Biofluid ligand library enrichment techniques and uses thereof

Abstract

The present disclosure provides, at least in part, methods for generating an aptamer library capable of binding a plurality of target molecules (e.g., one or more biomolecules) in a composite sample (e.g., a biological sample) using electrophoretic mobility shift (e.g., two-dimensional electrophoresis). The disclosure also provides databases containing information from one or more aptamer libraries (e.g., aptamer libraries generated by the methods described herein), as well as methods of utilizing such aptamer libraries (e.g., by comparing information of a reference aptamer library and a target aptamer library to discriminate differences).

Description

Technical Field

[0001] The present disclosure generally relates to a method for enriching a plurality of aptamers capable of binding one or more biomolecules in a biological sample. Background Art

[0002] Systematic evolution of ligands by exponential enrichment (SELEX) is currently a common method for isolating high-affinity aptamers for target molecules from large pools with random sequences. Aptamers can be selected for various targets. Aptamers have high affinity and specificity for their target molecules. However, many SELEX selection methods have been developed for isolating aptamers targeting single molecules. Due to the low success rate of aptamer selection, many factors in the selection process remain unclear.

[0003] Reference List

[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 SDAptamers: 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.

[0012] NPL 6: Klug and Famulok (1994). All you wanted to know about SELEX. Molecular Biology Reports, 20: 97-107.

[0013] NPL 7: Darmostuk et al. (2015). Current approaches in SELEX: An update to aptamer selection technology. Biotechnology Advances, 33(6): 1141-1161.

[0014] NPL 8: Morris et al., High affinity ligands from in vitro selection: Complex targets. Proc Natl Acad Sci USA. 1998 Mar 17;95(6):2902-2907.

[0015] NPL 9: Duffy et al., Modified nucleic acids: replication, evolution, and next-generation therapeutics, BMC Biology, vol. 18, article number: 112 (2020).

[0016] NPL 10: Hollenstein (2015) Generation of long, fully modified, and serum-resistant oligonucleotides by rolling circle amplification. Organic Biomolecular Chemistry, 13: 9829.

[0017] NPL 11: Kong et al. (2016). Generation of Synthetic Copolymer Libraries by Combinatorial Assembly on Nucleic Acid Templates. ACS Combinatorial Science, 18: 355-370.

[0018] NPL 12: 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.

[0019] NPL 13: Leonard et al., Basic Methods in Molecular Biology, 1986, Section 5-5 - Agarose Gel Electrophoresis, pp. 58-61.

[0020] NPL 14: Chai et al., Principle of Emulsion PCR and Its Applications in Biotechnology, J Anim Reprod Biotechnol 2019;34:259-266.

[0021] NPL 15: Shao et al., Emulsion PCR: A High Efficient Way of PCR Amplification of Random DNA Libraries in Aptamer Selection. PLoS ONE 6(9): e24910. Summary of the invention

[0022] Technical issues

[0023] It is an object of the present disclosure to provide a method for enriching a plurality of aptamers capable of binding one or more biomolecules in a biological sample.It is another object of the present disclosure to provide a method for analyzing a target aptamer library of a target biological sample.

[0024] Problem Solution

[0025] The present disclosure provides, at least in part, methods for generating aptamer libraries comprising multiple aptamers capable of binding multiple target molecules (e.g., one or more biomolecules) in a complex sample (e.g., a biological sample) using electrophoretic mobility shift (e.g., two-dimensional electrophoresis). In some embodiments, the methods described herein are described as biofluid ligand library enrichment technology (BIOLLET). The present disclosure also provides databases comprising information from one or more aptamer libraries (e.g., aptamer libraries generated by the methods described herein) and methods of utilizing such aptamer libraries (e.g., by comparing information from a reference aptamer library and a target aptamer library to discern differences).

[0026] In some aspects, the present disclosure provides a method for enriching multiple aptamers capable of binding to one or more biomolecules in a biological sample, the method comprising: (i) contacting a plurality of candidate aptamers with the biological sample to form a composition comprising a plurality of aptamer-biomolecule complexes; (ii) subjecting the composition to electrophoresis in a first direction in a first electrophoretic medium to obtain a portion of the first electrophoretic medium comprising the plurality of aptamer-biomolecule complexes; (iii) 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 comprising the plurality of aptamer-biomolecule complexes; and (iv) extracting the plurality of aptamers capable of binding to biomolecules in the biological sample from the plurality of aptamer-biomolecule complexes.

[0027] In some embodiments, the method further comprises (v) amplifying a plurality of aptamers capable of binding to a biomolecule in the biological sample to form an aptamer library.

[0028] In some embodiments, the plurality of aptamers capable of binding to biomolecules in a biological sample are amplified by polymerase chain reaction (PCR). In some embodiments, the PCR is emulsion PCR.

[0029] In some embodiments, the first electrophoretic medium is a first agarose gel. In some embodiments, the second electrophoretic medium is a second agarose gel.

[0030] In some embodiments, the first and second electrophoretic media contain sodium ions, potassium ions, lithium ions, ammonium ions, or any combination thereof, at a concentration between 100 mM and 200 mM. In some embodiments, the sodium ions are in the form of sodium chloride. 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.

[0031] In some embodiments, step (ii) further comprises cutting the portion of the first electrophoretic medium comprising the plurality of aptamer-biomolecule complexes from the remainder of the first electrophoretic medium. In some embodiments, the portion of the first electrophoretic medium is fitted into a well in a second electrophoretic medium to perform electrophoresis in a second direction.

[0032] In some embodiments, electrophoresis in the first direction and the second direction is performed at a temperature between 10°C and 20°C.

[0033] In some embodiments, the biological sample is 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 or cell lysate. In some embodiments, the biomolecules in the biological sample comprise nucleic acids, proteins, polypeptides, carbohydrates, lipids or combinations thereof. In some embodiments, the biological sample is not denatured. In some embodiments, the biological sample is diluted at a ratio between 1:200 and 1:2 prior to contacting with the plurality of candidate aptamers.

[0034] In some embodiments, the method further comprises contacting the biological sample with a plurality of competing nucleic acids prior to contacting with a plurality of candidate aptamers. In some embodiments, the plurality of competing nucleic acids is a set of random unrelated nucleic acids. In some embodiments, the plurality of competing nucleic acids is salmon sperm DNA.

[0035] In some embodiments, wherein 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).

[0036] 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.

[0037] In some embodiments, each of the plurality of candidate aptamers comprises a detectable label. In some embodiments, the detectable label is a fluorescent molecule.

[0038] In some embodiments, the method further comprises, prior to step (iv), cutting the portion of the second electrophoretic medium containing the plurality of aptamer-biomolecule complexes from the remainder of the second electrophoretic medium, and extracting the plurality of aptamer-biomolecule complexes from the portion of the second electrophoretic medium.

[0039] In some embodiments, steps (i)-(v) are repeated at least 4 times, and wherein when step (i) is repeated, the aptamer library obtained in step (v) is used as a starting plurality of candidate aptamers. In some embodiments, (i)-(iv) are repeated at least 9 times, and wherein when step (i) is repeated, the aptamer library obtained in step (iv) is used as a starting plurality of candidate aptamers.

[0040] In some embodiments, the method further comprises identifying sequences of the plurality of aptamers capable of binding to a biomolecule in the biological sample. In some embodiments, identifying sequences of the plurality of aptamers capable of binding to a biomolecule in the biological sample comprises next generation sequencing (NGS).

[0041] In some embodiments, the method further comprises identifying biomolecules bound to the plurality of aptamers that are capable of binding to the biomolecules in the biological sample.

