Protein capture techniques with surface linked aptamers

Abstract

An aptamer-based capture complex is described herein. The aptamer-based capture complex may include a solid surface and a plurality of aptamers coupled to a first portion the solid surface via a first plurality of linkers. The plurality of aptamers binds cognate targets. In some embodiments, the aptamer-based capture complex may include a second plurality of linkers coupled to a second portion of the solid surface. The first plurality of linkers comprises different functional groups than the second plurality of linkers.

Description

PROTEIN CAPTURE TECHNIQUES WITH SURFACE LINKED APTAMERSBACKGROUND

[0001] The technology disclosed relates to systems and methods for capturing and separating proteins, peptides, and other types of protein. In particular, the technology disclosed relates to capture, tagging, and separation using an aptamer-based capture complex.

[0002] The subject matter discussed in this section should not be assumed to be prior art merely as a result of its mention in this section. Similarly, a problem mentioned in this section or associated with the subject matter provided as background should not be assumed to have been previously recognized in the prior art. The subject matter in this section merely represents different approaches, which in and of themselves can also correspond to implementations of the claimed technology.

[0003] Protein expression patterns help define a cell’s identity and state. RNA transcripts are often used as a surrogate for protein expression, but the relationship between abundance of proteins and mRNA is not one-to-one. There are differences caused by regulation of posttranscriptional, translational and protein degradation. Therefore, direct nucleic acid sequencing of RNA transcripts may not provide an accurate estimation of protein expression.

[0004] Aptamers are nucleic acids that bind to molecular targets, such as proteins, with high affinity and specificity. Advancements in aptamer selection and design include Systematic Evolution of Ligands by Exponential enrichment (SELEX). In SELEX, high affinity nucleic acids for different analytes of interest can be isolated from a combinatorial library, permitting high throughput characterization of aptamer-target binding and multiplexed assays for analytes in a complex biological sample.BRIEF DESCRIPTION

[0005] In one embodiment, the present disclosure relates to an aptamer-based capture complex is described herein. The aptamer-based capture complex may include a solid surface and a plurality of aptamers coupled to a first portion the solid surface via a first plurality of linkers. The plurality of aptamers binds proteins. In some embodiments, the aptamer-based capture complex may include a second plurality of linkers coupled to a second portion of the solid surface. The first plurality of linkers comprises different functional groups than the second plurality of linkers.

[0006] In one embodiment, the present disclosure relates to a method of preparing an aptamer-based capture complex. The method includes providing a plurality of aptamer-based capture complexes into a solution, wherein each aptamer-based capture complex comprises a solid surface coupled to a first plurality of aptamers via a first plurality of linkers. The method also includes providing sample comprising cognate targets to the solution including the aptamer-based capture complex to form a plurality of aptamer-target capture complexes. Further, the method includes separating unbound aptamer complexes from the plurality of aptamer-target capture complexes to obtain a first plurality of bound aptamer complexes.

[0007] In one embodiment, the present disclosure relates to method of preparing an aptamer-based capture complex. The method includes (a) capturing an aptamer capable of binding a cognate target on a solid surface via a surface binding moiety to produce an aptamersurface complex. The method also includes (b) mixing a plurality of different aptamers- surface complexes formed by step (a) to produce a plurality of aptamer-surface complexes capable of binding a plurality of cognate targets, wherein each solid surface comprises an adapter specific to a single cognate targets. Further, the method includes (c) providing a sample comprising a plurality of cognate targets to the mixture. Further still, the method includes (d) capturing the cognate targets by binding to the plurality of aptamers on the aptamer-surface complexes. Even further, the method includes (e) releasing the aptamers bound to the solid surfaces to form a first plurality of cognate-target bound aptamers and a second plurality of unbound aptamers. Even further, the method includes (f) separating thefirst plurality of cognate-target bound aptamers from the second plurality of unbound aptamers to form a plurality of aptamer-based capture complexes bound to cognate targets.

[0008] In one embodiment, the present disclosure relates to a method of preparing an aptamer-based capture complex. The method includes (a) providing a plurality of aptamers capable of binding a plurality of cognate targets on a solid surface via a surface binding moiety to produce a plurality of aptamer-surface complexes. The method also includes (b) providing a sample comprising a plurality of cognate targets to the mixture. Further, the method includes (c) capturing the cognate targets by binding to the plurality of aptamers on the aptamer-surface complexes. Further still, the method includes (d) releasing the aptamers bound to the solid surfaces via the surface binding moiety to form a first plurality of cognate-target bound aptamers and a second plurality of unbound aptamers. Even further, the method includes (e) separating the first plurality of cognate-target bound aptamers from the second plurality of unbound aptamers to form a plurality of aptamer-based capture complexes bound to cognate targets.

