Techniques for enriching ligand libraries from biological fluids and uses thereof

Abstract

The present disclosure provides, at least in part, methods for generating aptamer libraries capable of binding to 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). The present disclosure also provides databases containing information from one or more aptamer libraries (e.g., aptamer libraries generated by the methods described herein) and methods for utilizing such aptamer libraries (e.g., by comparing the information in a reference aptamer library and a target aptamer library to identify differences).

Description

[Technical Field]

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

[0002] Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is a popular method currently used to isolate high-affinity aptamers for target molecules from large pools of random sequences. Aptamers can be selected against a wide range of targets. Aptamers have high affinity and specificity for their target molecules. However, most SELEX selection methods have been developed to isolate aptamers that target a single molecule. Due to the low success rate of aptamer selection, many factors in the selection process remain unclear. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] U.S. Patent Application Publication No. 2006 / 0160763 [Non-patent literature]

[0004] [Non-Patent Document 1] Keefe et al., Aptamers as therapeutics. Nat. Rev. Drug Discov. 2010, 9:537–550. [Non-patent document 2] Jayasena SDAptamers: An emerging class of molecules that rival antibodies in diagnostics.Clin.Chem.1999, 45:1628~1650 [Non-patent document 3] Tuerk et al., Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990, 249:505–510. [Non-patent document 4] Ellington et al., In vitro selection of RNA molecules that bind specific ligands.Nature.1990, 346:818~822. [Non-patent document 5] SELEX-A® evolutionary method to generate high-affinity nucleic acid ligands.Biomolecular Engineering, 24(4):381~403. [Non-patent document 6] Klug and Famulok (1994). All you wanted to know about SELEX. Molecular Biology Reports, 20:97~107. [Non-Patent Document 7] Darmostuk et al. (2015).Current approaches in SELEX:An update to aptamer selection technology.Biotechnology Advances, 33(6):1141~1161. [Non-patent document 8] Morris et al., High affinity ligands from in vitro selection: Complex targets, Proc Natl Acad Sci USA. 1998 March 17, 95(6):2902-2907. [Non-Patent Document 9] Duffy et al., Modified nucleic acids: replication, evolution, and next-generation therapeutics, BMC Biology, Vol. 18, Article No. 112 (2020). [Non-Patent Document 10] Hollenstein (2015) Generation of long, fully modified, and serum-resistant oligonucleotides by rolling circle amplification.Organic Biomolecular Chemistry, 13:9829. [Non-Patent Document 11] Kong et al. (2016).Generation of Synthetic Copolymer Libraries by Combinatorial Assembly on Nucleic Acid Templates.ACS Combinatorial Science, 18:355~370. [Non-Patent Document 12] Abraham et al., A quick and effective in-house method of DNA purification from agarose gel, suitable for sequencing, 3 Biotech. July 2017, 7(3):180. [Non-Patent Document 13] Leonard et al., Basic Methods in Molecular Biology, 1986, SECTION 5-5-Agarose Gel Electrophoresis, pp. 58-61. [Non-Patent Document 14] Chai et al., Principle of Emulsion PCR and Its Applications in Biotechnology, J Anim Reprod Biotechnol 2019, 34:259~266. [Non-Patent Document 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 [Problem to be solved by the invention]

[0005] An object of the present disclosure is to provide a method for enriching a plurality of aptamers that can bind to one or more biomolecules in a biological sample. Another object of the present disclosure is to provide a method for profiling a target aptamer library for a target biological sample. [Means for solving the problem]

[0006] The present disclosure provides 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), at least in part, using electrophoretic mobility shift (e.g., two-dimensional electrophoresis). In some embodiments, the methods described herein are described as BIO-fluid's Ligand Library Enrichment Technology (BIOLLET). The present 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 for utilizing such aptamer libraries (e.g., by comparing information from a reference aptamer library and a target aptamer library to identify differences).

[0007] 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 electrophoretic medium in a first direction 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 electrophoretic medium in a second direction to obtain a portion of a second electrophoretic medium comprising the plurality of aptamer-biomolecule complexes; and (iv) extracting a plurality of aptamers capable of binding to biomolecules in the biological sample from the plurality of aptamer-biomolecule complexes.

[0008] In some embodiments, the method further comprises (v) amplifying a plurality of aptamers capable of binding to biomolecules in the biological sample to form an aptamer library.

[0009] In some embodiments, the plurality of aptamers capable of binding to the biomolecule in the biological sample is amplified by polymerase chain reaction (PCR). In some embodiments, the PCR is emulsion PCR.

[0010] In some embodiments, the first electrophoresis medium is a first agarose gel. In some embodiments, the second electrophoresis medium is a second agarose gel.

[0011] 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.

[0012] In some embodiments, step (ii) further comprises cutting out a portion of the first electrophoresis medium comprising the plurality of aptamer-biomolecule complexes from the remainder of the first electrophoresis medium, hi some embodiments, the portion of the first electrophoresis medium is aligned with a well in a second electrophoresis medium for electrophoresis in a second direction.

[0013] In some embodiments, the electrophoresis in the first direction and the second direction is carried out at a temperature between 10°C and 20°C.

[0014] In some embodiments, the biological sample is serum, plasma, cerebrospinal fluid (CSF), urine, amniotic fluid, bone marrow, bronchoalveolar lavage fluid, oral swab, stool, 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 biological molecule in the biological sample comprises a nucleic acid, a protein, a polypeptide, a carbohydrate, a lipid, or a combination 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 before contacting with the plurality of candidate aptamers.

[0015] In some embodiments, the method further comprises contacting the biological sample with a plurality of competitor nucleic acids before contacting the biological sample with a plurality of candidate aptamers. In some embodiments, the plurality of competitor nucleic acids is a random set of unrelated nucleic acids. In some embodiments, the plurality of competitor nucleic acids is salmon sperm DNA.

[0016] In some embodiments, the plurality of candidate aptamers is single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA, or a peptide. In some embodiments, the plurality of candidate aptamers is single-stranded DNA (ssDNA).

[0017] In some embodiments, each of the plurality of candidate aptamers comprises a modified nucleotide, hi some embodiments, each of the plurality of candidate aptamers comprises one or more 5-tryptoamino-uracils in place of thymine.

[0018] In some embodiments, each of the plurality of candidate aptamers comprises a detectable label. In some embodiments, the detectable label is a fluorescent molecule.

[0019] In some embodiments, the method further comprises, prior to step (iv), cutting out a 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 said portion of the second electrophoretic medium.

[0020] In some embodiments, steps (i)-(v) are repeated at least four times, in which case the aptamer library obtained in step (v) is used as the plurality of starting candidate aptamers when repeating step (i). In some embodiments, steps (i)-(iv) are repeated at least nine times, in which case the aptamer library obtained in step (iv) is used as the plurality of starting candidate aptamers when repeating step (i).

[0021] In some embodiments, the method further comprises identifying a plurality of aptamer sequences capable of binding to the biomolecule in the biological sample. In some embodiments, the step of identifying a plurality of aptamer sequences capable of binding to the biomolecule in the biological sample comprises next-generation sequencing (NGS).

[0022] In some embodiments, the method further comprises identifying biomolecules bound to a plurality of aptamers capable of binding to the biomolecules in the biological sample.

[0023] In some embodiments, the method further comprises compiling and storing, in a computer-readable format, sequence information of the aptamer library that can bind to biomolecules in the biological sample and / or information of biomolecules bound to aptamers in the aptamer library.

[0024] In some aspects, the present disclosure further includes an aptamer database containing information of one or more aptamer libraries generated by the methods described herein, each aptamer library containing aptamers capable of binding to biomolecules in a biological sample.

[0025] In some aspects, the present disclosure provides a method for profiling a target aptamer library for a target biological sample, the method comprising: (i) obtaining information of a target aptamer library for the target biological sample from a first aptamer database; (ii) obtaining information of a reference aptamer library for a reference biological sample from a second aptamer database; and (iii) comparing the information of the target aptamer library for the target biological sample with the information of the reference aptamer library for the reference biological sample, and profiling the target aptamer library through this comparison.

[0026] In some aspects, the present disclosure provides a method for diagnosing a disease in a target subject, the method comprising: (i) obtaining target aptamer library information for a target biological sample from a first aptamer database; (ii) obtaining reference aptamer library information for a reference biological sample from a second aptamer database; and (iii) comparing the target aptamer library information for the target biological sample with the reference aptamer library information for the reference biological sample.

[0027] In some aspects, the present disclosure provides a method for aiding in the diagnosis of a disease in a target subject, the method comprising: (i) obtaining target aptamer library information for a target biological sample from a first aptamer database; (ii) obtaining reference aptamer library information for a reference biological sample from a second aptamer database; and (iii) comparing the target aptamer library information for the target biological sample with the reference aptamer library information for the reference biological sample.

[0028] In some aspects, the present disclosure provides a method for determining the status of a physical condition in a target subject, the method comprising the steps of: (i) obtaining target aptamer library information for a target biological sample from a first aptamer database; (ii) obtaining reference aptamer library information for a reference biological sample from a second aptamer database; and (iii) comparing the target aptamer library information for the target biological sample with the reference aptamer library information for the reference biological sample.

[0029] In some aspects, the present disclosure provides a non-transitory computer-readable medium that, when instructions stored on the medium are executed by at least one processor, causes the at least one processor to perform a method for profiling a target aptamer library for a target biological sample, the method comprising: (i) obtaining information of a target aptamer library for the target biological sample from a first aptamer database; (ii) obtaining information of a reference aptamer library for the reference biological sample from a second aptamer database; and (iii) comparing the information of the target aptamer library for the target biological sample with the information of the reference aptamer library for the reference biological sample, and profiling the target aptamer library through this comparison.

[0030] 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.

[0031] In some embodiments, step (i) comprises downloading target aptamer library information for the target biological sample from the aptamer database to a user's computer, or extracting target aptamer library information for the target biological sample from the aptamer database to a server on which the aptamer database is stored.

[0032] In some embodiments, step (ii) comprises downloading information of a reference aptamer library for the reference biological sample from the aptamer database to a user's computer, or extracting information of a reference aptamer library for the reference biological sample from the aptamer database to a server on which the aptamer database is stored.

[0033] In some embodiments, the method further comprises step (iv) of determining differences between the information of the target aptamer library and the information of the reference aptamer library, hi some embodiments, step (iv) comprises identifying biomolecules bound to aptamers of the target aptamer library or the reference aptamer library that exhibit differences between the information of the target aptamer library and the information of the reference aptamer library.

