

[0143] Emulsion PCR amplification of recovered ssDNA library

[0144] The recovered ssDNA was subjected to emulsion PCR amplification in a two-step process. It should be noted that all PCR reactions should be performed by emulsion PCR to retain the sequence order information in the library after removing high-frequency sequences. The preparation of the emulsion PCR solution is as follows: 100 μL of PCR solution is mixed with 250 μL of emulsified oil (4.5% Span80, 0.4% Tween 80, 0.05% Triton-X 100 and 95.05% mineral oil), and the solution is vigorously mixed with a magnetic stirring bar until completely mixed. The emulsion PCR reaction is tested in a small volume by different numbers of continuous PCR cycles. The PCR product of the specified cycle is recovered by chloroform extraction and analyzed by gel electrophoresis (6% polyacrylamide and 0.5x TBE buffer) to determine the appropriate PCR cycle, which can provide a clear single band of the DNA product without any concatemer and truncation. The remaining PCR samples are amplified by adopting a determined PCR cycle. The PCR product is recovered by chloroform extraction and purified by an Oligo Clean & Concentrator kit. The amplified DNA was eluted with 10 μL of deionized water and quantified by Qubit 4 Fluorometer (Invitrogen). This PCR amplification step was repeated until a sufficient amount of DNA was obtained.

[0145] Generation of aptamer libraries by primer extension

[0146] In some cases, 2 to 3 cycles are required to fully remove the target sequence from the library. The amplified DNA was diluted to a concentration of 1 ng / μL to prepare a template DNA sample for generating an aptamer library by primer extension. Then, 2 μL of solution was used for each PCR amplification of 100 μL. In this PCR amplification step, a 5'-biotinylated antisense strand primer was used, and the number of PCR cycles was fixed to 8 cycles. The PCR product was recovered by chloroform extraction and then purified by an Oligo Clean&Concentrator kit. Primer extension was performed on the purified DNA using a primer labeled with a 5'-TYE665 fluorophore. The product was directly fixed on streptavidin agarose beads filled with PBSN buffer (PBS containing 0.005% nonidet-P40). The beads were incubated at 16°C for 15 minutes at 1500 rpm for every 2 minutes. The beads were washed three times with PBS and filled with 40 μL of 20 mM NaOH solution to denature the product DNA to dissociate the 5'-TYE665 labeled aptamer. The bead solution was incubated at 37 ° C with 1500 rpm shaking for 1 min. The solution was centrifuged for 30 seconds by a desktop microcentrifuge. The supernatant was transferred to a new tube. The denaturation step was performed again, and the supernatant was combined in the same tube. The collected supernatant was quenched with 80 mM HCl to adjust the solution pH to about 7.0-8.0 and purified by Oligo Clean & Concentrator kit. The aptamer library was eluted with 10 μL deionized water and quantified by NanoDrop. The aptamer library was used for the next selection cycle.

[0147] Sequencing analysis of aptamer libraries

[0148] The PCR products obtained after 2D electrophoresis were subjected to emulsion PCR amplification in a 2-step process to prepare for next generation sequencing (NGS) analysis by MiSeq (illumina). The first step used an adapter sequence primer set, followed by an index sequence primer set in the second step. Thermal cycling was performed as described by the manufacturer. The PCR products were purified by NucleoSpin gel and PCR Clean-up (Macherey Nagel), quantified by Qubit 4 fluorometer in the first step, and quantified by a bioanalyzer in the second step. According to the manufacturer's instructions, the products were analyzed by MiSeq. All sequencing data for each round were generated as FASTAQ files. After extracting the aptamer domain by trimming the 5'- and 3'-primer regions, all sequences were used for frequency analysis.

[0149] Example 2: ASO depletion of high-abundance aptamers in aptamer library

[0150] This example describes a method for knocking down high-abundance aptamers in an aptamer library. In an aptamer library, high-abundance aptamers (e.g., aptamers with higher affinity for a target molecule) contain a higher number of NGS readings relative to low-abundance aptamers (e.g., aptamers with lower affinity for a target molecule). Furthermore, low-abundance aptamers exhibit lower detectability by NGS analysis, i.e., although aptamers in low-frequency regions are difficult to analyze due to lower readings, more sequences should be present in the detection limit region. When high-frequency aptamers are depleted from an aptamer library, the detectability of low-abundance aptamers that are able to bind to a target biomolecule increases, i.e., depletion of high-frequency sequences results in an increase in low-frequency sequences ( Figure 1 ).

[0151] The method further describes depleting high-abundance aptamers from an aptamer library by using an ASO. The ASO is designed to target a high-abundance aptamer (e.g., an aptamer with a percentage of reads exceeding a certain threshold). Hybridization of the ASO with the high-abundance aptamer induces a structural change that inactivates the high-abundance aptamer's ability to bind to the target molecule ( Figure 2 ).

[0152] Example 3: Manipulation of mouse serum aptamer library

[0153] The methods according to the present disclosure were used for the manipulation of aptamer libraries generated from mouse serum.

[0154] Aptamer libraries were generated and sequenced based on a method for generating aptamer libraries using separately developed unpurified biological samples.