[0009] In one embodiment, the present disclosure relates to a method. The method includes providing a plurality of cognate targets. The method also includes forming an aptamer-based capture complex comprising a plurality of aptamers that are bound to the plurality of cognate targets, wherein the plurality of aptamers is linked to a solid substrate of the aptamer-based capture complex via a detachable linker. Further, the method includes releasing the aptamers bound to the solid surfaces to form a first plurality of cognate-target bound aptamers and a second plurality of unbound aptamers. Further still, the method includes separating the first plurality of cognate-target bound aptamers from the second plurality of unbound aptamers to form a plurality of aptamer-based capture complexes bound to cognate targets.

[0010] The preceding description is presented to enable the making and use of the technology disclosed. Various modifications to the disclosed implementations will be apparent, and the general principles defined herein may be applied to other implementations and applications without departing from the spirit and scope of the technology disclosed. Thus, the technology disclosed is not intended to be limited to the implementations shown, but is tobe accorded the widest scope consistent with the principles and features disclosed herein. The scope of the technology disclosed is defined by the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] These and other features, aspects, and advantages of the present techniques will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

[0012] FIG. 1 is schematic diagram illustrating a first example of an aptamer-based capture complex (ABCC) having a first linker and a second linker, in accordance with aspects of the present disclosure;

[0013] FIG. 2 shows a method for capturing and separating proteins using the ABCC, in accordance with aspects of the present disclosure;

[0014] FIG. 3 shows a method for capturing proteins using a linker of the ABCC, in accordance with aspects of the present disclosure;

[0015] FIG. 4 shows a method for capturing a protein using the ABCC that includes a SNAP -tag, in accordance with aspects of the present disclosure;

[0016] FIG. 5 shows a method for capturing and separating proteins using the ABCC by performing a biotinylation concurrently with protein capture, in accordance with aspects of the present disclosure;

[0017] FIG. 6 shows a method for capturing and separating proteins using the ABCC by performing a biotinylation concurrently with cleavage of the first linker, in accordance with aspects of the present disclosure;

[0018] FIG. 7 shows another method of capturing and separating proteins using the aptamers that in accordance with aspects of the present disclosure;

[0019] FIG. 8A shows a method for pooling samples containing proteins, in accordance with aspects of the present disclosure;

[0020] FIG. 8B shows a first method for pooling samples containing proteins indexed using an ABCC, in accordance with aspects of the present disclosure;

[0021] FIG. 8C shows a second method for pooling samples containing proteins indexed using an ABCC, in accordance with aspects of the present disclosure;

[0022] FIG. 9 shows a first example of a method for attenuating a signal using an ABCC with modified aptamers, in accordance with aspects of the present disclosure; and

[0023] FIG. 10 shows a second example of a method for attenuating a signal using the ABCC, in accordance with aspects of the present disclosure.DETAILED DESCRIPTION

[0024] The following discussion is presented to enable any person skilled in the art to make and use the technology disclosed, and is provided in the context of a particular application and its requirements. Various modifications to the disclosed implementations will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the spirit and scope of the technology disclosed. Thus, the technology disclosed is not intended to be limited to the implementations shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

[0025] As used herein, the term “flow cell” is intended to mean a chamber having a surface across which one or more fluid reagents can be flowed. Generally, a flow cell will have an ingress opening and an egress opening to facilitate flow of fluid. Examples of flow cells and related fluidic systems and detection platforms that can be readily used in the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497; US 7,057,026; WO 91 / 06678; WO 07 / 123744; US 7,329,492; US 7,211,414; US7,315,019; US 7,405,281, and US 2008 / 0108082, each of which is incorporated herein by reference.

[0026] As used herein, the term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.

[0027] As used herein, the term “different", when used in reference to nucleic acids, means that the nucleic acids have nucleotide sequences that are not the same as each other. Two or more nucleic acids can have nucleotide sequences that are different along their entire length. Alternatively, two or more nucleic acids can have nucleotide sequences that are different along a substantial portion of their length. For example, two or more different nucleic acids can have target nucleotide sequence portions that are different from each other while also having a universal sequence region that is the same for the two or more different nucleic acids.

[0028] As used herein, the term “linker” is intended to mean a chemical bond or moiety that covalently bridges two other moieties. A linker can be, for example, the sugar-phosphate backbone that connects nucleotides in a nucleic acid moiety. The linker can include, for example, one or more of a nucleotide moiety, a nucleic acid moiety, a non-nucleotide chemical moiety, a nucleotide analogue moiety, amino acid moiety, polypeptide moiety, or protein moiety. A linker can be non-amplifiable, for example, by virtue of containing a non-nucleic acid moiety. Exemplary linkers are set forth in further detail below and in PCT Pub. No. WO 2012 / 061832; US Pat. App. Pub. No. 2012 / 0208724, US Pat. App. Pub. No. 2012 / 0208705 and PCT App. Ser. No. PCT / US2013 / 031023, each of which is incorporated herein by reference.