[0034] In some embodiments, the target biological sample is a biological sample derived from a subject with the same genetic background as the reference biological sample. In some embodiments, the target biological sample is a biological sample derived from a subject with a different genetic background than the reference biological sample. In some embodiments, the target biological sample is derived from a target subject having or suspected of having a disease. In some embodiments, the reference biological sample is derived from a healthy subject. In some embodiments, the comparison step results in a determination of whether the target subject has a disease.

[0035] In some embodiments, the target biological sample is a diseased sample. In some embodiments, the reference biological sample is a non-disease sample. In some embodiments, the reference biological sample is a diseased sample. In some embodiments, the comparing step results in the identification of biomarkers for the diseased sample.

[0036] In some embodiments, 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. [Effects of the Invention]

[0037] 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 allows for the selection of aptamers capable of binding to one or more biomolecules with a high success rate. Furthermore, the present disclosure provides a method for profiling a target aptamer library. The method allows for the determination of the status of a physical condition in a target subject or the diagnosis of a disease in a target subject. [Brief explanation of the drawings]

[0038] [Figure 1] Figure 1 shows gel imaging data after 1D electrophoresis and 2D electrophoresis after selection in round 1. In the gel imaging data of 1D electrophoresis, the left lane shows control data performed without a sample, and the right lane shows gel shift data. [Figure 2] Figure 1 shows gel imaging data after 1D electrophoresis in round 5. The left lane shows control gel shift data without competitor, and the center lane shows gel shift data. The arrows on the right side of the gel image indicate the gel excision points to separate the upper and lower gels to check for different sequence evolution in different gel shift regions. [Figure 3] Figure 1 shows the increasing curve of aptamer library enrichment after various rounds of selection. The normalized resulting size indicates the number of distinct aptamer sequences with a frequency of more than 3 reads seen in next-generation sequencing analysis. [Figure 4] Figure 1 shows the results of a comparative analysis of aptamer library information for serum samples from different C57BL / 6N mice. The top 30 sequences in the results for C57BL / 6N serum B were selected and shown in the figure. SEQ ID NO: 10 and SEQ ID NO: 15 showed specificity for C57BL / 6N serum D. [Figure 5]

[0023] Figure 1 shows the results of a comparative analysis of aptamer library information for C57BL / 6N serum samples and BALB / c serum samples. The top 30 sequences in the C57BL / 6N serum B results were selected and shown in the figure. SEQ ID NO: 14 and SEQ ID NO: 27 showed no reactivity with BALB / c serum. [Figure 6] Figure 1 shows SDS-PAGE data of an aptamer-based precipitation assay using the sd-10 aptamer. The sequence of the sd-10 aptamer is shown in the table. The lowercase "t" indicates 5-tryptoamino-uracil. The arrowhead indicates the target protein band of the sd10 aptamer. [Figure 7] Figure 1 shows SDS-PAGE data of an aptamer-based precipitation assay using the Nb-01 aptamer. The sequence of the Nb-01 aptamer is shown in the table. The lowercase "t" indicates 5-tryptoamino-uracil. The arrowhead indicates the target protein band of the Nb-01 aptamer. [Figure 8] FIG. 1 shows Western blot analysis of sd-10 aptamer target proteins using anti-mouse IgG antibody. [Figure 9] FIG. 1 shows Western blot analysis of Nb-01 aptamer target protein using anti-GPLD-1 antibody. [Figure 10] FIG. 1 shows a comparative analysis of the information in aptamer libraries for human CSF samples derived from HC and AD patients. DETAILED DESCRIPTION OF THE INVENTION

[0039] The present disclosure provides, at least in part, methods for generating an aptamer library 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., 2D electrophoresis). The present 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 for utilizing such aptamer libraries (e.g., by comparing the information in a reference aptamer library and a target aptamer library to identify differences).

[0040] In some aspects, the present disclosure relates to methods for enriching a plurality of aptamers capable of binding to one or more target molecules (e.g., biomolecules) present in a sample (e.g., a biological sample).

[0041] The term "aptamer," as used herein, refers to an oligonucleotide (e.g., a single-stranded DNA (ssDNA) molecule or a 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 between 20 and 60 nucleotides, between 25 and 55 nucleotides, between 30 and 50 nucleotides, between 35 and 45 nucleotides, between 20 and 50 nucleotides, between 20 and 40 nucleotides, between 25 and 40 nucleotides, between 20 and 30 nucleotides, between 30 and 40 nucleotides, between 30 and 60 nucleotides, between 40 and 60 nucleotides, or between 50 and 60 nucleotides. In some embodiments, target molecules for aptamers include proteins, peptides, carbohydrates, small molecules, toxins, and cells (e.g., living cells). Aptamers bind to their targets with high affinity, selectivity, and specificity (see, for example, Non-Patent Documents 1 and 2). Aptamer binding is determined by the tertiary structure of the aptamer rather than its primary sequence. Target recognition and binding involve three-dimensional, shape-dependent interactions as well as hydrophobic interactions, base stacking, and intercalation. Aptamers offer advantages over antibodies because they can be fully engineered in vitro, are easily produced by chemical synthesis, have desirable storage properties, and cause little or no immunogenicity in therapeutic applications.

[0042] The concept of in vitro evolution of aptamers was introduced in 1990 and named SELEX (Separate Enzyme-Activated Leukemia) (Non-Patent Documents 3-7). SELEX combines the rules of combinatorial library screening with in vitro evolution to enrich for aptamers (e.g., DNA aptamers) against a wide range of target molecules. The SELEX process involves three interconnected steps: (i) repeated incubation of multiple candidate aptamers with a single target molecule to bind high-affinity aptamers, (ii) separation of high-affinity binders from low-affinity binders and / or non-binders, and (iii) amplification of high-affinity binders using polymerase chain reaction (PCR). This process is repeated until high-affinity aptamers are enriched in the selection pool. Although SELEX successfully generates aptamers for purified proteins, these aptamers may not recognize the same protein at the protein's endogenous level or under intracellular conditions. A variant of SELEX, called "multiple-target SELEX," was developed to select aptamers targeting proteins in their native conformation on the erythrocyte membrane. The proteins and their associated aptamers were separated by one-dimensional SDS electrophoresis (8).

[0043] I. Biofluid Ligand Library Enrichment Technology (BIOLLET) 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 the steps of: (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 electrophoretic medium in a first direction 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 electrophoretic medium in a second direction to obtain a portion of the second electrophoretic medium comprising the aptamer-biomolecule complexes; and (iv) extracting a plurality of aptamers capable of binding to the biomolecules in the biological sample from the plurality of aptamer-biomolecule complexes. The term "aptamer-biomolecule complex," as used herein, refers to a molecular complex formed between an aptamer and the aptamer's 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 an affinity or avidity that allows the molecule to be used to distinguish the binding partner from an appropriate control in a binding assay or other binding environment. With respect to an aptamer, the term "specific binding" refers to the ability of the aptamer to bind to a target molecule with an affinity or avidity that allows the aptamer to be used to distinguish the specific target molecule from other molecules when compared to one or more appropriate reference target molecules. In some embodiments, the aptamer has a binding affinity of at least about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or less, K for binding the target molecule D If the aptamer has a specific binding to the target molecule,

[0044] The present disclosure recognizes the difficulty of concentrating aptamers capable of binding to target molecules in their native conformation from a complex sample (e.g., a biological sample). The present disclosure also identifies the disadvantages of using one-dimensional electrophoresis (1D electrophoresis) to concentrate aptamer-target molecule (e.g., biomolecule) complexes and / or performing electrophoresis under mild conditions (e.g., low-salt conditions). For example, and without wishing to be bound by any particular theory, 1D electrophoresis cannot separate aptamer-target molecule (e.g., biomolecule) complexes from aptamers that bind to target molecules through nonspecific binding. As used herein with respect to aptamers, the term "nonspecific binding" refers to the ability of an aptamer to bind to a molecule with an affinity or avidity that does not allow the aptamer to be used to distinguish one molecule from another when compared to an appropriate reference target molecule or molecules. In some embodiments, an aptamer is capable of binding to a molecule with an affinity or avidity that does not allow the aptamer to be used to distinguish one molecule from another when compared to an appropriate reference target molecule or molecules. -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M, 10 -1 M or more, K for binding molecules D If the aptamer has a non-specific binding domain, then the aptamer is non-specifically binding to the molecule.

[0045] In addition, the conditions of 1D electrophoresis may not be sufficient to disrupt non-specific binding between a particular aptamer and a target molecule. The present disclosure therefore aims to concentrate multiple aptamers that can bind to one or more biomolecules in a biological sample by concentrating aptamer-target molecule (e.g., biomolecule) complexes using 2D electrophoresis. 2D electrophoresis can be performed under salt conditions that are sufficient to disrupt non-specific interactions between the aptamer and the target molecule but not sufficient to disrupt the specific binding between the aptamer and its target molecule. Thus, the present disclosure enables the simultaneous concentration of multiple aptamers that can bind to multiple target molecules (e.g., biomolecules) in a complex sample (e.g., a biological sample).

[0046] The term "candidate aptamer," as used herein, refers to a pool of random aptamers or random oligonucleotides that may be aptamers, which are subjected to the methods described herein to enrich 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, in generating the initial plurality of candidate aptamers, the ratio of nucleotides in each aptamer is optimized, for example, with an A:C:G:T molar ratio of 1.0:1.0:1.0:1.0, 1.5:1.5:1.0:1.2, 1.30:1.25:1.45:1.00, or 1.50:1.25:1.15:1.00. In some embodiments, the candidate aptamers are an initial pool of unselected random aptamers or random oligonucleotides that may be aptamers. In some embodiments, the candidate aptamers are a plurality of aptamers that can bind to a plurality of one or more target molecules (e.g., biomolecules) in a sample (e.g., a biological sample) obtained as a result of the final enrichment round using the methods described herein. In some embodiments, the plurality of candidate aptamers are DNA or RNA. In some embodiments, the candidate aptamer is single-stranded DNA (ssDNA). In some embodiments, the plurality of candidate aptamers have folded into the correct tertiary structure of the candidate aptamer to bind the target molecule.

[0047] In some embodiments, the plurality of candidate aptamers comprises modified nucleotides. Modified nucleotides have been described previously, see, for example, Non-Patent Document 9. In some embodiments, each of the plurality of candidate aptamers comprises at least one modified nucleotide. In some embodiments, each of the plurality of candidate aptamers comprises a modified nucleotide (e.g., a modified nucleotide that can be used as a substrate by a DNA polymerase). Modified nucleotides are well known in the art (see, for example, Patent Document 1 and Non-Patent Documents 10 and 11). In some embodiments, each of the plurality of candidate aptamers may be selected from a group consisting of, but 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, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5 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-tryptoaminouracil. In some embodiments, the modified nucleotide is a 2'-modified nucleotide.For example, 2'-modified nucleotides are 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-tryptamino-uracils in place of thymine. In some embodiments, at least one thymine in each of the plurality of candidate aptamers is substituted with 5-tryptamino-uracil. In some embodiments, all thymines in each of the plurality of candidate aptamers are substituted with 5-tryptamino-uracil.