[0042] In some embodiments, the method further comprises compiling and storing sequence information of the aptamer library capable of binding to the biomolecule in the biological sample and / or information of the biomolecule binding to the aptamer in the aptamer library in a computer readable format.

[0043] In some aspects, the disclosure further comprises an aptamer database comprising information of one or more aptamer libraries generated by the methods described herein, wherein each aptamer library comprises aptamers capable of binding to a biomolecule in a biological sample.

[0044] In some aspects, the present disclosure provides a method for analyzing a target aptamer library of a target biological sample, the method comprising: (i) acquiring information of the target aptamer library of the target biological sample from a first aptamer database, (ii) acquiring information of a reference aptamer library of a reference biological sample from a second aptamer database, and (iii) comparing the information of the target aptamer library of the target biological sample with the information of the reference aptamer library of the reference biological sample, thereby analyzing the target aptamer library.

[0045] In some aspects, the present disclosure provides a method for diagnosing a disease in a target subject, the method comprising: (i) obtaining information of the target aptamer library of the target biological sample from a first aptamer database, (ii) obtaining information of a reference aptamer library of a reference biological sample from a second aptamer database, and (iii) comparing the information of the target aptamer library of the target biological sample and the reference aptamer library of the reference biological sample.

[0046] In some aspects, the present disclosure provides a method for assisting in the diagnosis of a disease in a target subject, the method comprising: (i) obtaining information of the target aptamer library of the target biological sample from a first aptamer database, (ii) obtaining information of a reference aptamer library of a reference biological sample from a second aptamer database, and (iii) comparing the information of the target aptamer library of the target biological sample and the reference aptamer library of the reference biological sample.

[0047] In some aspects, the present disclosure provides a method for determining a physical condition status of a target subject, the method comprising: (i) acquiring information of the target aptamer library of the target biological sample from a first aptamer database, (ii) acquiring information of a reference aptamer library of a reference biological sample from a second aptamer database, and (iii) comparing the information of the target aptamer library of the target biological sample and the reference aptamer library of the reference biological sample.

[0048] In some aspects, the present disclosure provides a non-transitory computer-readable medium having instructions stored thereon, which, when executed by at least one processor, cause the at least one processor to perform a method for analyzing a target aptamer library of a target biological sample, the method comprising: (i) obtaining information of the target aptamer library of the target biological sample from a first aptamer database, (ii) obtaining information of a reference aptamer library of a reference biological sample from a second aptamer database, and (iii) comparing the information of the target aptamer library of the target biological sample with the information of the reference aptamer library of the reference biological sample, thereby analyzing the target aptamer library.

[0049] In some embodiments, the first aptamer database in step (i) is an aptamer database described herein. In some embodiments, the second aptamer database in step (ii) is an aptamer database described herein. In some embodiments, the first aptamer database in step (i) is an aptamer database and the second aptamer database in step (ii) is an aptamer database described herein. In some embodiments, the first aptamer database and the second aptamer database are the same. In some embodiments, the first aptamer database and the second aptamer database are different.

[0050] In some embodiments, step (i) comprises downloading information of the target aptamer library of the target biological sample from the aptamer database to a user computer, or extracting information of the target aptamer library of the target biological sample from the aptamer database to a server storing the aptamer database.

[0051] In some embodiments, step (ii) comprises downloading information of the reference aptamer library of the reference biological sample from the aptamer database to a user computer, or extracting information of the reference aptamer library of the reference biological sample from the aptamer database to a server storing the aptamer database.

[0052] In some embodiments, the method further comprises step (iv) determining the difference between the information of the target aptamer library and the reference aptamer library. In some embodiments, step (iv) comprises identifying biomolecules that bind to aptamers in the target aptamer library or the reference aptamer library, which biomolecules show the difference between the information of the target aptamer library and the reference aptamer library.

[0053] In some embodiments, the target biological sample is a biological sample from a subject having the same genetic background as the reference biological sample. In some embodiments, the target biological sample is a biological sample from a subject having a different genetic background than the reference biological sample. In some embodiments, the target biological sample is from a target subject suffering from or suspected of suffering from a disease. In some embodiments, the reference biological sample is from a healthy subject. In some embodiments, the comparison step results in determining whether the target subject suffers from a disease.

[0054] In some embodiments, the target biological sample is a diseased sample. In some embodiments, the reference biological sample is a non-diseased sample. In some embodiments, the reference biological sample is a diseased sample. In some embodiments, the comparing step results in identifying biomarkers of the diseased sample.

[0055] In some embodiments, the biological sample is serum, plasma, cerebrospinal fluid (CSF), urine, amniotic fluid, bone marrow, bronchoalveolar lavage fluid, cheek swab, stool, gastrointestinal fluid, liposuction sample, saliva, milk, nasal swab, peritoneal fluid, semen, sputum, synovial fluid, tears, vaginal fluid, tissue biopsy, or cell lysate.

[0056] Beneficial Effects of the Invention

[0057] The present disclosure provides a method for enriching a plurality of aptamers capable of binding to one or more biomolecules in a biological sample. The method can select aptamers capable of binding to one or more biomolecules with a high success rate. In addition, the present disclosure provides a method for analyzing a target aptamer library. The method can determine the physical condition state of a target subject or diagnose a disease of a target subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] [ Figure 1 ] shows gel imaging data after 1D and 2D electrophoresis after selection round 1. In the gel imaging data of 1D electrophoresis, the left lane shows control data without sample running, and the right lane shows gel shift data.

[0059] [ Figure 2 ] shows gel imaging data after the fifth round of 1D electrophoresis. The left lane shows the control gel shift data without competitor, and the center lane shows the gel shift data. The arrows on the right side of the gel image show the gel excision points that separate the top gel and the bottom gel to examine different sequence evolution in different gel shift regions.

[0060] [ Figure 3 ] shows the growth curves of aptamer library enrichment after different rounds of selection. The normalized result size indicates the number of different aptamer sequences observed in the next generation sequencing analysis, where the frequency was more than 3 reads.

[0061] [ Figure 4 ] Comparative analysis results of aptamer library information from serum samples of different C57BL / 6N mice. The top 30 sequences in the results of C57BL / 6N serum B were selected and shown in the figure. Sequences No. 10 and No. 15 show C57BL / 6N serum D specificity.

[0062] [ Figure 5]Comparative analysis results of aptamer library information of C57BL / 6N and BALB / c serum samples. The top 30 sequences in the results of C57BL / 6N serum B were selected and shown in the figure. Sequences No. 14 and No. 27 showed no reactivity to BALB / c serum.

[0063] [ Figure 6 ] SDS-PAGE data of aptamer-based precipitation assay using sd-10 aptamer. The sequence of sd-10 aptamer is shown in the table. Lowercase letter "t" indicates 5-tryptamine-uracil. Arrows show the target protein band of sd10 aptamer.

[0064] [ Figure 7 ] SDS-PAGE data of aptamer-based precipitation assay using Nb-01 aptamer. The sequence of Nb-01 aptamer is shown in the table. Lowercase letter "t" indicates 5-tryptamine-uracil. Arrows show the target protein band of Nb-01 aptamer.

[0065] [ Figure 8 ] Western blot analysis of SD-10 aptamer target protein by using anti-mouse IgG antibody.

[0066] [ Fig. 9 ] Western blot analysis of Nb-01 aptamer target protein by using anti-GPLD-1 antibody.