[0029] As used herein the term “nucleic acid” can refer to at least two nucleotide monomers linked together. Examples include, but are not limited to DNA, such as genomic or cDNA; RNA, such as mRNA, sRNA or rRNA; or a hybrid of DNA and RNA. As apparent from the examples below and elsewhere herein, a nucleic acid can have a naturally occurring nucleicacid structure or a non-naturally occurring nucleic acid analog structure. A nucleic acid can contain phosphodiester bonds; however, in some embodiments, nucleic acids may have other types of backbones, comprising, for example, phosphoramide, phosphorothioate, phosphorodithioate, O-methylphosphoroamidite and peptide nucleic acid backbones and linkages. Nucleic acids can have positive backbones; non-ionic backbones, and non-ribose based backbones. Nucleic acids may also contain one or more carbocyclic sugars. The nucleic acids used in methods or compositions herein may be single stranded or, alternatively double stranded, as specified. In some embodiments a nucleic acid can contain portions of both double stranded and single stranded sequence, for example, as demonstrated by forked adapters. A nucleic acid can contain any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthanine, hypoxanthanine, isocytosine, isoguanine, and base analogs such as nitropyrrole (including 3 -nitropyrrole) and nitroindole (including 5-nitroindole), etc. In some embodiments, a nucleic acid can include at least one promiscuous base. A promiscuous base can base-pair with more than one different type of base and can be useful, for example, when included in oligonucleotide primers or inserts that are used for random hybridization in complex nucleic acid samples such as genomic DNA samples. An example of a promiscuous base includes inosine that may pair with adenine, thymine, or cytosine. Other examples include hypoxanthine, 5-nitroindole, acylic 5-nitroindole, 4-nitropyrazole, 4-nitroimidazole and 3 -nitropyrrole. Promiscuous bases that can base-pair with at least two, three, four or more types of bases can be used.

[0030] As used herein, the term “region,” when used in reference to a surface, means an area of the surface that is smaller than the entire area of the surface. The regions can be an area that is smaller than the entire area of a surface that is exposed or accessible to a fluid. Generally the term “region” is used to refer to a continuous, uninterrupted area of a surface, whether or not the region encompasses surface features, sites, contours etc. A region can encompass one or more locations to which a nucleic acid is attached or will be attached.

[0031] As used herein, the term “solid support” refers to a rigid substrate that is insoluble in general aqueous liquid. In some embodiments, the “solid support” may be soluble under certain conditions. The substrate can be non-porous or porous. The substrate can optionally be capable of taking up a liquid (e.g. due to porosity) but will typically be sufficiently rigid that the substrate does not swell substantially when taking up the liquid and does not contract substantially when the liquid is removed by drying. A nonporous solid support is generally impermeable to liquids or gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor, silica or silica- based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers. Particularly useful solid supports for some embodiments are located within a flow cell apparatus. Exemplary flow cells are set forth in further detail below.

[0032] Exemplary solid supports include, for example, arrays, beads, including streptavidin beads, polystyrene beads, silica beads, metal surfaces, flow cells and flow cell surfaces, wells (microtiter plates, tissue culture plates, glass slides, etc.), divits in solid surfaces including glass, plastics, among others. Any of a variety of solid supports can be used. Particularly useful solid supports are those used for nucleic acid arrays. Examples include glass, modified glass, functionalized glass, inorganic glasses, microspheres (e.g. inert and / or magnetic particles), plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, metals, an optical fiber or optical fiber bundles, polymers and multiwell (e.g. microtiter) plates. Exemplary plastics include acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes and Teflon™. Exemplary silica-based materials include silicon and various forms of modified silicon.

[0033] In particular embodiments, a solid support can be within or part of a vessel such as a well, tube, channel, cuvette, Petri plate, bottle or the like. A particularly useful vessel is aflow-cell, for example, as described in WO 2014 / 142841 Al; U.S. Pat. App. Pub. No. 2010 / 0111768 Al and U.S. Pat. No. 8,951,781 or Bentley et al., Nature 456:53-59 (2008), each of which is incorporated herein by reference. Exemplary flow-cells are those that are commercially available from Illumina, Inc. (San Diego, CA) for use with a sequencing platform such as a Genome Analyzer®, MiSeq®, NextSeq® or HiSeq® platform. Another particularly useful vessel is a well in a multiwell plate or microtiter plate.