[0048] In some embodiments, each of the plurality of candidate aptamers comprises a detectable label. The detectable label can facilitate detection of the aptamer-target molecule (e.g., biomolecule) complex. In some embodiments, the detectable label is a protein capable of generating a colorimetric change. In some embodiments, the protein capable of generating a colorimetric change is alkaline phosphatase, horseradish peroxidase, or luciferase. In some embodiments, the detectable label is a fluorescent molecule. In some embodiments, fluorescent molecules include, but are not limited to, fluorescent dyes, such as TYE665, Lucifer Yellow, dansyl, TruRed, fluorescein, Cy2, Cy3, Cy7, TRITC, X-rhodamine, or Texas Red, green fluorescent protein (GFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), mCherry, mTurquoise2, or mOrange. In some embodiments, each of the plurality of candidate aptamers is labeled with a phosphate group at the 5' end. In some embodiments, each of the plurality of candidate aptamers is labeled with a hydroxyl group at the 3' end of the candidate aptamer. In some embodiments, each of the plurality of candidate aptamers comprises one or more detectable labels. In some embodiments, each of the plurality of candidate aptamers comprises a TYE665 fluorophore at the 5' end.

[0049] In some embodiments, a plurality of candidate aptamers are prepared in a selection buffer prior to contacting target molecules (e.g., biomolecules) 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 may stabilize 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 has a concentration between 0.1 mM and 10 mM, between 1 mM and 10 mM, between 5 mM and 10 mM, between 8 mM and 10 mM, between 6 mM and 8 mM, between 2 mM and 8 mM, between 0.1 mM and 5 mM, between 0.5 mM and 4.5 mM, between 1 mM and 4 mM, between 2 mM and 3 mM, between 0.5 mM and 2 mM, between 0.6 mM and 1.5 mM, between 0.7 mM and 1.3 mM In some embodiments, the buffer of choice comprises magnesium ions at a concentration of between 0.8 mM and 1.2 mM, between 0.9 mM and 1.1 mM, between 0.6 mM and 1.2 mM, between 0.6 mM and 1.2 mM, between 0.7 mM and 1.2 mM, between 0.8 mM and 1.2 mM, between 0.9 mM and 1 mM, between 0.8 mM and 1 mM, between 0.9 mM and 1 mM, between 0.9 mM and 1.5 mM, between 0.9 mM and 1.2 mM, or between 1 mM and 2 mM. In some embodiments, the buffer of choice comprises magnesium at a concentration of 1 mM. In some embodiments, the magnesium ions are in the form of magnesium chloride.In some embodiments, the plurality of candidate aptamers have a concentration between 50 nM and 8000 nM, between 60 nM and 7000 nM, between 50 nM and 6000 nM, between 50 nM and 8000 nM, between 50 nM and 8000 nM, between 50 nM and 8000 nM, between 50 nM and 5000 nM, between 60 nM and 4000 nM, between 70 nM and 3000 nM, between 80 nM and 2000 nM, between 90 nM and 1000 nM, between 100 nM and 1000 nM. , 50nM-100nM, 100nM-200nM, 200nM-500nM, 500nM-1000nM, 1000nM-2000nM, 2000nM-3000nM, 3000nM-4000nM, 4000nM-5000nM, 1000nM-5000nM, 1000nM-2500nM, or 2500nM-5000nM.

[0050] In some embodiments, enriching a plurality of aptamers is also described as selecting a plurality of aptamers. The terms "enrich," "enrichment," or "enrichment process" are used interchangeably with selecting, selecting, or a selection process, respectively. In some embodiments, enriching a plurality of aptamers involves selecting aptamers that can bind to one or more target molecules (e.g., biomolecules) from aptamers that do not bind to the one or more target molecules (e.g., biomolecules). In some embodiments, enriching a plurality of aptamers involves selecting aptamers with higher affinity for one or more target molecules (e.g., biomolecules) over aptamers with lower affinity for one or more target molecules (e.g., biomolecules).

[0051] In some embodiments, the methods described herein include contacting a plurality of candidate aptamers with a complex sample (e.g., a biological sample) containing a plurality of different target molecules. In some embodiments, the complex sample includes a plurality of different target molecules of the same type (e.g., the complex sample contains a plurality of different proteins). In some embodiments, the complex sample contains a plurality of different types of target molecules (e.g., the complex sample contains proteins, nucleic acids, small molecules, toxins, etc.). In some embodiments, the complex sample contains different types of target molecules, and each type of target molecule further contains different individual target molecules (e.g., the complex sample contains a plurality of different proteins, a plurality of different nucleic acids, a plurality of different small molecules, and a plurality of different toxins, etc.). Examples of complex samples include, but are not limited to, biological samples (e.g., biological fluids such as serum), environmental samples (e.g., samples obtained from rivers, lakes, ponds, soil, the atmosphere, outer space, etc.), and manufacturing samples (e.g., samples obtained from bioreactors, samples obtained from HPLC flow-through fluids, samples containing small molecule drug intermediates, etc.). In some embodiments, the complex sample is a biological sample. The term "biological sample," as used herein, refers to a sample obtained from a biological subject.

[0052] Examples of biological samples include, but are not limited to, whole blood, interstitial fluid, skin, lymph, bile, serum, plasma, cerebrospinal fluid (CSF), urine, amniotic fluid, bone marrow, bronchoalveolar lavage fluid, oral swab, stool, gastrointestinal fluid, liposuction sample, saliva, milk, nasal swab, peritoneal fluid, semen, sputum, synovial fluid, tears, vaginal fluid, tissue biopsy, autopsy sample, cells or cell lysate, cultured cells, tissue sample (e.g., tissue sample from a human, non-human animal, plant, insect, or fungus), or in vivo endothelial cells. In some embodiments, the biological sample comprises target molecules (e.g., biomolecules), such as nucleic acids (e.g., DNA and RNA), proteins, peptides, lipids, polysaccharides, proteoglycans, and glycolipids. In some embodiments, the biological sample is serum. In some embodiments, the biological sample is obtained from a human subject. In some embodiments, the biological sample is obtained from a non-human subject. Examples of non-human subjects include, but are not limited to, monkeys, mice, rats, rabbits, goats, sheep, dogs, birds, and fish. In some embodiments, the subject is a healthy subject. In some embodiments, the subject is a subject suffering from, suspected of suffering from, or at risk for developing a disease.

[0053] In some embodiments, target molecules (e.g., biomolecules) in a sample (e.g., a biological sample) are denatured before being contacted with a plurality of candidate aptamers. In some embodiments, target molecules (e.g., biomolecules) in a sample (e.g., a biological sample) are not denatured before being contacted with a plurality of candidate aptamers. The terms "denaturing," "denaturing," or "denatured," as used herein, refer to subjecting a sample (e.g., a biological sample) to conditions that disrupt the linkages (e.g., disulfide bridges), bonds (e.g., hydrogen bonds, ionic bonds, etc.), and / or interactions (e.g., hydrophobic interactions) within one or more target molecules (e.g., biomolecules such as proteins) in the sample that provide the target molecules (e.g., biomolecules such as proteins and / or nucleic acids) in their natural (native) state with a highly ordered structure. In some embodiments, a denatured sample includes a sample in which biomolecules (e.g., proteins or nucleic acids) in the sample have lost their quaternary, tertiary, and secondary structure and thus retain only their primary structure (e.g., linear amino acid or nucleic acid sequence), such that the biomolecules no longer retain their respective structure and / or assigned function. In some embodiments, a denatured sample includes a sample in which biomolecules (e.g., proteins or nucleic acids) have been degraded into fragments, such that the biomolecules no longer retain their structure and / or assigned function. Thus, a denatured sample includes biomolecules (e.g., proteins or nucleic acids) that have not retained their assigned function (e.g., binding ability, biological activity, etc.). Conversely, an undenatured sample is a sample containing target molecules (e.g., biomolecules) that retain their native conformation and their respective assigned function. Methods for denaturing target molecules in a sample are known in the art, such as by heating, by treatment with alkali, acid, urea, or detergent, or by vigorous shaking. In some embodiments, the target molecules (e.g., biomolecules) are in their native conformation.In some embodiments, contacting multiple candidate aptamers with target molecules (e.g., biomolecules) in their native conformations is advantageous in that selected aptamers can bind to their target molecules (e.g., biomolecules) under conditions other than those for which the aptamers were selected (e.g., in vivo in a subject). Those skilled in the art will understand that denaturation includes subjecting target molecules (e.g., biomolecules) in a sample (e.g., biological sample) to conditions (e.g., heating, sonication, incubation in the presence of detergents, etc.) sufficient to cause some change in the native or natural structure of the target molecule (e.g., biomolecule, e.g., protein, DNA, RNA, toxin, or small molecule) that prevents the target molecule from performing its assigned function. The term "undenatured" as used herein refers to maintaining sufficient structure of target molecules (e.g., biomolecules) in a sample (e.g., biological sample) to perform their assigned function. In some embodiments, 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 assigned functions.

[0054] In some embodiments, the sample (e.g., biological sample) is not diluted before contacting with the plurality of candidate aptamers. In some embodiments, the sample (e.g., biological sample) is diluted before contacting with the plurality of candidate aptamers. In some embodiments, the biological 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, or between 1:50 and 1:2, or between 1:10 ...100 and 1:2, or between 1:100 and 1:2, or between 1:100 and 1:2, or between 1:100 and 1:2, or between 1:100 and 1:2, or between 1:100 and 1:2, or between 1:100 and 1:2, or between 1:100 and 1:2, or between 1:100 and 1:2, or between 1:1 Diluted at ratios between 1:25 and 1:2, between 1:10 and 1:2, between 1:5 and 1:2, between 1:500 and 1:50, between 1:500 and 1:100, between 1:500 and 1:200, between 1:200 and 1:100, between 1:200 and 1:50, between 1:200 and 1:10, between 1:100 and 1:50, between 1:100 and 1:10, between 1:100 and 1:5, or between 1:100 and 1:2. 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 1:200, between 1:200 and 1:100, between 1:200 and 1:150, between 1:200 and 1:50, between 1:250 and 1:200, or between 1:200 and 1:100, between 1:200 and 1:150, or between 1:200. In some embodiments, the serum is diluted at a ratio of 1:200 before contacting with the plurality of candidate aptamers. In some embodiments, the biological sample is CSF, and the CSF is diluted at a ratio of 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 before contacting the biological sample with the plurality of candidate aptamers.Non-limiting examples of dilution buffers include phosphate-buffered saline (PBS), Dulbecco's phosphate-buffered saline (DPBS), Hank's balanced salt solution (HBSS), and Dulbecco's modified Eagle's medium (DMEM). Those skilled in the art can select an appropriate dilution buffer according to the biological sample used.