[0067] [ Fig.10 ] Comparative analysis of aptamer library information from human CSF samples derived from HC and AD patients. DETAILED DESCRIPTION

[0068] The present disclosure provides, at least in part, methods for generating aptamer libraries comprising a plurality of aptamers capable of binding to a plurality of target molecules (e.g., one or more biomolecules) in a complex sample (e.g., a biological sample) using two-dimensional electrophoretic mobility shift (e.g., 2-D electrophoresis). The present disclosure also provides databases comprising information from one or more aptamer libraries (e.g., aptamer libraries generated by the methods described herein) and methods of utilizing such aptamer libraries (e.g., by comparing information of a reference aptamer library and a target aptamer library to discern differences).

[0069] In some aspects, the disclosure relates to methods for enriching a plurality of aptamers capable of binding one or more target molecules (eg, biomolecules) present in a sample (eg, a biological sample).

[0070] 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 selectively bind to a target molecule. In some embodiments, the aptamer is a nucleic acid aptamer. In some embodiments, the aptamer is a single-stranded DNA aptamer. In some embodiments, the nucleic acid 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 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.

[0071] The concept of aptamer in vitro evolution was introduced in 1990 and is called exponential enrichment ligand systematic evolution or SELEX (non-patent literature 3-7). 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) multiple candidate aptamers are repeatedly incubated with target molecules to allow binding of high-affinity aptamers; (ii) high-affinity binders are separated from low-affinity binders and / or non-binding substances; and (iii) high-affinity binders are amplified using polymerase chain reaction (PCR). The process is repeated until high-affinity aptamers are enriched in the selection pool. Although SELEX successfully produces aptamers for purified proteins, these aptamers may not recognize the same protein at endogenous levels or conditions in cells. A variant of SELEX, called "complex target SELEX", has been developed to select aptamers that target proteins in their native conformation on the erythrocyte membrane. The protein and its related aptamer were separated by one-dimensional SDS electrophoresis (Non-Patent Document 8).

[0072] I. Biofluid Ligand Library Enrichment Technology (BIOLLET)

[0073] In some aspects, the present disclosure provides a method for enriching a plurality of aptamers capable of binding to one or more biomolecules in a biological sample, the method comprising: (i) contacting a plurality of candidate aptamers with the biological sample to form a composition comprising a plurality of aptamer-biomolecule complexes; (ii) subjecting the composition to electrophoresis in a first direction in a first electrophoretic medium to obtain a portion of the first electrophoretic medium comprising the aptamer-biomolecule complexes; (iii) 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 comprising the aptamer-biomolecule complexes; and (iv) extracting the plurality of aptamers capable of binding to biomolecules in the biological sample from the plurality of aptamer-biomolecule complexes. As used herein, the term "aptamer-biomolecule complex" refers to a molecular complex formed between an aptamer and its target molecule (e.g., a biomolecule) 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 particular target molecule from other target molecules. In some embodiments, if the K of an aptamer binding to a target molecule is greater than 100,000, the aptamer may be used to distinguish a particular target molecule from other target molecules. D is at least about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or lower, the aptamer specifically binds to the target molecule.

[0074] 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., biomolecule) complexes. For example, and without wishing to be bound by any particular theory, 1D electrophoresis may not be able to separate aptamer-target molecule (e.g., biomolecule) 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.

[0075] 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 aptamer-target molecule (e.g., biomolecule) complexes 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).

[0076] As used herein, the term "candidate aptamer" refers to a pool of random aptamers or random oligonucleotides, which may 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). In some embodiments, when generating an initial plurality of candidate aptamers, the ratio of nucleotides in each aptamer is optimized, for example, the A:C:G:T molar ratio 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 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 that are capable of binding to one or more target molecules (e.g., biomolecules) in a sample (e.g., a biological sample) from the last round of enrichment using the methods described herein. In some embodiments, the plurality of candidate aptamers is DNA or RNA. In some embodiments, the candidate aptamer is a single-stranded DNA (ssDNA).In some embodiments, the plurality of candidate aptamers fold in their proper tertiary structure to bind to a target molecule.

[0077] In some embodiments, a plurality of candidate aptamers include modified nucleotides. Modified nucleotides have been previously described, see, e.g., non-patent literature 9. In some embodiments, each of a plurality of candidate aptamers includes at least one modified nucleotide. In some embodiments, each of a plurality of candidate aptamers includes 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 literature 1 and non-patent literature 10 and 11). 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, the 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 includes, 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 a 5-tryptamine-uracil. In some embodiments, all thymines in each of the plurality of candidate aptamers are replaced with a 5-tryptamine-uracil.

[0078] 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.

[0079] 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. In some embodiments, the salt is capable of stabilizing the tertiary structure of the aptamer. In some embodiments, the salt is a divalent ion, including but not limited to magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof. In some embodiments, the selection buffer comprises a concentration between 0.1 mM and 10 mM, between 1 mM and 10 mM, between 5 mM and 10 mM, between 8 mM and 10 mM, between 6 mM and 8 mM, between 2 mM and 8 mM, between 0.1 mM and 5 mM, between 0.5 mM and 4.5 mM, between 1 mM and 4 mM, between 2 mM and 3 mM, between 0.5 mM and 2 mM, between 0.6 mM and 1.5 mM, between 0.7 mM and 1.5 mM, between 0.8 mM and 2 ... In some embodiments, the buffer comprises magnesium at a concentration of 1 mM. In some embodiments, the magnesium ion is 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.

[0080] 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).

[0081] 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.

[0082] 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.

[0083] In some embodiments, the target molecules (e.g., biomolecules) in the sample (e.g., biological sample) are denatured before contacting with a plurality of candidate aptamers. In some embodiments, the target molecules (e.g., biomolecules) in the sample (e.g., biological sample) are 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 bonds), 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) in the sample, which are responsible for the highly ordered structure of the target molecules (e.g., biomolecules such as proteins and / or nucleic acids) in their natural (native) state. In some embodiments, the denatured sample comprises a sample in which the biomolecules (e.g., proteins or nucleic acids) within 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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 electrophoretic medium in a first direction. In some embodiments, the size difference between the aptamer-target molecule (e.g., biomolecule) complex and the aptamer that binds 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 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-biomolecule complex, and the same portion may also contain the aptamer that binds the target molecule (e.g., biomolecule) due to non-specific binding. Figure 2 C-2D).

[0089] Thus, in some embodiments, the present disclosure seeks 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-biomolecule 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-biomolecule 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-biomolecule 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 nonspecific 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-biomolecule complex from the remainder of the second electrophoretic medium.

[0090] 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 the aptamer-biomolecule 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 OligoClean & 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 12.

[0091] 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 13. 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 comprising 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%.

[0092] 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 contains 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 contains 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, 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 certain 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 with 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 with concentrations between X and Y means that the total concentration of the three ions is within the range of X and Y, etc. Any units known in the art may be used to describe concentrations, 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 comprises a combination of ions (e.g., sodium ions, potassium ions, lithium ions, and / or ammonium ions) having 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.

[0093] 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 sufficient to disrupt non-specific binding of the aptamer to the target molecule. 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).

[0094] In some embodiments, the second electrophoretic medium comprises sodium ions (e.g., sodium chloride), potassium ions, lithium ions, ammonium ions, or any combination thereof, at a concentration between 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 second electrophoretic medium comprises sodium ions (e.g., sodium chloride), potassium ions, lithium ions, ammonium ions, or any combination thereof, at a concentration between 100 mM and 200 mM (e.g., any concentration between 100 mM and 200 mM). In some embodiments, the ions are sodium ions. In some embodiments, the sodium ions can be any sodium salt known in the art (e.g., sodium chloride).

[0095] 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 one or more divalent ions (e.g., magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof) at a concentration sufficient to stabilize the structure of the aptamer 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 structure and / or 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. In some embodiments, the divalent ions are magnesium ions. 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.

[0096] 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.

[0097] 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.