[0034] As used herein, the term “surface,” when used in reference to a material, is intended to mean an external part or external layer of the material. The surface can be in contact with another material such as a gas, liquid, gel, polymer, organic polymer, second surface of a similar or different material, metal, or coat. The surface, or regions thereof, can be substantially flat. The surface can have surface features such as wells, pits, channels, ridges, raised regions, pegs, posts or the like. The material can be, for example, a solid support, gel, or the like.

[0035] The term “analyte-binding region” refers to an oligonucleotides (ssDNA or ssRNA) that will form a cognate target by binding to an analyte of interest. The “analyte-binding region” may include one or more portions that permit binding of the analyte-binding region to the analyte. For example, the one or more portions may include an aptamer region that will bind to the analyte of interest with high affinity. It should be noted that the analyte-binding region may include other sequences (e.g., identification sequences) to enable downstream sequencing and analysis.

[0036] As used herein, an aptamer may refer to a non-naturally occurring nucleic acid that has specific binding affinity for a target molecule. The binding of the aptamer to the target molecule can result in catalytically changing the target molecule, reacting with the target molecule in a way that modifies or alters the target molecule or the functional activity of the target molecule, covalently attaching to the target molecule (as in a suicide inhibitor), and facilitating the reaction between the target molecule and another molecule. In one embodiment, the target molecule is a three-dimensional chemical structure, other than a polynucleotide, that binds to the aptamer through a mechanism which is predominantlyindependent of Watson / Crick base pairing or triple helix binding. In an embodiment, the aptamer is not a nucleic acid having the known physiological function of being bound by the target molecule.

[0037] Aptamers include nucleic acids that are identified from a candidate mixture of nucleic acids. A specific binding affinity of an aptamer for its target may refer to aptamer binding to its target generally with a much higher degree of affinity than it binds to other, nontarget, components in a mixture or sample. Different aptamers may have either the same number or a different number of nucleotides. Aptamers may be DNA or RNA and may be single stranded, double stranded, or contain double stranded regions. The aptamers discussed herein can be used in any diagnostic, imaging, high throughput screening or target validation techniques or procedures or assays for which aptamers, oligonucleotides, antibodies and ligands, without limitation can be used.

[0038] Aptamers are short single stranded nucleic acid molecules (ssDNA or ssRNA) that can bind to their specific target molecules with high affinity. Accordingly, aptamers can be used for multiomic applications, such as proteome characterization of a sample in a high- throughput manner. In some cases, an aptamer-based assay may be performed using two separate bead-based captures. In a first capture step, aptamer-analyte complexes can be attached to a solid support, e.g., using a releasable first tag included on the aptamer. The aptamer-analyte complexes bound to the solid support in the first capture step are treated with an agent that introduces a second tag, e.g., biotin, to the target molecule component of the aptamer-analyte complexes. Partitioning is completed by releasing of uncomplexed aptamers and aptamer-analyte complexes from the first solid support, for example by photocleavage. In a second capture step, the aptamer-analyte complexes contained in the mixture can be captured by the introduced second tag present on the analyte. Thus, while a first capture step is mediated by a tag on the aptamer, the second capture step is mediated by a tag on the analyte. Accordingly, some aptamer-based assays are performed using a two-step capture and separation. After the second capture, the aptamers can be disassociated from bound analytes for downstream detection.

[0039] This disclosure relates to methods and compositions for capturing cognate targets (e.g., analytes, target proteins, sample proteins, sample) using surface-linked aptamers on an aptamer-based capture complex (ABCC) (e.g., protein capture complex (PCC)). In general, the ABCC includes aptamers that are coupled to a substrate (e.g., streptavidin beads, polystyrene beads, silica beads, metal surfaces, flow cells) via a detachable linker (e.g., photo- cleavable linker (UV-cleavable) and / or enzyme-cleavable linker). The aptamers on the ABCC may bind or capture cognate targets, such as proteins, in solution. The detachable linker may be cleaved, which may release the protein-bound aptamers into solution. The protein-bound aptamers may be recaptured via a tag or other functionalization on the aptamer, while aptamers not bound to protein may be washed away or removed from the solution. In some embodiments, one or more binding competitors can be added to the solution that facilitate removal of unbound aptamers. Accordingly, the ABCC may be used to capture and separate proteins that bind to the aptamers from other proteins. In some embodiments, capturing and separating proteins may be performed with the ABCC. In some embodiments, an additional substrate (e.g., an additional bead) may be used to recapture the aptamers bound to protein via the tag or other functionalization. In another example, the aptamer in the ABCC can be mixed with the cognate targets, e.g. proteins, in solution prior to the aptamer being in contact with and bound to a solid surface to form the ABCC complex via the aptamer-linker, e.g. a detachable linker. In some embodiments, one or more dummy aptamers can be added to the mixture of aptamer and cognate targets in solution prior to the ABCC complex being contacted with a solid surface (bead, well, flow cell surface, etc.). Suitable ways of separating the ABCC complex once bound to a surface are described in more detail herein.