[0055] In some embodiments, the method further comprises contacting the sample (e.g., biological sample) with a plurality of competitor nucleic acids before contacting the sample (e.g., biological sample) with a plurality of candidate aptamers. The competitor nucleic acids are used to block non-specific binding between the aptamer and the target molecule. In some embodiments, the competitor nucleic acids are a random set of unrelated nucleic acids. In some embodiments, the competitor nucleic acid is salmon sperm DNA.

[0056] In some embodiments, a plurality of candidate aptamers are contacted with a sample (e.g., a biological sample), whereby the aptamers bind to the aptamer's target molecule (e.g., a biomolecule), generating 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., an 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., an unbound biomolecule). In some embodiments, the composition also comprises an aptamer bound to a biomolecule by non-specific binding.

[0057] In some embodiments, the methods described herein comprise separating aptamer-target molecule (e.g., aptamer-biomolecule) complexes from unbound aptamers. In some embodiments, the methods described herein comprise separating aptamer-target molecule (e.g., aptamer-biomolecule) complexes from unbound aptamers. In some embodiments, the methods described herein comprise separating aptamer-target molecule (e.g., aptamer-biomolecule) complexes from aptamers bound to biomolecules by non-specific binding. In some embodiments, the methods comprise concentrating aptamer-target molecules (e.g., aptamer-biomolecule complexes) from a composition that also includes unbound aptamers and / or aptamers bound to target molecules by non-specific binding.

[0058] In some embodiments, concentrating aptamer-target molecules (e.g., aptamer-biomolecule complexes) from a composition that also contains unbound aptamers and / or aptamers bound to target molecules by non-specific binding comprises subjecting the composition to electrophoresis. The term "electrophoresis," as used herein, refers to a technique used to separate molecules (e.g., DNA, RNA, proteins, aptamer-target molecule complexes) based on their size and charge. An electrophoresis system includes two oppositely charged electrodes (anode and cathode) connected by a conductive electrophoretic medium. An electric current is used to move and separate molecules within the electrophoretic medium. In some embodiments, a negative charge is applied, resulting in molecules migrating toward the positive charge. Furthermore, electrophoretic media typically have pores that allow smaller molecules to migrate faster than larger molecules. In some embodiments, the present disclosure is based on the theory that multiple aptamer-target molecule (e.g., biomolecule) complexes differ in size from unbound aptamers (e.g., biomolecules). In some embodiments, unbound aptamers are smaller in size than the aptamer-target molecules (e.g., biomolecules) and therefore migrate faster during electrophoresis. In some embodiments, the individual aptamer-target molecules (e.g., biomolecules) in a plurality of aptamer-target molecule (e.g., biomolecule) complexes differ in size from one another. In some embodiments, the individual aptamer-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 a 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 bound to the target molecule (e.g., biomolecule) by non-specific binding is not sufficient to separate the aptamer-target molecule (e.g., biomolecule) complex from the aptamer bound to the target molecule (e.g., biomolecule) by 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 an aptamer bound to a target molecule (e.g., a biomolecule) by non-specific binding (Figures 2C-2D).

[0059] 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 concentrating 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 the aptamer-biomolecule complexes, which may also contain aptamers bound to target molecules (e.g., biomolecules) by non-specific binding, to electrophoresis in a second electrophoretic medium in a second direction. As used herein, the term "second direction" refers to a direction different from the first direction. In some embodiments, the second direction is perpendicular to the first direction. In some embodiments, the method further includes excising a portion of the first electrophoretic medium containing the aptamer-biomolecule complexes (which may also contain aptamers bound to target molecules (e.g., biomolecules) by non-specific binding) from the remainder of the first electrophoretic medium. In some embodiments, the method further comprises aligning the excised portion of the first electrophoretic medium containing the aptamer-biomolecule complexes (which portion may also contain aptamers bound to target molecules (e.g., biomolecules) by non-specific binding) with a well in a second electrophoretic medium. Subjecting the composition to electrophoresis in two dimensions disrupts weak non-specific binding between the aptamers and target molecules (e.g., biomolecules), resulting in unbound aptamers that migrate faster than the aptamer-target molecule (e.g., biomolecule) complexes. In some embodiments, the aptamer-target molecule (e.g., biomolecule) complexes reside in an oblique region within the second electrophoretic medium after electrophoresis in the second direction. In some embodiments, the method further comprises excising the portion of the second electrophoretic medium containing the aptamer-biomolecule complexes from the remainder of the second electrophoretic medium.

[0060] In some embodiments, the method further comprises extracting aptamers capable of binding to one or more target molecules (e.g., biomolecules) from a portion of the second electrophoretic medium containing the aptamer-biomolecule complexes. In some embodiments, the aptamers are separated from the aptamer-target molecule complexes during this step. Methods for extracting aptamers from a portion of the electrophoretic medium have been previously described, for example, by commercially available kits such as Oligo Clean and Concentrator Kits by Zymo Research, QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany), Wizard SV Gel and PCR Clean Up System (Promega, Madison, WI, USA), and GENECLEAN® II kit (MP BIOMEDICALS, Solon, OH, USA), or by the method described in Non-Patent Document 12.

[0061] In some embodiments, the electrophoretic medium contains pores that allow migration of molecules subjected to electrophoresis. Non-limiting examples of electrophoretic media include agarose, polyacrylamide, a silica 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. Methods for preparing agarose gels for electrophoresis are known in the art, for example, as described by

[13] . In some embodiments, the agarose gel is prepared from dry agarose powder (e.g., by dissolving the agarose powder in an appropriate buffer, such as Tris buffer, by heating and solidifying the agarose by cooling to room temperature). In some embodiments, the agarose gel is a pre-made gel purchased from a vendor. In some embodiments, the first electrophoresis medium is an agarose gel containing any concentration of agarose 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%), between 1% and 2%, between 0.5% and 1%, between 1.5% and 2%, or between 2% and 2.5%.

[0062] In some aspects, the present disclosure also considers optimal conditions (e.g., salt, temperature) for performing electrophoresis. In some embodiments, 2D electrophoresis is performed under salt conditions (e.g., an electrophoresis medium containing a certain concentration of ions) that can alleviate the electrostatic effects of biomolecules (e.g., proteins or DNA). In some embodiments, 2D electrophoresis is performed under salt conditions (e.g., an electrophoresis medium containing a certain concentration of ions) that are sufficient to disrupt nonspecific interactions between aptamers and target molecules but not sufficient to disrupt specific binding between aptamers and their target molecules. 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 and potassium ions, a combination of sodium and lithium ions, a combination of sodium and ammonium ions, a combination of potassium and lithium ions, a combination of potassium and ammonium ions, a combination of lithium ions and ammonium ions, a combination of sodium, potassium, and lithium ions, a combination of sodium, potassium, and ammonium ions, a combination of potassium, lithium, and ammonium ions, or a combination of sodium, potassium, lithium, and ammonium ions). The term "any of these combinations at a concentration," as used herein, refers to the total concentration of an ion(s) in a combination (e.g., any of the combinations described herein). For example, a combination of two ions at concentrations between X and Y refers to the total concentration of the two ions being in the range of X to Y; a combination of three ions at concentrations between X and Y refers to the total concentration of the three ions being in the range of X to Y; and so forth.Concentrations can be described using any units known in the art, e.g., 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 / mL, μg / μL, μg / nL, ng / L, ng / dL, ng / mL, ng / μL, ng / nL, pg / L, pg / dL, pg / mL, pg / μL, or pg / nL. For example, if an electrophoretic medium includes a combination of ions (e.g., sodium ions, potassium ions, lithium ions, and / or ammonium ions) at a total concentration between 100 mM and 200 mM, this means that the total concentration of the combination of ions is between 100 mM and 200 mM. It is within the skill of one of ordinary skill in the art to select the concentration of each ion in the combination to reach a desired range of total concentration.

[0063] 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 sufficient to disrupt non-specific binding of aptamers to target molecules. In some embodiments, the first electrophoretic medium has a concentration between 50 mM and 500 mM, between 80 mM and 450 mM, between 100 mM and 400 mM, between 150 mM and 350 mM, between 200 mM and 300 mM, between 100 mM and 400 mM, between 100 mM and 300 mM, between 100 mM and 200 mM, between 100 mM and 150 mM, between 150 mM and 200 mM, between 110 mM and 190 mM, between 120 mM and 180 mM, between 130 mM and 170 mM, between 140 mM and 160 mM, between 120 mM and 150 mM, between 120 mM and 160 mM, between 120 mM and 170 mM, between 120 mM and 170 mM, between 120 mM and 180 mM, between 130 mM and 170 mM, between 140 mM and 160 mM, between 120 mM and 150 mM, between 120 mM and 160 mM, between 120 mM and 170 mM, between 120 mM and 180 mM, between 120 mM and 180 mM, between 130 mM and 170 mM, between 120 mM and 180 mM, between 140 mM and 160 mM, between 120 mM and 18 ... and 130 mM, 150 mM to 160 mM, 150 mM to 170 mM, 150 mM to 180 mM, 150 mM to 190 mM, 100 mM to 120 mM, 120 mM to 130 mM, 130 mM to 140 mM, 140 mM to 150 mM, 150 mM to 160 mM, 160 mM to 170 mM, 170 mM to 180 mM, 180 mM to 190 mM, or 190 mM to 200 mM. In some embodiments, the first electrophoretic medium comprises sodium ions (e.g., sodium chloride), potassium ions, lithium ions, ammonium ions, or any combination thereof, at a concentration between 100 mM and 200 mM (e.g., any concentration between 100 mM and 200 mM).

[0064] In some embodiments, the second electrophoretic medium is between 50 mM and 500 mM, between 80 mM and 450 mM, between 100 mM and 400 mM, between 150 mM and 350 mM, between 200 mM and 300 mM, between 100 mM and 400 mM, between 100 mM and 300 mM, between 100 mM and 200 mM, between 100 mM and 150 mM, between 150 mM and 200 mM, between 110 mM and 190 mM, between 120 mM and 180 mM, between 130 mM and 170 mM, between 140 mM and 160 mM, between 120 mM and 150 mM, between 120 mM and 160 mM, between 120 mM and 170 mM, between 120 mM and 170 mM, between 120 mM and 120 mM and 130 mM, 150 mM to 160 mM, 150 mM to 170 mM, 150 mM to 180 mM, 150 mM to 190 mM, 100 mM to 120 mM, 120 mM to 130 mM, 130 mM to 140 mM, 140 mM to 150 mM, 150 mM to 160 mM, 160 mM to 170 mM, 170 mM to 180 mM, 180 mM to 190 mM, or 190 mM to 200 mM. In some embodiments, the second electrophoretic medium comprises sodium ions (e.g., sodium chloride), potassium ions, lithium ions, ammonium ions, or any combination thereof, at a concentration between 100 mM and 200 mM (e.g., any concentration between 100 mM and 200 mM). 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).