[0098] 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 the second electrophoretic medium portion containing the aptamer-biomolecule complex. In some embodiments, the method further comprises amplifying a plurality of aptamers extracted from the 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.

[0099] 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-biomolecule 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, any suitable amplification method known in the art can be employed by the methods described herein, as long as the amplification method (e.g., 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.

[0100] In some embodiments, the present disclosure is based at least in part on the following discovery: emulsion PCR is able to retain sequence order information in multiple aptamers that can bind to one or more target molecules (e.g., biomolecules) after forming an aptamer library by amplification. 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 droplets act as a miniaturized "reactor" for each PCR reaction and are physically separated from each other without exchanging macromolecules, especially PCR products. In some embodiments, individual DNA molecules are compartmentalized into these different reaction droplets, 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 bias is reduced. (See, e.g., non-patent literature 14 and 15).

[0101] In some embodiments, enriching a plurality of aptamers is achieved by performing multiple rounds of selection for aptamers that can bind to one or more target molecules (e.g., biomolecules) in a sample (e.g., a biological sample). In some embodiments, the steps of the methods described herein are repeated at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least eleven times, at least twelve times, at least thirteen times, at least fourteen times, at least fifteen times, or more. In some embodiments, the steps of the methods described herein are repeated four times. In some embodiments, the steps of the methods described herein are repeated nine times. In some embodiments, the aptamer library obtained after amplification (e.g., emulsion PCR) is used as a starting plurality of candidate aptamers for the next round of contact with target molecules (e.g., biomolecules) in a sample (e.g., a biological sample).

[0102] In some embodiments, the method further comprises identifying the sequences of a plurality of aptamers capable of binding to a target molecule (e.g., a biomolecule) in a sample (e.g., a biological sample). 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 systems). In some embodiments, the aptamer is sequenced by next generation sequencing. In some embodiments, the aptamer is sequenced by MiSeq.

[0103] In some embodiments, the method further includes identifying biomolecules bound to a plurality of aptamers, the plurality of aptamers being capable of binding to these biomolecules in a biological sample. In some embodiments, biomolecules (e.g., proteins) can be separated from bound aptamers. Methods for identifying biomolecules are well known to those of ordinary skill in the art. In short, methods for identifying biomolecules include, but are not limited to, DNA sequencing (e.g., NGS), Northern blotting, Southern blotting, 1D or 2D gel electrophoresis, mass spectrometry, enzyme-linked immunosorbent assay (ELISA), SDS-PAGE, protein blotting, thin layer chromatography, high performance liquid chromatography (HPLC), gas chromatography, or any combination thereof. In some embodiments, biomolecules (e.g., proteins) are identified by mass spectrometry.

[0104] In some embodiments, the methods described herein further include compiling and storing the sequence information of the aptamer library capable of binding to the biomolecule in the biological sample and / or the information of the biomolecule bound to the aptamer in the aptamer library in a computer-readable format. In some embodiments, the sequence information of the aptamer in the aptamer library and / or the information of the biomolecule bound to the aptamer can be uploaded to a server to form a database. In some embodiments, the database includes information from more than one aptamer library. When the aptamer library is generated, more information of other different aptamer libraries can be added to the database (for example, the database can include the aptamer library information of mouse serum, the aptamer library information of mouse CSF, the aptamer library information of human serum, the aptamer library information of human CSF, the aptamer library information of cancer tissue, etc.). Once the aptamer library information is identified and added to the database, such information can be easily downloaded and used without further performing the steps in the methods described herein. In some embodiments, the aptamer database is stored in a server. In some embodiments, the server storing the aptamer database further provides means for downloading the aptamer library information stored therein. In some embodiments, the server storing the aptamer database further provides means for exporting aptamer library information for comparison with another aptamer library on the server.

[0105] II. Methods of using aptamer library information

[0106] In some aspects, the present disclosure also provides a method for analyzing a target aptamer library of a target biological sample, the method comprising: (i) obtaining information of the target aptamer library of the target biological sample from a first aptamer database, (ii) obtaining information of a reference aptamer library of a reference biological sample from a second aptamer database, (iii) comparing the information of the target aptamer library of the target biological sample and the reference aptamer library of the reference biological sample, thereby analyzing the target aptamer library. Those skilled in the art will recognize that the terms "obtain", "provide", "retrieve" or "obtain" are used interchangeably and refer to steps including: accessing an aptamer database, locating information corresponding to an aptamer library (e.g., aptamer sequence identity, aptamer modification, aptamer relative abundance, aptamer purity, etc.), so as to provide the information in a subsequent step (e.g., step (iii), wherein the information of the target aptamer library and the reference aptamer library are compared). As used herein, the term "analyzing a target aptamer library" refers to analyzing biological information or characteristics of a target subject based on information (e.g., sequence information, including but not limited to the sequence frequency or amount of aptamers in the aptamer library that are capable of binding to biological molecules in the biological sample) of the target aptamer library of the target biological sample and the reference aptamer library of the reference biological sample.

[0107] "A method for analyzing a target aptamer library" may provide a method for determining a physical condition state of a target subject or diagnosing a disease of a target subject.

[0108] The diagnosis of a disease can be based on, for example, the difference between the information of an aptamer library from a biological sample of a healthy subject and the information of an aptamer library from a biological sample of a subject suffering from or suspected of suffering from the disease. When the information of the target aptamer library of the target biological sample is compared with the information of the reference aptamer library of a reference biological sample of a subject suffering from or suspected of suffering from the disease, if the target subject has at least one feature representing the characteristic information of the reference aptamer library (e.g., high / low sequence frequency or amount of one or more specific aptamers), the target subject is diagnosed as suffering from the disease or having a risk of the disease.

[0109] Diseases can be diagnosed by comparing the information of the target aptamer library of the target biological sample with the information of the reference aptamer library of the reference biological sample from a healthy subject. If the information of the target aptamer library of the target biological sample is substantially the same as the information of the reference aptamer library of the reference biological sample from a healthy subject, the target subject is considered to be healthy. If the information of the target aptamer library of the target biological sample shows any difference relative to the information of the reference aptamer library of the reference biological sample from a healthy subject, it can be determined that the target subject suffers from any disease or has a risk of any disease.

[0110] Alternatively or additionally, the information of the target aptamer library of the target biological sample can be compared with the information of the reference aptamer library of the reference biological sample from the subject independently diagnosed with the disease or having the risk of the disease. In this case, if the target subject has at least one feature (e.g., high / low sequence frequency or amount of one or more specific aptamers) representing the characteristic information of the reference aptamer library, then the target subject is diagnosed as having the disease or having the risk of the disease.

[0111] "A method for analyzing a target aptamer library" may include identifying biomolecules that bind to aptamers in a target aptamer library or a reference aptamer library, which biomolecules show differences between information of the target aptamer library of a target biological sample and information of a reference aptamer library of a reference biological sample.

[0112] In some aspects, the present disclosure also provides a non-transitory computer-readable medium having instructions stored thereon, which, when executed by at least one processor, cause the at least one processor to perform a method for analyzing a target aptamer library of a target biological sample, the method comprising: (i) obtaining information of the target aptamer library of the target biological sample from a first aptamer database, (ii) obtaining information of a reference aptamer library of a reference biological sample from a second aptamer database, and (iii) comparing the information of the target aptamer library of the target biological sample with the information of the reference aptamer library of the reference biological sample, thereby analyzing the target aptamer library.