[0040] It is presently recognized that using the ABCC may reduce the amount of sample preparation materials. For example, indexes may be added to the aptamer before and / or after capture by the ABCC. By providing multiple instances to index of the aptamers, and ultimately the protein, samples may be pooled. In this way, the disclosed techniques may reduce the amount of plastic or other materials used in sample preparation, such as dilution protocols. Accordingly, the disclosed techniques may reduce the amount of plastic or other materials used in sample preparation.

[0041] Further, it is presently recognized that using modified ABCCs with “dummy” or otherwise inactive aptamers, may reduce the amount of dilutions utilized to obtain a concentration of analytes within the dynamic range of instruments. The techniques include forming dummy aptamers by eliminating the cleavage group on the aptamer (e.g., UV or enzymatic-cleavage). Eliminating the cleavage group prevents the aptamer from releasing captured proteins, and ultimately, attenuating the signal indicating the concentration of the number of proteins that were captured. It may be advantageous to form ABCCs with varying amounts of dummy aptamers based on target protein for the ABCCs. For example, ABCCs that target high abundance proteins may have a larger number or percentage of dummy aptamers as compared to ABCCs that target low abundance proteins. Accordingly, the disclosed techniques may be used to provide quantitative information regarding target cognates in a sample.

[0042] With the foregoing in mind, FIG. 1 shows an exemplary schematic diagram of an aptamer-based capture complex (ABCC) 10 (e.g., protein capture complex). The ABCC 10 generally includes a solid substrate 12 that is coupled to aptamers 14 via a detachable linker 16 (e.g., a first linker, an aptamer linker). The aptamer 14 and the detachable linker 16 may be coupled via a covalent bond that is UV cleavable, enzyme-cleavable, or both, as discussed in more detail herein. In general, the aptamer 14 of the ABCC 10 may bind or capture a protein, or another cognate target, based on an affinity of the aptamer 14 for the protein. In some embodiments, the ABCC 10 may also include a recapture linker 18 (e.g., a second linker, a protein linker, an additional linker) that is coupled to the solid substrate 12. In some embodiments, the ABCC 10 may be a monoclonal capture complex having aptamers 14 all bind to one protein. In some embodiments, the ABCC may be a polyclonal capture complex having different aptamers 14 that bind to different proteins. The recapture linker 18 may be used to recapture aptamers 14 that are released into solution after the detachable linker 16 is cleaved. However, in some embodiments, the aptamers 14 released into the solution may be captured with an additional solid substrate having linkers or other moieties that may bind aptamers. Again, this embodiment may be modified as to the order of the steps, e.g., the aptamer in the ABCC can be mixed with the cognate targets, e.g. proteins, in solution prior tothe aptamer being in contact with and bound to a solid surface to form the ABCC complex via the aptamer-linker. In some embodiments, one or more dummy aptamers can be added to the mixture of aptamer and cognate targets in solution prior to the ABCC complex being contacted with a solid surface (bead, well, flow cell surface, etc.).

[0043] In some embodiments, the solid substrate 12 may be a polymeric particle, nanoparticle, metal oxide particles, metal oxide nanoparticles, metal nanoparticles, metal particles, polystyrene bead, a silica bead, or a magnetic bead. For example, the solid substrate may be streptavidin beads, polystyrene beads, silica beads, a metal surfaces, and other substrates that may be functionalized with the detachable linker 16 disclosed herein. In some embodiments, the solid substrate 12 may be a hydrogel, such as a chemical hydrogel that includes covalent cross-linking bonds or a physical hydrogel that does not include covalent bonds. The hydrogels may be formed using natural polymers (e.g., chitosan, alginate, gelatin, fibrin, hyaluronic acid, and so on) and / or synthetic polymers (e.g., polyethylene glycol (PEG), acrylate polymers, polyvinylpyrrolidone (PVP), polyvinyl alcohol, among others).

[0044] As described herein, the aptamer 14 may be DNA or RNA and may be single stranded, double stranded, or contain double stranded regions. In some embodiments, the aptamer 14 may be functionalized with a linker (e.g., a protein functionalized with a polyethylene glycol (PEG) linker) that is capable of reacting with a surface functional group on the solid substrate to form the detachable linker 16. However, in some embodiments, the linker may be capable of reacting with the recapture linker 18.