[0065] 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 that may stabilize 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 electrophoresis 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 has a concentration between 0.1 mM and 10 mM, between 0.1 mM and 9 mM, between 0.1 mM and 8 mM, between 0.1 mM and 7 mM, between 0.1 mM and 6 mM, between 0.1 mM and 4 mM, between 0.1 mM and 3 mM, between 0.1 mM and 2 mM, between 0.1 mM and 1 mM, between 0.1 mM and 0.5 mM, between 0.5 mM and 10 mM, between 0.5 mM and 9 mM, between 0.5 mM and 8 mM, between 0.5 mM and 7 mM, between 0.5 mM and 6 mM, between 0.5 mM and 5 mM, between 0.5 mM and 4 mM, between 0.5 mM and 3 mM, between 0.5 ... Between 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 10 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 The present invention also includes magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof, at a concentration of between 0.5 mM and 3 mM, between 0.5 mM and 2 mM, between 0.6 mM and 1.5 mM, between 0.7 mM and 1.3 mM, between 0.8 mM and 1.2 mM, between 0.9 mM and 1.1 mM, between 0.6 mM and 1.2 mM, between 0.6 mM and 1.2 mM, between 0.7 mM and 1.2 mM, between 0.8 mM and 1.2 mM, between 0.9 mM and 1 mM, between 0.8 mM and 1 mM, between 0.9 mM and 1.5 mM, between 0.9 mM and 1.2 mM, or between 1 mM and 2 mM.In some embodiments, the first electrophoretic medium comprises magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof, at a concentration of 1 mM. In some embodiments, the second electrophoretic medium comprises magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof, at a concentration sufficient to stabilize the structure of the aptamer 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 0.5 mM).In some embodiments, the first electrophoretic medium has a concentration between 0.1 mM and 10 mM, between 0.1 mM and 9 mM, between 0.1 mM and 8 mM, between 0.1 mM and 7 mM, between 0.1 mM and 6 mM, between 0.1 mM and 4 mM, between 0.1 mM and 3 mM, between 0.1 mM and 2 mM, between 0.1 mM and 1 mM, between 0.1 mM and 0.5 mM, between 0.5 mM and 10 mM, between 0.5 mM and 9 mM, between 0.5 mM and 8 mM, between 0.5 mM and 7 mM, between 0.5 mM and 6 mM, between 0.5 mM and 5 mM, between 0.5 mM and 4 mM, between 0.5 mM and 3 mM, between 0.5 ... Between 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 10 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 The present invention also includes magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof, at a concentration of between 0.5 mM and 3 mM, between 0.5 mM and 2 mM, between 0.6 mM and 1.5 mM, between 0.7 mM and 1.3 mM, between 0.8 mM and 1.2 mM, between 0.9 mM and 1.1 mM, between 0.6 mM and 1.2 mM, between 0.6 mM and 1.2 mM, between 0.7 mM and 1.2 mM, between 0.8 mM and 1.2 mM, between 0.9 mM and 1 mM, between 0.8 mM and 1 mM, between 0.9 mM and 1.5 mM, between 0.9 mM and 1.2 mM, or between 1 mM and 2 mM.In some embodiments, the second electrophoretic medium contains magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof at a concentration of 1 mM. In some embodiments, the divalent ion is magnesium ion. Magnesium can be added to the first and / or second electrophoretic medium in the form of any known magnesium salt, such as magnesium chloride and magnesium sulfate. In some embodiments, the magnesium ions are in the form of magnesium chloride.

[0066] In some embodiments, the running buffer for electrophoresis in the first direction comprises boric acid at a concentration between 40 mM and 100 mM. In some embodiments, the running buffer for electrophoresis in the first direction comprises tris(hydroxymethyl)aminomethane at a concentration between 40 mM and 100 mM. In some embodiments, the running buffer for electrophoresis in the second direction comprises boric acid at a concentration between 40 mM and 100 mM. In some embodiments, the running buffer for electrophoresis in the second direction comprises tris(hydroxymethyl)aminomethane at a concentration between 40 mM and 100 mM.

[0067] In some embodiments, 2D electrophoresis is performed at a temperature optimal for migration and separation of molecules in the composition (e.g., aptamer-biomolecule complexes and unbound aptamers). In some embodiments, electrophoresis in the first direction is performed at a temperature between 8°C and 22°C, between 9°C and 21°C, 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, or between 12°C and 13°C. In some embodiments, electrophoresis in the first direction is performed at 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, or 22°C. In some embodiments, electrophoresis in the second direction is performed at a temperature between 8°C and 22°C, between 9°C and 21°C, or between 10°C and 20°C, between 11°C and 19°C, between 12°C and 18°C, between 13°C and 17°C, between 14°C and 16°C, between 10°C and 15°C, between 11°C and 14°C, or between 12°C and 13°C. In some embodiments, electrophoresis in the second direction is performed at 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C. In some embodiments, the amount of salt (e.g., sodium chloride and / or magnesium chloride) in the first and second electrophoresis media raises the temperature of the electrophoresis media to a temperature that may interfere with the stability of the aptamer-target molecule complex. In some embodiments, the electrophoresis unit is placed in a cold water bath filled with ice to maintain the temperature within the optimal temperature ranges described herein.

[0068] As described herein, in some embodiments, the method further comprises extracting aptamers capable of binding to one or more target molecules (e.g., biomolecules) from a portion of the second electrophoretic medium containing the aptamer-biomolecule complexes. In some embodiments, the method further comprises amplifying the plurality of aptamers extracted from the aptamer-target molecule (e.g., biomolecule) complexes to form an aptamer library. In some embodiments, the amplification of aptamers capable of binding to one or more target molecules (e.g., biomolecules) is performed by polymerase chain reaction (PCR). Polymerase chain reaction (PCR) is a laboratory technique used to amplify DNA sequences by using short DNA sequences called primers. The temperature of the sample is repeatedly raised and lowered to promote DNA replication enzymes to copy the target DNA sequences. Non-limiting examples of PCR include emulsion PCR, asymmetric PCR, convection PCR, dial-out PCR, digital PCR, helicase-dependent amplification, hot-start PCR, in silico PCR, inverse PCR, ligation-based PCR, miniprimer PCR, multiplex ligation-dependent probe amplification, multiplex PCR, nanoparticle-assisted PCR, nested PCR, overlap extension PCR, quantitative PCR, reverse complement PCR, single specific primer-PCR, and solid-phase PCR.

[0069] In some embodiments, the plurality of aptamers capable of binding to one or more target molecules (e.g., biomolecules) extracted from the portion of the second electrophoretic medium containing the aptamer-biomolecule complexes includes different amounts of aptamers (i.e., sequence order), e.g., aptamers that bind to the target molecule with high affinity are present at high levels, while aptamers that bind to the target molecule with low affinity are present at low levels. In some aspects, the present disclosure contemplates preserving the sequence order information of the plurality of aptamers capable of binding to one or more target molecules (e.g., biomolecules) after generating an aptamer library by amplification. In some embodiments, any suitable amplification method known in the art (e.g., PCR) can be utilized by the methods described herein, provided that the amplification method can preserve the sequence order information of the plurality of aptamers capable of binding to one or more target molecules (e.g., biomolecules) after generating an aptamer library by amplification.

[0070] In some embodiments, the present disclosure is based, at least in part, on the discovery that emulsion PCR can preserve sequence order information in multiple aptamers that can bind to one or more target molecules (e.g., biomolecules) after amplification to generate an aptamer library. The term "emulsion PCR," as used herein, refers to a PCR reaction performed in aqueous droplets emulsified in the oil phase of a water-in-oil emulsion. In some embodiments, the aqueous droplets function as miniaturized "reactors" for each PCR reaction, are physically separated from one another, and do not involve the exchange of macromolecules, particularly PCR products. In some embodiments, individual DNA molecules are compartmentalized in their own separate reaction droplets, allowing for the amplification of these molecules independently of one another. Emulsion PCR avoids the formation of unproductive chimeras and other by-products, reducing overall amplification bias (see, e.g., Non-Patent Documents 14 and 15).

[0071] In some embodiments, enrichment of the plurality of aptamers is performed through multiple rounds of selecting 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 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or more times. In some embodiments, the steps of the methods described herein are repeated four times. In some embodiments, the steps of the methods described herein are repeated nine times. In some embodiments, the aptamer library obtained after amplification (e.g., emulsion PCR) is used as a plurality of starting candidate aptamers for contacting target molecules (e.g., biomolecules) in a sample (e.g., a biological sample) in the next round.

[0072] In some embodiments, the method further includes identifying sequences of a plurality of aptamers capable of binding to target molecules (e.g., biomolecules) in the sample (e.g., biological sample). Methods of sequencing nucleic acids (e.g., aptamers) are known in the art, including, but not limited to, basic sequencing (e.g., Maxam-Gilbert sequencing), chain termination sequencing (e.g., Sanger sequencing), deep sequencing and de novo sequencing (e.g., shotgun sequencing), next-generation sequencing (e.g., single-molecule real-time sequencing), ion torrent sequencing, pyrosequencing, sequencing-by-synthesis (e.g., MiSeq), combinatorial sequencing, and the like. These include real-probe anchor synthesis, sequencing by ligation (SOLiD sequencing), nanopore sequencing, GenapSys sequencing, or chain termination (Sanger sequencing), long-read sequencing, and short-read sequencing methods (e.g., massively parallel signature sequencing (MPSS), polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, DNA nanoball sequencing, Heliscope single-molecule sequencing, and microfluidics systems). In some embodiments, the aptamer is sequenced by next-generation sequencing. In some embodiments, the aptamer is sequenced by MiSeq.

[0073] In some embodiments, the method further comprises identifying biomolecules bound to a plurality of aptamers capable of binding to the biomolecule in the biological sample. In some embodiments, the biomolecules (e.g., proteins) can be separated from the bound aptamers. Methods for identifying biomolecules are well known to those skilled in the art. Briefly, methods for identifying biomolecules include, but are not limited to, DNA sequencing (e.g., NGS), Northern blot, Southern blot, 1D or 2D gel electrophoresis, mass spectrometry, enzyme-linked immunosorbent assay (ELISA), SDS-PAGE, Western blot, thin-layer chromatography, high-performance liquid chromatography (HPLC), gas chromatography, or any combination thereof. In some embodiments, the biomolecules (e.g., proteins) are identified by mass spectrometry.