[0113] In some embodiments, the first aptamer database is generated by the methods described herein (e.g., a method of enriching a plurality of aptamers that can bind to one or more biomolecules in a biological sample). In some embodiments, the second aptamer database is generated by the methods described herein (e.g., a method of enriching a plurality of aptamers that can bind to one or more biomolecules in a biological sample). In some embodiments, the first aptamer database and the second aptamer database are generated by the methods described herein (e.g., a method of enriching a plurality of aptamers that can bind to one or more biomolecules in a biological sample). In some embodiments, the first aptamer database and the second aptamer database are the same. In some embodiments, the first aptamer database and the second aptamer database are different.

[0114] In some embodiments, the aptamer library information is initially obtained by the methods described herein (e.g., by BIOLLET) and stored in an aptamer database. In some embodiments, obtaining information of an aptamer library (e.g., a target aptamer library and / or a reference aptamer library) from an aptamer database does not require performing the step of obtaining the aptamer library information again (e.g., by BIOLLET).

[0115] In some embodiments, the method further comprises downloading information of a target aptamer library of a target biological sample from an aptamer database to a user computer, or extracting information of a target aptamer library of a target biological sample from an aptamer database to a server storing the aptamer database. In some embodiments, the method further comprises downloading information of a reference aptamer library of a reference biological sample from an aptamer database to a user computer, or extracting information of a reference aptamer library of a reference biological sample from an aptamer database to a server storing the aptamer database.

[0116] In some embodiments, the method further comprises determining differences between the information of the target aptamer library and the reference aptamer library.

[0117] The target aptamer library and / or the reference aptamer library may be selected for comparison purposes.

[0118] In certain embodiments, the target biological sample is a biological sample from a subject having the same genetic background as the reference biological sample. For example, the target biological sample is the serum from C57BL / 6 mice, and the reference biological sample is the serum from another C57BL / 6 mouse. In certain embodiments, the target biological sample is the serum from a human subject of a certain race, and the reference biological sample is the serum from a human subject of the same race. By comparing the target aptamer library with the reference aptamer library, the difference of serum molecules (for example, protein) can be identified, and the difference can indicate that factors may have affected serum molecules (for example, protein). Such factors may include but are not limited to environmental factors, dietary factors, epigenetic modifications, disease conditions, age or sex.

[0119] In some embodiments, the target biological sample is a biological sample from a subject having a different genetic background from the reference biological sample. For example, the target biological sample is serum from C57BL / 6 mice, and the reference biological sample is serum from BALB / c mice. In some embodiments, the target biological sample is serum from a human subject of a certain race, and the reference biological sample is serum from human subjects of different races. By comparing the target aptamer library and the reference aptamer library, the difference in serum molecules (e.g., proteins) can be identified, and the difference can indicate that a certain genetic background is prone to a certain disease or is at risk of suffering from a certain disease.

[0120] In some embodiments, the target biological sample and the reference biological sample are from the same subject but obtained at different times. For example, the target biological sample is serum from a human subject before treatment, and the reference biological sample is serum from the same human subject after treatment. By comparing the target aptamer library and the reference aptamer library, the difference in serum molecules (e.g., proteins) can be identified, and the difference can indicate the therapeutic efficacy. In other embodiments, when the subject suffers from or is suspected of having a disease, the target biological sample is serum from a human subject, and when the subject is healthy, the reference biological sample is serum from the same human subject. By comparing the target aptamer library and the reference aptamer library, the difference in serum molecules (e.g., proteins) can be identified, and the difference can indicate that the subject suffers from a suspected disease or has a risk of a suspected disease.

[0121] In some embodiments, the target biological sample is obtained from a subject suffering from or suspected of having a disease, and the reference biological sample is obtained from a healthy individual. For example, the target biological sample is serum from a human subject suffering from or suspected of having a disease, and the reference biological sample is serum from a healthy human subject. By comparing the target aptamer library and the reference aptamer library, differences in serum molecules (e.g., proteins) can be identified, and the differences can indicate that the target subject suffers from or is at risk of a suspected disease.

[0122] In some embodiments, the target biological sample is obtained from a subject suffering from or suspected of having a disease, and the reference biological sample is obtained from a subject suffering from or suspected of having a certain disease. For example, the target biological sample is serum from a human subject suffering from or suspected of having a disease, and the reference biological sample is serum from a subject suffering from or suspected of having a certain disease. By comparing the target aptamer library and the reference aptamer library, the disease of the human subject can be diagnosed.

[0123] The above embodiments can be implemented in any of a variety of ways. One or more aspects and embodiments of the present disclosure related to the execution of a process or method can utilize program instructions that can be executed by a device (e.g., a computer, a processor, or other device) to execute these processes or methods or control their execution. In this regard, various inventive concepts can be embodied as a computer-readable storage medium (or multiple computer-readable storage media) encoded with one or more programs (e.g., a computer memory, one or more floppy disks, compressed disks, optical disks, tapes, flash memory, circuit configurations in field programmable gate arrays or other semiconductor devices, or other tangible computer storage media), which, when executed on one or more computers or other processors, executes one or more methods of implementing the various embodiments described above. One or more computer-readable media can be removable, so that one or more programs stored thereon can be loaded onto one or more different computers or other processors to implement various aspects of the above aspects. In some embodiments, the computer-readable medium can be a non-transient medium.

[0124] The term "program" or "software" is used in this article in a general sense to refer to any type of computer code or computer executable instruction set that can be used to program a computer or other processor to implement various aspects as described above. In addition, it should be understood that according to one aspect, one or more computer programs that perform the methods of the present disclosure when executed do not need to reside on a single computer or processor, but can be distributed in a modular manner among multiple different computers or processors to implement various aspects of the present disclosure.

[0125] Computer executable instructions can be in many forms, such as program modules, that are executed by one or more computers or other devices. Typically, program modules include routines, programs, objects, components, data structures, etc. that perform specific tasks or implement specific abstract data types. Typically, in various embodiments, the functionality of program modules can be combined or distributed as needed.

[0126] In addition, the data structure may be stored in a computer-readable medium in any suitable form. To simplify the description, the data structure may be shown as having fields that are related by position in the data structure. Such relationships can also be achieved by assigning storage in a computer-readable medium with positions that convey the relationships between the fields to the fields. However, any suitable mechanism may be used to establish the relationships between the information in the fields of the data structure, including by using pointers, tags or other mechanisms that establish relationships between data elements.

[0127] When implemented in software, the software code can be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers.

[0128] Furthermore, it should be understood that a computer may be embodied in any of a variety of forms, such as, by way of non-limiting example, a rack-mounted computer, a desktop computer, a laptop computer, or a tablet computer. Additionally, a computer may be embedded in a device not generally regarded as a computer but having suitable processing capabilities, including a personal digital assistant (PDA), a smart phone, or any other suitable portable or fixed electronic device.

[0129] In addition, the computer can have one or more input and output devices. These devices are particularly useful for presenting a user interface. Examples of output devices that can be used to provide a user interface include printers or display screens for visual presentation outputs, and loudspeakers or other sound generating devices for auditory presentation outputs. Examples of input devices that can be used for user interfaces include keyboards and pointing devices, such as mice, touch pads, and digitized tablets. As another example, a computer can receive input information by speech recognition or in other audible formats.

[0130] Such computers may be interconnected by one or more networks of any suitable form, including local or wide area networks (such as enterprise networks) and intelligent networks (IN) or the Internet. Such networks may be based on any suitable technology and may operate according to any suitable protocol, and may include wireless networks, wired networks, or fiber optic networks.

[0131] In addition, as described, some aspects can be embodied as one or more methods. The actions performed as part of the method can be ordered in any suitable manner. Therefore, the following embodiments can be constructed: wherein the actions are performed in an order different from the order shown, thereby including performing some actions simultaneously, even if these actions are shown as sequential actions in the illustrative embodiments.