[0045] The detachable linker 16 is generally a chemical species that includes a covalent bond formed by a reaction between a functional group of the aptamer 14 and a functional group disposed on the surface of the substrate 12. For example, the detachable linker 16 may include an amino-carboxyl bond formed based on a reaction between a carboxylic acid terminated functional group on the substrate 12 and an amine group on the aptamer 14. As another nonlimiting example, the detachable linker 16 may include a biotin-amino bond formed based on a reaction between biotin and an amine group on the aptamer 14. As described herein, the detachable linker 16 may be UV-cleavable, enzyme-cleavable, or both. In some embodiments,the detachable linker 16 may be a biotin linker this is both UV-cleavable and enzyme- cleavable. For example, the detachable linker 16 may be configured to cleave using a base excision repair enzyme, a restriction endonuclease, or both.

[0046] The recapture linker 18 is generally a linker capable of directly or indirectly binding a protein or other cognate bound to the aptamer 14. For example, the recapture linker 18 may be an alkyne linker capable of binding an azido tag bound to a protein. In some embodiments, the recapture linker 18 may be a different linker than the detachable linker 16. In some embodiments, after the recapture linker 18 binds the protein, the protein may be tagged or otherwise functionalized (e.g., biotinylated), which may facilitate recapture of the protein after it is released or shifting the protein to a different linker. In some embodiments, both the detachable linker 16 and the recapture linker 18 may include a similar chemical moiety, such as a biotin. For example, the detachable linker 16 on the substrate 12 may include biotin, and an additional substrate may include biotin used to recapture the protein-bound aptamer after it is released from the substrate 12 (e.g., via cleavage of the detachable linker 16). In some embodiments, the detachable linker 16 and the recapture linker 18 may be different chemical species or moi eties. For example, the recapture linker 18 may include a carboxy group or an alkyne group that binds an amino group on the protein, and the detachable linker 16 may include a different species, such as biotin, as described herein. In some embodiments, detachable linker 16 may include an alkyne group that’s coupled to substrate 12 via a carboxyl functional group.

[0047] In the embodiments described in the FIGS. 1-10 and description herein, it is noted that the disclosure covers both methods in which the aptamer 14 are first bound to a substrate 12 and then contacted with the sample comprising proteins, and methods in which the aptamer 14 is first contacted and bound to proteins within a sample, and subsequently contacted and bound to the substrate 12. In other words, while some examples are demonstrated for the first method of binding the substrate then protein, it is contemplated that the method steps may be reversed to first bind the protein and then subsequently the surface. In some embodiments, the aptamers 14 may be substantially the same aptamers, thereby forming a monoclonalcapture complex (e.g., an ABCC 10 having the same aptamer). In some embodiments, two or more of the aptamers 14 may be different, thereby forming a polyclonal capture complex (e.g., an ABCC 10 having different aptamers). In some embodiments, two or more monoclonal capture complexes having different aptamers may be combined to form a mixture capable of binding different cognate targets.

[0048] FIG. 2 shows a process 30 for capturing and separating proteins 32 using aptamers 14. In general, the process 30, at block 34, includes providing aptamers bound to a substrate. At block 36, the process 30 includes providing proteins 32 that bind to the aptamers 14 to form a protein-bound aptamer 38. The unbound proteins 32 and other material, such as plasma, may be washed away. At block 40, the process 30 includes biotinylating the protein of the protein-bound aptamer 38. In some embodiments, block 40 includes washing the biotinylated protein, quenching, and performing buffer exchange to remove unbound biotin. At block 42, the process 30 includes cleaving (e.g., UV cleavage or enzymatic cleavage) the protein-bound aptamers 38 from the substrate. At block 44, the process 30 includes eluting the protein-bound aptamers 38 and binding competitors, which may facilitate removing aptamers 14 that are not bound to proteins 32. At block 46, the process 30 includes washing away unbound aptamers and non-specific aptamers 14 that may bind to the binding competitors as compared to the protein. At block 48, the process 30 includes collecting the protein-bound aptamers 38, thereby obtaining the proteins 32 that bound to aptamers. Accordingly, the aptamers that were bound to the proteins 32 may be utilized in a Next-generation sequencing (NGS) protocol or microarray for detection and relative quantification of the amount of protein.