[0074] In some embodiments, the methods described herein further include compiling and storing, in a computer-readable format, sequence information of an aptamer library capable of binding to a biomolecule in a biological sample and / or information on biomolecules bound to the aptamers in the aptamer library. In some embodiments, sequence information of aptamers in the aptamer library and / or information on biomolecules bound to the aptamers can be uploaded from a database to a server. In some embodiments, the database includes information from two or more aptamer libraries. Once an aptamer library is created, further information on additional, different aptamer libraries can be added to the database (e.g., the database can include information on an aptamer library for mouse serum, an aptamer library for mouse CSF, an aptamer library for human serum, an aptamer library for human CSF, an aptamer library for cancer tissue, etc.). Once aptamer library information is identified and added to the database, such information can be easily downloaded and used without the need to perform further steps of the methods described herein. In some embodiments, the aptamer database is stored on a server. In some embodiments, the server storing the aptamer database further provides means for downloading the aptamer library information stored on the server, hi some embodiments, the server storing the aptamer database further provides means for exporting the aptamer library information for comparison with other aptamer libraries on the server.

[0075] II. How to Use Information from an Aptamer Library In some aspects, the present disclosure also provides a method for profiling a target aptamer library for a target biological sample, the method comprising: (i) obtaining information about the target aptamer library for the target biological sample from a first aptamer database; (ii) obtaining information about a reference aptamer library for a reference biological sample from a second aptamer database; and (iii) comparing the information about the target aptamer library for the target biological sample with the information about the reference aptamer library for the reference biological sample, thereby profiling the target aptamer library. Those skilled in the art will recognize that the terms "obtaining," "providing," "deriving," and "obtaining" are used interchangeably and refer to steps including accessing an aptamer database, finding information corresponding to the aptamer library (e.g., aptamer sequence identity, aptamer modifications, relative aptamer amounts, aptamer purity, etc.), and providing the information in a subsequent step (e.g., step (iii) of comparing the information about the target aptamer library and the reference aptamer library). The term "profiling a target aptamer library," as used herein, refers to analyzing biological information or characteristics of a target subject based on information from a target aptamer library for a target biological sample and information from a reference aptamer library for a reference biological sample (e.g., sequence information, including but not limited to, the frequency or amount of aptamer sequences in an aptamer library that can bind to biomolecules in a biological sample).

[0076] The "method for profiling a target aptamer library" may provide a method for determining the status of a physical condition in a target subject or for diagnosing a disease in a target subject.

[0077] Diagnosis of disease can be based on, for example, the difference between the information of an aptamer library for a biological sample derived from a healthy subject and the information of an aptamer library for a biological sample derived from a subject with or suspected of having a disease. When the information of the target aptamer library for the target biological sample is compared with the information of a reference aptamer library for a reference biological sample derived from a subject with or suspected of having a disease, if the target subject has at least one characteristic (e.g., a high / low sequence frequency or amount of a particular aptamer(s)) corresponding to the characteristic information of the reference aptamer library, the target subject is diagnosed as having a disease or a risk of a disease.

[0078] Diseases can be diagnosed by comparing the information of the target aptamer library for a target biological sample with the information of the reference aptamer library for a reference biological sample derived from a healthy subject. If the information of the target aptamer library for a target biological sample is essentially the same as the information of the reference aptamer library for a reference biological sample derived from a healthy subject, the target subject is considered to be healthy. If the information of the target aptamer library for a target biological sample shows any difference compared to the information of the reference aptamer library for a reference biological sample derived from a healthy subject, it can be determined that the target subject has some disease or some risk of disease.

[0079] Alternatively, or in addition, the information in the target aptamer library for the target biological sample may be compared with the information in the reference aptamer library for a reference biological sample from a subject independently diagnosed with a disease or at risk of a disease, in which case the target subject is diagnosed with a disease or at risk of a disease if the target subject has at least one characteristic (e.g., a high / low sequence frequency or amount of a particular aptamer(s)) that corresponds to the characteristic information in the reference aptamer library.

[0080] A "method for profiling a target aptamer library" may include identifying biomolecules bound to aptamers in the target aptamer library or the reference aptamer library that exhibit differences between information in the target aptamer library for the target biological sample and information in the reference aptamer library for the reference biological sample.

[0081] In some aspects, the present disclosure also provides a non-transitory computer-readable medium that, when instructions stored on the medium are executed by at least one processor, causes the at least one processor to perform a method for profiling a target aptamer library for a target biological sample, the method comprising: (i) obtaining information of a target aptamer library for the target biological sample from a first aptamer database; (ii) obtaining information of a reference aptamer library for a reference biological sample from a second aptamer database; and (iii) comparing the information of the target aptamer library for the target biological sample with the information of the reference aptamer library for the reference biological sample, and profiling the target aptamer library through this comparison.

[0082] In some embodiments, the first aptamer database is generated by a method described herein (e.g., a method for 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 a method described herein (e.g., a method for 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 a method described herein (e.g., a method for 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.

[0083] In some embodiments, the aptamer library information is initially obtained by a method described herein (e.g., by BIOLLET) and stored in an aptamer database. In some embodiments, obtaining aptamer library (e.g., target aptamer library and / or reference aptamer library) information from the aptamer database does not require obtaining the aptamer library information again (e.g., by BIOLLET).

[0084] In some embodiments, the method further comprises downloading information of a target aptamer library for the target biological sample from the aptamer database to a user's computer or extracting information of the target aptamer library for the target biological sample from the aptamer database to a server on which the aptamer database is stored, In some embodiments, the method further comprises downloading information of a reference aptamer library for a reference biological sample from the aptamer database to a user's computer or extracting information of a reference aptamer library for the reference biological sample from the aptamer database to a server on which the aptamer database is stored.

[0085] In some embodiments, the method further comprises determining the difference between the information of the target aptamer library and the information of the reference aptamer library.

[0086] The target aptamer library and / or the reference aptamer library can be selected based on the purpose of the comparison.

[0087] In some embodiments, the target biological sample is a biological sample from a subject with the same genetic background as the reference biological sample. For example, the target biological sample is serum from a C57BL / 6 mouse, and the reference biological sample is serum from another C57BL / 6 mouse. 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 a human subject of the same race. By comparing the target aptamer library and the reference aptamer library, differences in molecules (e.g., proteins) in the serum can be identified, and the differences can suggest that a certain factor may have affected the molecules (e.g., proteins) in the serum. Such factors may include, but are not limited to, environmental factors, dietary factors, epigenetic modifications, disease states, age, or gender.

[0088] In some embodiments, the target biological sample is a biological sample from a subject with a different genetic background than the reference biological sample. For example, the target biological sample is serum from a C57BL / 6 mouse, and the reference biological sample is serum from a BALB / c mouse. 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 a human subject of a different race. By comparing the target aptamer library and the reference aptamer library, differences in molecules (e.g., proteins) in the serum can be identified, and these differences can suggest that a certain genetic background is prone to or at risk of having a certain disease.

[0089] In some embodiments, the target biological sample and the reference biological sample are derived from the same subject but obtained at different time points. 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, differences in serum molecules (e.g., proteins) can be identified, which may indicate the effectiveness of the treatment. In other embodiments, the target biological sample is serum from a human subject when the subject has or is suspected of having a disease, and the reference biological sample is serum from the same human subject when the subject is healthy. By comparing the target aptamer library and the reference aptamer library, differences in serum molecules (e.g., proteins) can be identified, which may indicate that the subject has the suspected disease or is at risk for the suspected disease.

[0090] In some embodiments, the target biological sample is obtained from a subject having 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 having 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 molecules (e.g., proteins) in the serum can be identified, and the differences can suggest that the target subject has the suspected disease or is at risk for the suspected disease.

[0091] In some embodiments, the target biological sample is obtained from a subject having or suspected of having a disease, and the reference biological sample is obtained from a subject having or suspected of having a particular disease. For example, the target biological sample is serum from a human subject having or suspected of having a disease, and the reference biological sample is serum from a subject having or suspected of having a particular disease. By comparing the target aptamer library and the reference aptamer library, it is possible to diagnose a disease in a human subject.

[0092] The above embodiments can be implemented in any of numerous ways. One or more aspects and embodiments of the present disclosure involving the implementation of a process or method may utilize program instructions executable by a device (e.g., a computer, processor, or other device) to perform or control the implementation of the process or method. In this regard, various inventive concepts may be embodied as a computer-readable recording medium (or multiple computer-readable recording media) (e.g., computer memory, one or more floppy disks, compact disks, optical disks, magnetic tapes, flash memory, circuitry in field programmable gate arrays or other semiconductor devices, or other tangible computer recording media) encoded with one or more programs that, when implemented on one or more computers or other processors, perform methods for implementing one or more of the various embodiments described above. The one or more computer-readable media may be transportable, such that one or more programs stored on the computer-readable media can be loaded into one or more different computers or other processors to implement the various programs of the above aspects. In some embodiments, the computer readable medium may be a non-primary medium.

[0093] The terms "program" or "software" are used herein in a general sense to refer to any type of computer code or set of computer-executable instructions that can be used to program a computer or other processor to implement the various aspects described above. Furthermore, according to one aspect, one or more computer programs that, when executed, perform the methods of the present disclosure need not reside on a single computer or processor, but may be distributed in a modular manner among many different computers or processors to implement various aspects of the present disclosure.

[0094] Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically, the functionality of the program modules may be combined or distributed as desired in various embodiments.

[0095] Additionally, the data structure may be stored on a computer-readable medium in any suitable form. For ease of explanation, the data structure may be depicted as having fields that are related through their location within the data structure. Such associations are in turn achieved by assigning records to the fields with locations within the computer-readable medium that convey the association between the fields. However, any suitable mechanism may be used to establish associations between information in fields of the data structure, including through the use of pointers, tags, or other mechanisms that establish associations between data elements.

[0096] When implemented in software, the software code may be executed on any suitable processor or collection of processors, whether provided on a single computer or distributed among multiple computers.

[0097] It will further be understood that a computer may be embodied in any of many 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 also be embodied in devices not generally considered computers but having suitable processing capabilities, including a personal digital assistant (PDA), a smartphone, or any other suitable portable or fixed electronic device.