[0132] Other embodiments

[0133] 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.

[0134] 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.

[0135] Equivalent

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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".

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] Examples

[0145] Materials and methods

[0146] Preparation of agarose gels for 1D and 2D electrophoresis

[0147] 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 a 100-200mM NaCl concentration, depending on the selected conditions. The mixture was heated by microwave to completely dissolve the agarose powder. After cooling to about 60°C, a molar magnesium chloride solution was added to the agarose solution at a concentration of 1mM and mixed thoroughly. 11 ml of the solution was poured into a gel tray (55mm(W)x60 mm(L)) with a hole comb. The number of holes for 1D electrophoresis was 3-4 holes (10mm(W)x1 mm(L)), and 2D electrophoresis was 1 hole (45mm(W)x2 mm(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.

[0148] Preparation of DNA libraries for binding reactions

[0149] Selection was performed using a single-stranded DNA (ssDNA) library with a random region of 43 nucleotides and a TYE665 fluorophore at the 5' end. The ssDNA library was diluted in a selection buffer (PBS containing 0.005% nonidet-P40 and 1 mM magnesium chloride) at a concentration ranging from 100 nM to 5000 nM, depending on the selection conditions. The ssDNA library was denatured at 95°C for 3 min and then slowly cooled to room temperature within 30 min to form a stable ssDNA structure. The renatured ssDNA library solution was mixed with an appropriately diluted biological fluid sample solution in a 10 μL reaction volume. The dilution range of the biological fluid sample depends on the type of biological fluid; for example, serum samples use 2 to 200 times dilution values ​​and cerebrospinal fluid (CSF) samples use 1 to 2 times dilution values. Before mixing with the ssDNA library solution, the biological fluid sample is mixed with a competitor (such as salmon sperm DNA and any unrelated DNA oligomers). Dextran sulfate was also used to reduce the charge-dependent nonspecific interactions of ssDNA with biomolecules. The mixture was incubated at room temperature for 10 min, and then 2 μL of 6× loading buffer containing 36% glycerol and 6 mM magnesium chloride was quickly added to the reaction mixture, and the samples were then loaded on an agarose gel.

[0150] 1D electrophoresis separation

[0151] Fill the electrophoresis unit, such as Mupid2, with fresh 1xTB 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. The running time depends on the selection round. 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.

[0152] 2D electrophoresis separation

[0153] Another electrophoresis unit is filled with fresh 1xTB buffer, and the gel for 2D electrophoresis is placed in a gel box. As with 1D electrophoresis, the device is placed in a cold water bath, as with 1D electrophoresis, and before performing 2D electrophoresis, the gel is pre-run at 100V for 10min. This pre-run step is performed during the 1D electrophoresis run so that the 2D electrophoresis can be started immediately after the gel is excised. After the pre-run, the gel is removed from the gel box and the excised gel is carefully inserted into the hole. The gel is placed back in the gel box and 2D electrophoresis is started at 100V in the dark for 55-60min. The running time depends on the selection round. After the run, the gel is removed from the gel box, visualized by the ChemiDoc MP imaging system (Bio-Rad), and only the diagonal area formed by the aptamer-biomolecule complex is excised. The excised gel slice is placed in a microtube.

[0154] Extraction of DNA from agarose gel

[0155] 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.

[0156] PCR amplification of recovered ssDNA libraries

[0157] The recovered ssDNA was PCR amplified in a two-step process. In the first step, a test PCR reaction containing different numbers of continuous PCR cycles was performed in a small volume. It should be noted that all PCR amplifications were performed as emulsion PCR procedures, in which 100 μL of PCR solution was mixed with 250 μL of emulsified oil (4.5% Span80, 0.4% Tween 80, 0.05% Triton-X100 and 95.05% mineral oil), and the solution was vigorously mixed with a magnetic stirring bar until completely mixed. The PCR products of the specified cycles were 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 concatemers and truncations. The remaining PCR samples were amplified by adopting a determined PCR cycle. The PCR products were recovered by chloroform extraction and purified by an OligoClean & 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.

[0158] Generation of aptamer libraries by primer extension

[0159] 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. 100 μL of each PCR amplification used 2 microliters of solution. 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 the OligoClean&Concentrator kit. The purified DNA was primer extended by 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 3 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 shaking at 1500rpm for 1min. The solution was centrifuged for 30 seconds by a tabletop 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 80mM 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.

[0160] Sequencing analysis of aptamer libraries

[0161] The PCR products obtained in each round of selection were PCR amplified in a 2-step process to prepare for next generation sequencing analysis by MiSeq (illumina). The first step uses an adapter sequence primer set, followed by an index sequence primer set in the second step. Thermal cycling is performed as described by the manufacturer. The PCR product is purified by NucleoSpin gel and PCRClean-up (Macherey Nagel), and the first step is quantified by Qubit 4 fluorometers, and the second step is quantified by a bioanalyzer. According to the manufacturer's instructions, the product is analyzed by MiSeq. All sequencing data of each round are generated as FASTQ files. After extracting the aptamer domain by trimming the 5'- and 3'-primer regions, all sequences are used for frequency analysis to study the generation of aptamers during selection.

[0162] Comparative analysis of information from aptamer libraries

[0163] The method disclosed herein is used for comparative analysis of information of aptamer libraries. An aptamer library is selected based on the sequence analysis results of the aptamer library. The aptamer library is renatured to form a stable structure and then mixed with the sample. The conditions are the same as those for preparing the aptamer library. Multiple aptamer-biomolecule complexes are purified by a 1D electrophoresis procedure. The ssDNA recovered from the gel is amplified using fixed PCR cycles and analyzed directly by next-generation sequencing without 2D electrophoresis. The information of the aptamer library is obtained by calculating the frequency % of each sequence in the total number of reads for each sample. The frequency % information (information of the aptamer library) is compared in various reference biological samples to evaluate changes in the state of the biomolecules.

[0164] Example 1: Aptamer library generation from mouse serum

[0165] Methods according to the present disclosure were used to generate an aptamer library targeting mouse serum samples.

[0166] Five rounds of selection were performed using a completely randomized DNA library. The starting library contained a random region of 43 nucleotides consisting of adenine, guanine, cytosine, and 5-tryptamine-uracil instead of thymine. Representative imaging data of 1D and 2D electrophoresis are shown in Figure 1 A particular indication of a successful selection is the observation that certain sequences increase in number during the selection rounds. Another indication is the observation that different sequence variations appear in different regions of the gel after electrophoresis. For this purpose, additional rounds 5 and 6 were performed using a modified procedure in which the gel after 1D electrophoresis was divided into 2 parts, as Figure 2 The top and bottom images are shown for analysis of specific sequence evolution. In the next generation sequencing analysis, the average sequencing read count was 50,933. The library growth curve is shown in Figure 3 where the result size indicates the number of sequences with more than 3 counts. For comparison, Figure 3 Also shown are growth curves for normal SELEX data and 1D electrophoresis-based biofluid SELEX data. The top twenty sequences in frequency in the library from the entire gel are listed in Table 1 below. The top twenty sequences from different regions of the gel (bottom and top) are listed in Tables 2 and 3 below, respectively.

[0167] [Table 1]

[0168]

[0169] Table 1. Shows a list of the top 20 aptamer sequences obtained from the whole gel analysis. The lower case letter "t" represents 5-tryptamine-uracil.

[0170] [Table 2]

[0171]

[0172] Table 2 shows the Figure 2 List of the top 20 aptamer sequences obtained for the indicated bottom gel portion. The frequencies of these aptamers were compared to the entire gel data and the top gel data to investigate specificity. The lower case letter "t" indicates 5-tryptamine-uracil.