[0155] According to the present disclosure, emulsion PCR is used to preserve sequence order information after target sequence depletion. Figure 5 and 6 Shown are exemplary data comparing PCR products between emulsion PCR and universal PCR methods obtained by simultaneously replicating the same aptamer library. Figure 7 Data for PAGE detection of PCR products are shown. Emulsion conditions were as follows: 1; combination of sonication and vortexing, 2; vortexing, 3; stirring by magnetic stir bar, 4; universal PCR.

[0156] In the validation experiment, the target aptamer sequence was selected from the high frequency sequences observed in the next generation sequencing analysis of the aptamer library. Two sequences were removed from the target list to study the specificity of the present invention. The antisense oligonucleotide library was systematically designed based only on the sequence information obtained from the next generation sequencing. The length was fixed at 30 nucleotides and the target sequence was set from position 7 of the aptamer domain. The DNA oligonucleotides were chemically synthesized without any specific chemical modification. Figure 3 and Figure 8Results are shown for the simultaneous inactivation of 18 aptamers by 18 different ASOs. In this experiment, aptamers #10 and #15 were not targeted by any ASO to study ASO specificity. Fig. 9 Results are shown for the simultaneous inactivation of 52 aptamers by 52 different ASOs. The changes in sequence frequencies upon addition of ASOs were investigated. Fig.10 The change in sequence frequency in the aptamer library is shown, where an ASO is added during aptamer denaturation to allow the ASO to hybridize with the target aptamer. Sequence comparison analysis of the nonspecific inactivation sequences by the added ASOs shows that nonspecific inactivation occurs in sequences with relatively high homology to the ASOs. Fig.11 Shown are the changes in sequence frequencies in an aptamer library where ASOs were added after aptamer structure formation to reduce unexpected hybridization between aptamers and ASOs due to high homology sequences. Fig.12 Analysis of the number of available sequences after target depletion is shown. It was found that approximately 30% or more of the sequences observed had sufficient sequence frequency for sequencing analysis ( Figure 4 ).

Claims

1. A method for sequencing low-abundance aptamers in 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; as well as (iii) contacting a plurality of antisense oligonucleotides (ASOs) targeting the high-abundance aptamers of the aptamer library with the aptamer library to form a mixture; wherein contacting the ASOs with the aptamer library results in inactivation of the high-abundance aptamers of the aptamer library.

2. The method of claim 1, further comprising selecting the plurality of aptamers capable of binding to one or more target molecules in the sample in 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 one or more target molecules from these aptamer-target molecule complexes.

3. The method of claim 2, further comprising repeating steps (a)-(c) and (i)-(iii), and wherein when step (a) is repeated, the mixture obtained from step (iii) comprises the plurality of candidate aptamers.

4. The method of claim 3, wherein the method is repeated at least 3 times.

5. The method according to any one of claims 1 to 4, further comprising sequencing the aptamer library obtained from step (iii).

6. The method of any one of claims 1-5, wherein the ASOs comprise modified nucleotides.

7. The method of any one of claims 1-6, wherein sequencing low-abundance aptamers in the aptamer library comprises next generation sequencing (NGS).

8. The method of any one of claims 1-7, wherein the sample is a biological sample.

9. The method of claim 8, wherein the biological sample is serum, plasma, cerebrospinal fluid (CSF), urine, amniotic fluid, bone marrow, bronchoalveolar lavage fluid, buccal swab, stool, 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 of claim 8 or 9, wherein the biological sample comprises target molecules comprising nucleic acids, proteins, polypeptides, carbohydrates, lipids or a combination thereof.

11. The method of any one of claims 8-10, wherein the biological sample is not denatured. 12 . The method of claim 1 , 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 of 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 of 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 of 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 of 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 of any one of claims 2 to 16, wherein step (b) comprises electrophoresing the composition in a first electrophoretic medium in a first direction to obtain a portion of the first electrophoretic medium containing the aptamer-target molecule complexes.

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

19. The method of claim 17 or 18, wherein the first electrophoretic medium is a first agarose gel.

20. The method of claim 18 or 19, wherein the second electrophoretic medium is a second agarose gel.

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

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

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

24. The method of any one of claims 19-23, wherein the first and the second electrophoretic media comprise 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 of any one of claims 19-24, wherein the first and the second electrophoretic media comprise magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof at a concentration of 1 mM.

26. The method of any one of claims 23-25, wherein the magnesium ion is in the form of magnesium chloride.

27. The method of any one of claims 19-26, wherein step (b) further comprises cutting the portion of the first electrophoretic medium comprising the aptamer-target molecule complexes from the rest of the first electrophoretic medium.

28. The method of claim 27, wherein the portion of the first electrophoretic medium is fitted into a hole in the second electrophoretic medium to perform electrophoresis in the second direction.

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

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

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

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

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

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

35. The method of any one of claims 1-34, further comprising, before step (c), cutting out the portion of the second electrophoretic medium containing the aptamer-target molecule complexes from the rest of the second electrophoretic medium, and extracting the aptamer-target molecule complexes from the portion of the second electrophoretic medium.

36. The method of any one of claims 1-35, further comprising denaturing and renaturing the aptamer library prior to contacting the ASOs with the aptamer library.

37. The method of any one of claims 1-36, further comprising denaturing and renaturing the aptamer library after contacting the ASOs with the aptamer library.

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