[0098] A computer may also have one or more input and output devices. These devices may be used, among other things, to provide a user interface. Examples of output devices that may be used to provide a user interface include a printer or display screen for visual display of output, and a speaker or other sound-generating device for audible display of output. Examples of input devices that may be used in a user interface include keyboards and pointing devices such as mice, touchpads, and digitizing tablets. As another example, a computer may receive input information through voice recognition or in other audible formats.

[0099] Such computers may be interconnected by one or more networks of any suitable form, including local area networks or wide area networks, e.g., 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.

[0100] Also, as described, some aspects may be embodied as one or more methods. The actions performed as part of the method may be ordered in any suitable manner. Thus, embodiments may be constructed in which actions are performed in an order different from that shown, and may include performing some actions simultaneously even though they are shown as sequential actions in the exemplary embodiment.

[0101] Other embodiments All of the features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0102] From the above description, those skilled in the art can easily ascertain the essential features of the present disclosure, and can make various changes and modifications to the present invention so that it is adapted to various uses and conditions without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are also within the scope of the claims.

[0103] equivalent While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision numerous other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each such variation and / or modification is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application or applications in which one or more teachings of the present invention are used. Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. Accordingly, it will be understood that the foregoing embodiments are described by way of example only, and that, within the scope of the appended claims and their equivalents, the inventive embodiments may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure are directed 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 is within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not inconsistent.

[0104] All definitions defined and used herein are understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0105] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each document is cited, which in some cases may include the document in its entirety.

[0106] The indefinite articles "a" and "an," as used herein in the specification and claims, unless expressly indicated otherwise, should be understood to mean "at least one."

[0107] The term "and / or," as used herein in the specification and claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and non-conjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so conjoined. Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with an open-ended term such as "comprising," may, for example, in one embodiment refer to A only (optionally including elements other than B), in another embodiment refer to B only (optionally including elements other than A), and in yet another embodiment refer to both A and B (optionally including other elements).

[0108] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items within a list, "or" or "and / or" should be interpreted as inclusive, i.e., including not only at least one, but two or more of a number or list of elements, and optionally additional unlisted items. Only terms clearly indicated to the contrary, such as "only one" or "exactly one," or "consisting of," when used in the claims, refer to the inclusion of exactly one element of a number or list of elements. Generally, as used herein, the term "or" should be interpreted as indicating exclusive alternatives only (i.e., "one or the other, but not both") when preceded by exclusive terms such as "either," "one," "only one," or "exactly one." "Consisting essentially of," when used in the claims, has its ordinary meaning as used in the field of patent law.

[0109] As used herein in the specification and claims, the phrase "at least one," in reference to a list of one or more elements, means at least one element selected from any one or more of the elements in the list of elements, but is understood to not necessarily include at least one of each and every element specifically listed in the list of elements, and does not exclude any combinations of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether or not related to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, similarly, "at least one of A or B," or, similarly, "at least one of A and / or B") may refer in one embodiment to, for example, at least one A, optionally including two or more, with no B present (and optionally including elements other than B); in another embodiment to at least one B, optionally including two or more, with no A present (and optionally including elements other than A); and in yet another embodiment to at least one A, optionally including two or more, and at least one B, optionally including two or more (and optionally including other elements).

[0110] It should also be understood that, unless expressly indicated to the contrary, in any method claimed herein that includes more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are recited. [Example]

[0111] Materials and Methods Preparation of agarose gels for 1D and 2D electrophoresis Agarose gels for 1D and 2D electrophoresis were prepared as follows: Agarose powder was mixed at a concentration of 1.0–1.2 wt% in 1x TB buffer (80 mM Tris base, 80 mM boric acid) containing 100–200 mM NaCl depending on the conditions selected. The mixture was heated in a microwave to completely dissolve the agarose powder. After cooling to approximately 60°C, 1 molar magnesium chloride solution was added to the agarose solution at a concentration of 1 mM and mixed thoroughly. 11 milliliters of the solution was poured into a gel tray (55 mm (W) x 60 mm (L)) with a well comb in place. The number of wells was 3–4 wells (10 mm (W) x 1 mm (L)) for 1D electrophoresis and 1 well (45 mm (W) x 2 mm (L)) for 2D electrophoresis. The thickness of the agarose gel was adjusted to be relatively thin to reduce heat generated from the high-salt agarose gel during electrophoresis. The gel was left at room temperature for at least 3 hours, but not more than 6 hours, before use.

[0112] Preparation of DNA libraries for ligation reactions A single-stranded DNA (ssDNA) library containing a 43-nucleotide random region and a TYE665 fluorophore at the 5' end was used for selection. The ssDNA library was diluted in selection buffer (PBS with 0.005% Nonidet-P40 and 1 mM magnesium chloride) to a specific 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 over 30 min to allow the formation of stable ssDNA structures. The reconstituted ssDNA library solution was mixed with a 10 μL reaction volume of an appropriately diluted biofluid sample solution. The dilution range of the biofluid sample depends on the type of biofluid; for example, a 2- to 200-fold dilution is used for serum samples, and a 1- to 2-fold dilution is used for cerebrospinal fluid (CSF) samples. The biofluid sample was mixed with a competitor, such as salmon sperm DNA and any unrelated DNA oligo, and then mixed with the ssDNA library solution. Dextran sulfate was also used to reduce charge-dependent nonspecific interactions between ssDNA and biomolecules. The mixture was incubated at room temperature for 10 minutes, and then 2 μL of 6× loading buffer containing 36% glycerol and 6 mM magnesium chloride was quickly added to the reaction mixture. The sample was then loaded onto an agarose gel.

[0113] 1D electrophoretic separation An electrophoresis unit, such as Mupid2, was filled with fresh 1x TB buffer. The gel for 1D electrophoresis prepared above was placed in a gel box. For high-salt agarose gels, the electrophoresis unit was placed in a cold water bath filled with crushed ice to maintain the buffer temperature between 10 and 20°C during electrophoresis. The gel was pre-run at 100 V for 10 minutes, after which the samples were run. The sample mixture was carefully loaded into the wells of the gel. 1D electrophoresis was performed in the dark at 100 V for 55 to 60 minutes. The run time depended on the selection round. After the run, the gel was removed from the gel box and visualized using a ChemiDoc MP Imaging System (Bio-Rad). The entire region of the aptamer-biomolecule complex, present entirely above the free aptamer region, was excised for 2D electrophoresis. The size of the excised gel was adjusted to fit the wells of the 2D electrophoresis gel.

[0114] 2D electrophoretic separation The other electrophoresis unit was filled with fresh 1x TB buffer, and the gel for 2D electrophoresis was placed in the gel box. As with 1D electrophoresis, the instrument was placed in the same cold water bath as for 1D electrophoresis, and the gel was pre-run at 100 V for 10 minutes. Then, 2D electrophoresis was performed. This pre-run step was performed between 1D electrophoresis runs, so that 2D electrophoresis could begin immediately after gel excision. After the pre-run, the gel was removed from the gel box, and the excised gel was carefully inserted into the well. The gel was returned to the gel box, and 2D electrophoresis was initiated at 100 V for 55–60 minutes in the dark. The run time depended on the selection round. After the run, the gel was removed from the gel box for visualization using a ChemiDoc MP Imaging System (Bio-Rad). Only the diagonal region formed by the aptamer-biomolecule complex was excised. The excised gel pieces were placed in a microfuge tube.

[0115] Extraction of DNA from agarose gel The excised gel pieces were melted at 95°C for 3 minutes. The melted agarose solution was distributed into several tubes with 110 μL aliquots. The tubes were incubated at 55°C for 1 minute. 2 μL of heat-stable β-agarase was added to each tube, and the tubes were incubated at 55°C for 15 minutes to enzymatically digest the agarose. The ssDNA library was recovered using Oligo Clean and Concentrator Kits (Zymo Research) according to the manufacturer's instructions. The recovered ssDNA was eluted with 30 μL of deionized water.

[0116] PCR amplification of the recovered ssDNA library The recovered ssDNA was subjected to PCR amplification in a two-step process. In the first step, test PCR reactions containing different numbers of consecutive PCR cycles were performed in small volumes. Note that all PCR amplifications were performed as an emulsion PCR procedure, in which 100 μL of PCR solution was mixed with 250 μL of emulsion oil (4.5% Span 80, 0.4% Tween 80, 0.05% Triton-X 100, and 95.05% mineral oil), and the solution was vigorously mixed with a magnetic stir bar until completely mixed. PCR products at specified cycles were recovered by chloroform extraction and analyzed by gel electrophoresis (6% polyacrylamide gel with 0.5× TBE buffer) to determine the appropriate PCR cycle that could provide a clear single band for the DNA product without any concatemers or truncations. The remaining PCR samples were amplified using the determined PCR cycle. The PCR product was recovered by chloroform extraction and purified using Oligo Clean and Concentrator Kits. The amplified DNA was eluted with 10 μL of deionized water and quantified using a Qubit 4 fluorometer (Invitrogen). This PCR amplification step was repeated until sufficient DNA was obtained.

[0117] Generation of aptamer libraries by primer extension The amplified DNA was diluted to a concentration of 1 ng / μL to prepare a template DNA sample for primer extension to generate an aptamer library. Two microliters of the solution were used for each 100 μL PCR amplification. A 5'-biotinylated antisense strand primer was used in this PCR amplification step, and the number of PCR cycles was fixed at 8. The PCR product was recovered by chloroform extraction and then purified using Oligo Clean and Concentrator Kits. The purified DNA was subjected to primer extension using a 5'-TYE665 fluorophore-labeled primer. The product was directly immobilized on streptavidin agarose beads filled with PBSN buffer (PBS with 0.005% Nonidet-P40). The beads were incubated at 16°C and 1500 rpm for 15 minutes with shaking every 2 minutes. The beads were washed three times with PBS and filled with 40 μL of 20 mM NaOH solution to denature the product DNA and release the 5'-TYE665-labeled aptamer. The bead solution was incubated at 37°C and 1500 rpm for 1 minute with shaking. The solution was centrifuged for 30 seconds in a benchtop microcentrifuge. The supernatant was transferred to a new tube. The denaturation step was performed once more, and the supernatants were combined in the same tube. The collected supernatant was quenched with 80 mM HCl to adjust the pH of the solution to approximately 7.0-8.0 and purified using the Oligo Clean and Concentrator Kit. The aptamer library was eluted with 10 μL of deionized water and quantified using a NanoDrop. The aptamer library was used for the next selection cycle.