[0173] [Table 3]

[0174]

[0175] Table 3 shows the Figure 2 List of the top 20 aptamer sequences obtained for the indicated top gel portion. The frequencies of these aptamers were compared to the entire gel data and the bottom gel data to investigate specificity. The lower case letter "t" indicates 5-tryptamine-uracil.

[0176] Example 2: Comparative analysis of mouse serum aptamer library information

[0177] The methods according to the present disclosure were used to comparatively analyze the information of aptamer libraries from serum samples of mice of the same or different strains.

[0178] Serum samples were prepared from C57BL / 6N (B6 / N) and BALB / c mouse strains. The aptamer library was generated using one of the C57BL / 6N mouse sera. After 5 rounds of selection, the aptamer library was selected based on the sequencing results. A gradual slope of sequence frequency, with approximately 1%-2% being the top-ranked sequence frequency, was the basic selection criterion. The aptamer library was interacted with several serum samples from C57BL / 6N or BALB / c strains, and the aptamer-protein complexes were purified by 1D electrophoresis and then subjected to next-generation sequencing analysis using MiSeq (Illumina). Figure 4 and Figure 5 The comparative analysis results of aptamer library information between samples of the same mouse strain (C57BL / 6N vs. C57BL / 6N) or samples of different mouse strains (C57BL / 6N vs. BALB / c) are shown respectively. It should be noted that different aptamer libraries were used for each comparative analysis.

[0179] Some aptamers showing sample-specific patterns were selected to test target specificity by aptamer-based precipitation assay. One was the sd-10 aptamer, which showed high specificity only for serum D in the same strain analysis, and the other was the Nb-01 aptamer, which showed specificity only for C57BL / 6N serum obtained from a different mouse strain analysis. Both aptamers were modified with biotin at the 5' end to precipitate the target molecules. These aptamers were allowed to interact with the serum samples, followed by a pull-down assay by using streptavidin beads (either agarose beads or magnetic FG beads). After extensive washing, the beads were analyzed by non-denaturing, stain-free SDS-PAGE. The gel was visualized by the ChemiDoc MP imaging system (Bio-Rad), as shown in Figure 2. Figure 6 and Figure 7 As shown, the pull-down results of sd-10 aptamer and Nb-01 aptamer are shown, respectively.

[0180] The target proteins of sd-10 and Nb-01 aptamers were identified to further confirm the molecular specificity of these aptamers. The target bands were excised and used for MS analysis. The results indicated that sd-10 aptamer recognized mouse IgG and Nb-01 aptamer recognized GPLD-1. Mouse IgG antibody ( Figure 8 ) and GPLD-1 antibody ( Fig. 9 Western blot analysis performed by ) confirmed the results of MS analysis. Figure 8 As shown, the SD-10 aptamer recognized only a small fraction of the total antibodies in the serum, indicating a specific interaction with a certain type of differentially expressed antibodies.

[0181] Example 3: Comparative analysis of aptamer library information of human cerebrospinal fluid samples

[0182] The methods according to the present disclosure were used to comparatively analyze the information of aptamer libraries of human cerebrospinal fluid (CSF) samples.

[0183] CSF samples were purchased from PrecisionMed, LLC. A mixture of 5 CSF samples derived from 5 AD patients was used to generate an aptamer library targeting human CSF samples. The aptamer library was selected based on the same procedure as in Example 2. The aptamer library was interacted with each CSF sample derived from 7 healthy volunteers (HC) and 10 AD patients. The aptamer-protein complex was purified by 1D electrophoresis and then subjected to next generation sequencing analysis using HiSeq X (Illumina). Each sequence frequency was obtained as an analysis score. The average analysis score of the HC was used as a baseline, and the fold change (FC) of each sequence relative to the baseline was calculated and plotted as Fig.10Comparative analysis of the aptamer library information showed that the response patterns of the aptamers to HC and AD CSF samples were different. Some aptamers showing differences between the information of the aptamer library for AD CSF samples and the aptamer library for HC CSF samples can be selected, and biomolecules that bind to the aptamers in the aptamer library for AD CSF samples or the aptamer library for HC CSF samples can be identified as described in Example 2.

Claims

1. A method for enriching a plurality of aptamers capable of binding one or more biomolecules in a biological sample, the method comprising: (i) contacting a plurality of candidate aptamers with a biological sample to form a composition comprising a plurality of aptamer-biomolecule complexes; (ii) subjecting the composition to electrophoresis in a first electrophoretic medium in a first direction to obtain a portion of the first electrophoretic medium containing the plurality of aptamer-biomolecule complexes; (iii) 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 plurality of aptamer-biomolecule complexes; as well as (iv) extracting the plurality of aptamers capable of binding to biomolecules in the biological sample from the plurality of aptamer-biomolecule complexes. 2 . The method of claim 1 , further comprising (v) amplifying the plurality of aptamers capable of binding to the biomolecules in the biological samples to form an aptamer library. 3 . The method of claim 2 , wherein the plurality of aptamers capable of binding to biomolecules in the biological samples are amplified by polymerase chain reaction (PCR). The method of claim 3 , wherein the PCR is emulsion PCR.

5. The method of any one of claims 1-4, wherein step (ii) further comprises cutting the portion of the first electrophoretic medium comprising the plurality of aptamer-biomolecule complexes from the rest of the first electrophoretic medium.

6. The method of claim 5, 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.

7. The method of any one of claims 1 to 6, wherein the first electrophoretic medium is a first agarose gel.

8. The method of any one of claims 1-7, wherein the second electrophoretic medium is a second agarose gel.

9. The method of any one of claims 1-8, 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.

10. The method of claim 9, wherein the sodium ions are in the form of sodium chloride.

11. The method of any one of claims 1-10, 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.

12. The method of any one of claims 1-11, 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.

13. The method of any one of claims 1-12, 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.

14. The method of any one of claims 11-13, wherein the magnesium ions are in the form of magnesium chloride.

15. The method of any one of claims 1-12, wherein electrophoresis in the first direction and the second direction is performed at a temperature between 10°C and 20°C.

16. The method of any one of claims 1-15, 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 or cell lysate.

17. The method of any one of claims 1-16, wherein the biomolecules in the biological sample comprise nucleic acids, proteins, polypeptides, carbohydrates, lipids, or a combination thereof.

18. The method of any one of claims 1-17, wherein the biological sample is not denatured.

19. The method of any one of claims 1-18, wherein the biological sample is diluted at a ratio between 1:200 and 1:2 prior to contacting with the plurality of candidate aptamers.

20. The method of any one of claims 1-19, further comprising contacting the biological sample with a plurality of competitor nucleic acids prior to contacting with the plurality of candidate aptamers.

21. The method of claim 20, wherein the plurality of competing nucleic acids is a group of random unrelated nucleic acids.

22. The method of claim 20 or 21, wherein the plurality of competing nucleic acids is salmon sperm DNA.

23. The method of any one of claims 1-22, wherein the plurality of candidate aptamers are single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA, or peptides.

24. The method of claim 23, wherein the plurality of candidate aptamers are single-stranded DNA (ssDNA).

25. The method of claim 24, wherein each of the plurality of candidate aptamers comprises a modified nucleotide.

26. The method of any one of claims 1-25, wherein each of the plurality of candidate aptamers is labeled.

27. The method of claim 26, wherein each of the plurality of candidate aptamers is fluorescently labeled.

28. The method of any one of claims 1-27, further comprising, before step (iv), cutting the portion of the second electrophoretic medium containing the plurality of aptamer-biomolecule complexes from the rest of the second electrophoretic medium, and extracting the plurality of aptamer-biomolecule complexes from the portion of the second electrophoretic medium.