[0049] In some embodiments, the ABCC 10 may include the recapture linker 18. It is presently recognized that including the recapture linker 18 may provide capture and separation of proteins or other cognate targets using a single type of substrate, as compared to providing a first type of substrate for capture and a second type of substrate for separation (e.g., corresponding to blocks 46 and 48 of the process 30 of FIG. 2). In some embodiments, the substrate may be functionalized with an additional linker that continues to bind the aptamers after the detachable linker is cleaved. To illustrate this, FIG. 3 shows a method 80 for capturingproteins using a linker of the ABCC 10, in accordance with aspects of the present disclosure. As described above, with respect to FIG. 1, the ABCC 10 includes aptamers 14, a detachable linker 16, and a recapture linker 18. At block 82, the process 80 includes providing the ABCC 10. At block 84, the process 80 includes providing proteins 32 to the ABCC 10, thereby forming protein-bound aptamers 38. At block 86, the process 80 includes tagging the proteins 32 with a tag 87, which may facilitate recapture of the aptamers 14 after they released into solution and / or react with the recapture linker 18 to form a bond that remains intact after the detachable linker 16 is cleaved. For example, the proteins 32 may be tagged with a PEG linker that is capable of reacting with a surface functional group of the recapture linker 18 on the solid substrate to form the detachable linker 16. Further, block 86 may include providing a binding competitor 89 that may prevent measurement of signals related to binding of proteins 32 by non-specific aptamers 14. At block 88, the process 80 includes capturing the proteinbound aptamers 38 tagged with the tag 87 based on a reaction between the tag 87 and the recapture linker 18. At block 90, the process 80 includes cleaving the detachable linker 16. Accordingly, protein-bound aptamers 38 tagged with the tag 87 may remain coupled to the substrate 12 via the recapture linker 18.

[0050] At block 92, the process 80 includes collecting the protein-bound aptamers 38, thereby obtaining the proteins 32 that bound to aptamers. Accordingly, the aptamers that were bound to the proteins 32 may be utilized in a Next-generation sequencing (NGS) protocol or microarray for detection and relative quantification of the amount of protein.

[0051] As described above, the protein 32 may be tagged with a tag 87, such as a PEG linker. To illustrate this, FIG. 4 shows a method 120 for binding a protein 32 functionalized with the tag 87. In general, the process 120 includes providing tags 87 to a solution that includes protein 32 and the substrate 12. As shown, the tag 87 is a PEG linker that includes an azido and an N-hydroxysuccinimide (NHS) terminal groups. In some embodiments, the tags 87 may be provided in excess of the protein 32. In any case, the tag 87 may bind to an amine group of the protein 32, such as an N-terminus of the protein 32 or a side chain on two amino acids of the protein 32. At least in some instances, Azido may be added after attachingthe alkyne-terminated PEG linker, and quickly reacted otherwise the azido may reduce in reactivity. In some instances, CLICK chemistry may be protein compatible. It may be advantageous to utilize copper CLICK chemistry as this may facilitate separating the two binding steps of the PEG linkers.

[0052] In some embodiments, the ABCC 10 may be utilized to tag proteins 32 after they are captured with the aptamer 14. FIG. 5 shows a method 160 for capturing a protein using the ABCC that includes a SNAP-tag, in accordance with aspects of the present disclosure. At block 162, the process 160 includes providing the ABCC 10. As shown, the substrate 12 is coupled to a detachable linker 16 that includes a SNAP linker that is bound to the aptamer 14. Further, the substrate 12 is coupled to a recapture linker 18 that is a SNAP linker. For example, the recapture linker 18 may be bound to a streptavidin surface.

[0053] At block 164, the process 160 includes providing proteins 32 to the ABCC 10, thereby forming protein-bound aptamers 38. At block 166, the process 160 includes tagging the proteins 32 with a tag 87. For example, the proteins 32 may be tagged with a PEG linker that is capable of reacting with a surface functional group of the recapture linker 18 on the solid substrate to form the detachable linker 16. Further, block 166 may include providing a binding competitor 89 that may bind to non-specific aptamers 14, thereby preventing measurement of signals related to binding of proteins 32 by non-specific aptamers 14. At block 168, the process 160 includes capturing the protein-bound aptamers 38 tagged with the tag 87 based on a reaction between the tag 87 and the recapture linker 18. In the illustrated embodiment, the tag 87 binds the SNAP linker (e.g., the recapture linker 18). At block 170, the process 160 includes cleaving the detachable linker 16. Accordingly, protein-bound aptamers 38 tagged with the tag 87 may remain coupled to the substrate 12 via the recapture linker 18. The tag may be a flexible PEG linker with NHS group for protein reactivity. In the illustrated embodiment, the T group on the other end of the linker a modified nitrogenous base (e.g., benzylguanine), which may react rapidly and specifically with the SNAP tag on the streptavidin. At block 172, the process 160 includes collecting the protein-bound aptamers 38, thereby obtaining the proteins 32 that bound to aptamers. Accordingly, the aptamers thatwere bound to the proteins 32 may be utilized in a Next-generation sequencing (NGS) protocol or microarray for detection and relative quantification of the amount of protein.

[0054] As described above with respect to FIG. 2, the protein 32 may be biotinylated after capture by the aptamer 14. However, it is presently recognized that the biotinylating step (e.g., block 40 of FIG. 2) and the protein capturing step (e g., block 36 of FIG. 2) may be performed concurrently. FIG. 6 shows a method 200 for capturing and separating proteins using the ABCC 10 by performing a biotinylation concurrently with protein capture, in accordance with aspects of the present disclosure.