[0118] Sequencing analysis of aptamer libraries The PCR products obtained from each selection round were PCR-amplified in a two-step process and prepared for next-generation sequencing analysis using a MiSeq (Illumina). An adapter sequence primer set was used in the first step, followed by an index sequence primer set in the second step. Thermal cycling was performed according to the manufacturer's instructions. PCR products were purified using NucleoSpin Gel and PCR Clean-up (Macherey Nagel) and quantified using a Qubit 4 fluorometer in the first step and a Bioanalyzer in the second step. Products were analyzed using a MiSeq according to the manufacturer's instructions. All sequencing data from each round were generated as FASTQ files. After extracting aptamer domains by trimming the 5'- and 3'-primer regions, all sequences were used for frequency analysis to examine aptamer generation during selection.

[0119] Comparative analysis of aptamer library information The method according to the present disclosure was applied to the comparative analysis of aptamer library information. The aptamer library was selected based on the results of sequence analysis of the aptamer library. The aptamer library was reconstituted to form a stable structure and then mixed with the sample. The conditions were identical to those prepared for the aptamer library. Multiple aptamer-biomolecule complexes were purified by 1D electrophoresis. The ssDNA recovered from the gel was amplified with a certain PCR cycle and directly analyzed by next-generation sequencing without 2D electrophoresis. The aptamer library information was obtained by calculating the percentage frequency of each sequence in the total number of reads of each sample. The frequency percentage information (aptamer library information) was compared between various reference biological samples to evaluate changes in the state of the biomolecules.

[0120] Example 1: Generation of an aptamer library for mouse serum The method according to the present disclosure was applied to the generation of an aptamer library targeting mouse serum samples.

[0121] Selection was performed in five selection rounds using a completely random DNA library. The starting library had a 43-nucleotide random region consisting of 5-tryptoamino-uracil instead of adenine, guanine, cytosine, and thymine. Representative imaging data from 1D and 2D electrophoresis are shown in Figure 1. The success of the selection is demonstrated by the observation that the number of specific sequences increases with each selection round. Another observation is that different sequence variations appear in different regions of the electrophoretic gel. To this end, a modified procedure was performed in additional rounds 5 and 6, in which the 1D electrophoretic gel was divided into two parts, upper and lower, as shown in Figure 2, for analysis of specific sequence evolution. Next-generation sequencing analysis yielded an average sequencing read of 50,933. The library growth curve is shown in Figure 3, where the resulting size indicates the number of sequences exceeding 3 counts. For comparison, the growth curves of the conventional SELEX data and the 1D electrophoresis-based biofluid SELEX data are also shown in Figure 3. The top 20 sequences in terms of frequency in the library obtained from the entire gel are shown below in Table 1. The top 20 sequences obtained from different regions in the lower and upper regions of the gel are listed below in Tables 2 and 3, respectively.

[0122] [Table 1]

[0123] Table 1. A list of the top 20 aptamer sequences obtained from the whole gel analysis is shown. The lowercase "t" stands for 5-tryptoamino-uracil.

[0124] [Table 2]

[0125] Table 2. Shown is a list of the top 20 aptamer sequences obtained from the lower gel portion shown in Figure 2. The frequencies of these aptamers are compared with the full gel data and the upper gel data to determine specificity. The lowercase "t" indicates 5-tryptoamino-uracil.

[0126] [Table 3]

[0127] Table 3. Shown is a list of the top 20 aptamer sequences obtained from the upper gel portion shown in Figure 2. The frequencies of these aptamers are compared with the full gel data and the lower gel data to determine specificity. The lowercase "t" indicates 5-tryptoamino-uracil.

[0128] Example 2: Comparative analysis of the information in aptamer libraries for mouse serum The method according to the present disclosure was applied to the comparative analysis of aptamer library information for mouse serum samples from the same or different strains.

[0129] Serum samples were prepared from C57BL / 6N (B6 / N) and BALB / c mouse strains. One of the C57BL / 6N mouse sera was used to generate an aptamer library. The aptamer library was selected based on the results of sequencing after five selection rounds. A gentle gradient in sequence frequency, with a top sequence frequency of approximately 1-2%, was the basic selection criterion. The aptamer library was interacted with several serum samples from either the C57BL / 6N or BALB / c strains. The aptamer-protein complexes were purified by 1D electrophoresis and then subjected to next-generation sequencing analysis using MiSeq (Illumina). The results of comparative analyses of aptamer library information between samples from the same mouse strain (C57BL / 6N vs. C57BL / 6N) or different mouse strains (C57BL / 6N vs. BALB / c) are shown in Figures 4 and 5, respectively. Note that different aptamer libraries were used for each comparative analysis.

[0130] Some aptamers that showed sample-specific patterns were selected for target specificity testing by aptamer-based precipitation assays. One aptamer, the sd-10 aptamer, showed high specificity only for serum D in the same strain, while the other, the Nb-01 aptamer, showed specificity only for C57BL / 6N serum obtained from a different mouse strain. Both aptamers were modified with biotin at the 5' end to precipitate the target molecules. These aptamers were allowed to interact with serum samples, followed by a pull-down assay using streptavidin beads (agarose beads or magnetic FG beads). After extensive washing, the beads were analyzed by non-denaturing, stain-free SDS-PAGE. The gels were visualized using a ChemiDoc MP Imaging System (Bio-Rad). The pull-down results for the sd-10 aptamer and Nb-01 aptamer are shown in Figures 6 and 7, respectively.

[0131] The target proteins of the 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 showed that the sd-10 aptamer recognized mouse IgG, and the Nb-01 aptamer recognized GPLD-1. Western blot analysis with mouse IgG antibody (Figure 8) and GPLD-1 antibody (Figure 9) confirmed the results of MS analysis. As shown in Figure 8, the sd-10 aptamer recognized only a small fraction of the total antibodies in serum, indicating a specific interaction with a specific type of differentially expressed antibody.

[0132] Example 3: Comparative analysis of the information in aptamer libraries for human cerebrospinal fluid samples The method according to the present disclosure was applied to the comparative analysis of information from an aptamer library for human cerebrospinal fluid (CSF) samples.

[0133] CSF samples were purchased from PrecisionMed, LLC. A mixture of five CSF samples from five AD patients was used to create an aptamer library targeting human CSF samples. The aptamer library was selected based on the same procedure as in Example 2. This aptamer library was interacted with CSF samples from seven healthy individuals (HCs) and ten AD patients. The aptamer-protein complexes were purified by 1D electrophoresis and then subjected to next-generation sequencing analysis using HiSeq X (Illumina). The frequency of each sequence was obtained as a profiling score. The average profiling score of HCs was taken as the baseline, and the fold change (FC) of each sequence from the baseline was calculated and plotted in Figure 10. Comparative analysis of the aptamer library information showed that the patterns of aptamer reactivity to CSF samples from HCs and AD patients were different. Some aptamers that show differences between the information of the aptamer library for AD CSF samples and the information of the aptamer library for HC CSF samples can be selected, and biomolecules that bind to aptamers in the aptamer library for AD CSF samples or aptamers in the aptamer library for HC CSF samples can be identified by performing as described in Example 2.

Claims

1. 1. A method for enriching a plurality of aptamers capable of binding to one or more biomolecules in a biological sample, 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 comprising a plurality of aptamer-biomolecule complexes; (iii) subjecting a portion of the first electrophoretic medium to electrophoresis in a second electrophoretic medium in a second direction to obtain a portion of the second electrophoretic medium comprising a plurality of aptamer-biomolecule complexes; and (iv) extracting a plurality of aptamers capable of binding to biomolecules in the biological sample from the plurality of aptamer-biomolecule complexes; A method comprising:

2. The method of claim 1, further comprising the step of (v) amplifying a plurality of aptamers capable of binding to biomolecules in the biological sample to form an aptamer library.

3. The method of claim 2, wherein a plurality of aptamers capable of binding to biomolecules in a biological sample are amplified by polymerase chain reaction (PCR).

4. The method of claim 3, wherein the PCR is emulsion PCR.

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

6. 6. The method of claim 5, wherein a portion of the first electrophoresis medium is aligned with a well in a second electrophoresis medium to effect electrophoresis in a second direction.

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

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

9. 9. The method of any one of claims 1 to 8, wherein the first and second electrophoretic media comprise sodium ions, potassium ions, lithium ions, ammonium ions, or any combination thereof, at a concentration of between 100 mM and 200 mM.

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

11. 11. The method of any one of claims 1 to 10, wherein the first and 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. 12. The method of any one of claims 1 to 11, wherein the first and 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. 13. The method of any one of claims 1 to 12, wherein the first and second electrophoretic media comprise magnesium ions, calcium ions, copper ions, zinc ions, or any combination thereof at a concentration of 1 mM.

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

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

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

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

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

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

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

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

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

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

25. 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 to 25, wherein each of the plurality of candidate aptamers is labeled.

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

28. 28. The method of any one of claims 1 to 27, further comprising, prior to step (iv), cutting out a 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 said portion of the second electrophoretic medium.

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

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

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

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

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

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

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

36. 1. A method for profiling a target aptamer library for a target biological sample, comprising: (i) obtaining information of a target aptamer library for a target biological sample from a first aptamer database; (ii) obtaining information of a reference aptamer library for a reference biological sample from a second aptamer database; and (iii) comparing the information of the target aptamer library for the target biological sample with the information of the reference aptamer library for the reference biological sample, and profiling the target aptamer library by this comparison; Including, 36. A method wherein the first aptamer database in step (i) is the aptamer database of claim 35.

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

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

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

40. 40. The method of any one of claims 36 to 39, wherein step (i) comprises downloading information of a target aptamer library for the target biological sample from the aptamer database to a user's computer, or extracting information of a target aptamer library for the target biological sample from the aptamer database to a server on which the aptamer database is stored.

41. 41. The method of any one of claims 36 to 40, wherein step (ii) comprises downloading information of a reference aptamer library for the reference biological sample from the aptamer database to a user's computer, or extracting information of a reference aptamer library for the reference biological sample from the aptamer database to a server on which the aptamer database is stored.

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

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

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

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

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

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

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

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

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

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

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

53. 52. The method of claim 50 or 51, wherein the comparing step results in the identification of biomarkers for the diseased sample.

54. 54. The method of any one of claims 36 to 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, the instructions stored on the medium, when executed by at least one processor, causing the at least one processor to: (i) obtaining target aptamer library information for a target biological sample from a first aptamer database; (ii) obtaining information of a reference aptamer library for a reference biological sample from a second aptamer database; and (iii) comparing the information of the target aptamer library for the target biological sample with the information of the reference aptamer library for the reference biological sample, and profiling the target aptamer library by this comparison; Including, The first aptamer database in step (i) is the aptamer database of claim 35. Method for profiling a target aptamer library for a target biological sample - Patent Application 20070122997 A non-transitory computer-readable medium for performing the above.

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

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

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

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

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

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

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

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

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

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

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

68. 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 a disease or is at risk for a disease.

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

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

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

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

73. 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, 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.

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