29. The method of any one of claims 2-28, wherein steps (i)-(v) are repeated at least 4 times, and wherein when step (i) is repeated, the plurality of aptamers capable of binding to biomolecules in a biological sample obtained in step (v) are used as a starting plurality of candidate aptamers.

30. The method of any one of claims 1-29, wherein (i)-(iv) is repeated at least 9 times, and wherein when step (i) is repeated, the plurality of aptamers capable of binding to biomolecules in a biological sample obtained in step (iv) are used as a starting plurality of candidate aptamers.

31. The method of any one of claims 1-30, further comprising identifying sequences of the plurality of aptamers capable of binding to biomolecules in the biological sample.

32. The method of claim 31 , wherein identifying sequences of the plurality of aptamers capable of binding to biomolecules in the biological sample comprises next generation sequencing (NGS).

33. The method of any one of claims 1-32, further comprising identifying biomolecules bound to the plurality of aptamers, the plurality of aptamers being capable of binding to biomolecules in the biological sample.

34. The method of claim 32 or 33, further comprising compiling and storing sequence information of the plurality of aptamers capable of binding to biomolecules in the biological sample and / or information of the biomolecules bound to the plurality of aptamers into a computer-readable format.

35. An aptamer database comprising information of one or more aptamer libraries generated by the method of any one of claims 1 to 34, wherein each aptamer library comprises aptamers capable of binding to biomolecules in a biological sample.

36. A method for analyzing a target aptamer library of a target biological sample, the method comprising: (i) acquiring information of a target aptamer library of the target biological sample from a first aptamer database, (ii) acquiring information of a reference aptamer library of a reference biological sample from a second aptamer database, and (iii) comparing the information of the target aptamer library of the target biological sample with the information of the reference aptamer library of the reference biological sample, thereby analyzing the target aptamer library, The first aptamer database in step (i) is the aptamer database as claimed in claim 35.

37. The method of claim 36, wherein the second aptamer database in step (ii) is the aptamer database of claim 35.

38. The method of claim 36 or 37, wherein the first aptamer database and the second aptamer database are the same.

39. The method of claim 36 or 37, wherein the first aptamer database and the second aptamer database are different.

40. The method of any one of claims 36-39, wherein step (i) comprises downloading the information of the target aptamer library of the target biological sample from the aptamer database to a user computer, or extracting the information of the target aptamer library of the target biological sample from the aptamer database to a server storing the aptamer database.

41. The method of any one of claims 36-40, wherein step (ii) comprises downloading the information of the reference aptamer library of the reference biological sample from the aptamer database to a user computer, or extracting the information of the reference aptamer library of the reference biological sample from the aptamer database to a server storing the aptamer database.

42. The method of any one of claims 36-41, further comprising (iv) determining the difference between the information of the target aptamer library and the reference aptamer library.

43. The method of claim 42, wherein step (iv) comprises identifying biomolecules that bind to aptamers in the target aptamer library or the reference aptamer library, the biomolecules showing differences between the information of the target aptamer library and the reference aptamer library.

44. The method of any one of claims 36-43, wherein the target biological sample is a biological sample from a subject having the same genetic background as the reference biological sample.

45. The method of any one of claims 36-43, wherein the target biological sample is a biological sample from a subject having a different genetic background than the reference biological sample.

46. ​​The method of any one of claims 36-45, wherein the target biological sample is from a target subject having or suspected of having a disease.

47. The method of claim 46, wherein the reference biological sample is from a healthy subject.

48. The method of claim 46, wherein the reference biological sample is from a subject having or suspected of having a disease.

49. The method of any one of claims 46-48, wherein the comparing step results in a determination of whether the target subject has the disease or is at risk for the disease.

50. The method of any one of claims 36-49, wherein the target biological sample is a diseased sample.

51. The method of claim 50, wherein the reference biological sample is a non-diseased sample.

52. The method of claim 50, wherein the reference biological sample is a diseased sample.

53. The method of claim 50 or 51, wherein the comparing step results in identifying a biomarker for the diseased sample.

54. The method of any one of claims 36-53, 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 or cell lysate.

55. A non-transitory computer readable medium having stored thereon instructions which, when executed by at least one processor, cause the at least one processor to perform a method for analyzing a target aptamer library of a target biological sample, the method comprising: (i) acquiring information of a target aptamer library of the target biological sample from a first aptamer database, (ii) acquiring information of a reference aptamer library of a reference biological sample from a second aptamer database, and (iii) comparing the information of the target aptamer library of the target biological sample with the information of the reference aptamer library of the reference biological sample, thereby analyzing the target aptamer library, The first aptamer database in step (i) is the aptamer database as claimed in claim 35.

56. The non-transitory computer readable medium of claim 55, wherein the second aptamer database in step (ii) is the aptamer database of claim 35.

57. The non-transitory computer readable medium of claim 55 or 56, wherein the first aptamer database and the second aptamer database are the same.

58. The non-transitory computer readable medium of claim 55 or 56, wherein the first aptamer database and the second aptamer database are different.

59. The non-transitory computer-readable medium of any one of claims 55-58, wherein step (i) comprises downloading the information of the target aptamer library of the target biological sample from the aptamer database to a user computer, or extracting the information of the target aptamer library of the target biological sample from the aptamer database to a server storing the aptamer database.

60. The non-transitory computer-readable medium of any one of claims 55-59, wherein step (ii) comprises downloading the information of the reference aptamer library of the reference biological sample from the aptamer database to a user computer, or extracting the information of the reference aptamer library of the reference biological sample from the aptamer database to a server storing the aptamer database.

61. The non-transitory computer readable medium of any one of claims 55-60, further comprising (iv) determining differences between the information of the target aptamer library and the reference aptamer library.

62. The non-transitory computer readable medium of claim 61, wherein step (iv) comprises identifying biomolecules that bind to aptamers in the target aptamer library or the reference aptamer library, the biomolecules showing differences between the information of the target aptamer library and the reference aptamer library.

63. The non-transitory computer readable medium of any one of claims 55-62, wherein the target biological sample is a biological sample from a subject having the same genetic background as the reference biological sample.

64. The non-transitory computer readable medium of any one of claims 55-62, wherein the target biological sample is a biological sample from a subject having a different genetic background than the reference biological sample.

65. The non-transitory computer readable medium of any one of claims 55-64, wherein the target biological sample is from a target subject having or suspected of having a disease.

66. The non-transitory computer readable medium of claim 65, wherein the reference biological sample is from a healthy subject.

67. The non-transitory computer readable medium of claim 65, wherein the reference biological sample is from a subject having or suspected of having a disease.

68. The non-transitory computer readable medium of any one of claims 65-67, wherein the comparing step results in a determination of whether the target subject has the disease or is at risk for the disease.

69. The non-transitory computer readable medium of any one of claims 55-68, wherein the target biological sample is a diseased sample.

70. The non-transitory computer readable medium of claim 69, wherein the reference biological sample is a non-diseased sample.

71. The non-transitory computer readable medium of claim 69, wherein the reference biological sample is a diseased sample.

72. The non-transitory computer readable medium of claim 69 or 70, wherein the comparing step results in identifying a biomarker for the diseased sample.

73. The non-transitory computer-readable medium of any one of claims 55-72, wherein the biological sample is serum, plasma, cerebrospinal fluid (CSF), urine, amniotic fluid, bone marrow, bronchoalveolar lavage fluid, cheek swab, stool, gastrointestinal fluid, liposuction sample, saliva, milk, nasal swab, peritoneal fluid, semen, sputum, synovial fluid, tears, vaginal fluid, tissue biopsy, or cell lysate.

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