[0055] At block 202, the process 200 includes providing the ABCC 10. As described above, with respect to FIG. 1, the ABCC 10 includes a substrate 12, aptamers 14, and a detachable linker 16. At block 204, the process 200 includes providing proteins 32 concurrently with biotin. Accordingly, some biotin may bind to the substrate 12, which may then bind an untargeted protein to the substrate 12. The unbound proteins 32 and other material, such as plasma, may be washed away. At block 206, the process 200 includes cleaving (e.g., UV cleavage or enzymatic cleavage) the protein-bound aptamers 38 from the substrate. Untargeted proteins coupled to the substrate 12 via biotin may remain bound to the surface. At block 208, the process 200 includes removing the substrate 12 and eluting the protein-bound aptamers 38 and binding competitors, which may facilitate removing aptamers 14 that are not bound to proteins 32. At block 210, the process 200 includes washing away unbound aptamers and non-specific aptamers 14 that may bind to the binding competitors as compared to the protein. Further, at block 210, the process includes capturing the proteinbound aptamers 38 onto a substrate (e.g., a new substrate or the same substrate) (e.g., ‘catch 2’). At block 212, the process 200 includes collecting the protein-bound aptamers 38, thereby obtaining the proteins 32 that bound to aptamers. Accordingly, the aptamers that were bound to the proteins 32 may be utilized in a Next-generation sequencing (NGS) protocol or microarray for detection and relative quantification of the amount of protein.

[0056] As described above with respect to FIG. 2, the protein 32 may be biotinylated before cleavage of the detachable linker 16. However, it is presently recognized that the biotinylatingstep (e.g., block 40 of FIG. 2) and the detachable linker cleavage step (e.g., block 42 of FIG. 2) may be performed concurrently. FIG. 7 shows a method 240 for capturing and separating proteins using the ABCC 10 by performing a biotinylation concurrently with cleavage of the detachable linker 16, in accordance with aspects of the present disclosure.

[0057] As shown in FIG. 7, at block 242, the process 240 includes providing the ABCC 10. As described above, with respect to FIG. 1, the ABCC 10 includes a substrate 12, aptamers 14, and a detachable linker 16. At block 244, the process 240 includes providing proteins 32 to the ABCC 10, thereby forming protein-bound aptamers 38. At block 246, the process 240 includes coupling protein 32 biotinylation with enzyme cleavage. The enzyme that cleaves the detachable linker 16 may be a base excision repair enzyme, a restriction endonuclease, or both. At block 248, the process 240 includes removing the substrate 12 and eluting the proteinbound aptamers 38 and binding competitors, which may facilitate removing aptamers 14 that are not bound to proteins 32. At block 250, the process 240 includes washing away unbound aptamers and non-specific aptamers 14 that may bind to the binding competitors as compared to the protein. At block 252, the process 240 includes collecting the protein-bound aptamers 38, thereby obtaining the proteins 32 that bound to aptamers. Accordingly, the aptamers 14 that were bound to the proteins 32 may be utilized in a Next-generation sequencing (NGS) protocol or microarray for detection and relative quantification of the amount of protein. This methodology eliminates the need for UV cleavable linkers and provides more options for detatching the linkers depending on the type of base excision repair enzymes or a restriction endonucleases used.

[0058] As described herein, the disclosed techniques may be used to reduce the amount of material used during library preparation. To illustrate this, FIG. 8A shows a method 280 for pooling samples containing proteins, in accordance with aspects of the present disclosure. In general, the method 280 include generally similar steps as described in FIG. 2. For example, the method 280 includes binding proteins; biotinylating proteins; cleaving proteins, eluting the proteins, aptamers, and competitors; capturing the protein-bound aptamers with an additional substrate; and performing elution to collected the aptamers that were bound to the proteins.

[0059] By providing an index to the proteins 32 after they are bound to the aptamers 1, subsequent pooling steps may be reduced. This is generally illustrated in FIG. 8B. Additionally or alternatively, the index or an additional index may be provided to the aptamers before they capture the protein. This is generally illustrated in FIG. 8C. Accordingly, indexed aptamers 14 may be pooled, thereby reducing the number of wells or other containers used during elution or washing steps.

[0060] As described herein, it may be advantageous to form ABCCs with varying amounts of dummy aptamers based on target protein for the ABCCs. To illustrate this, FIG. 9 shows a method 400 for attenuating a signal using an ABCC with modified aptamers. In general, the ABCC 10 may include modified or otherwise inactive aptamers 410. In general, the inactive aptamers 410 may be capable of binding protein 32. However, the inactive aptamers 410 may not cleave from the substrate 12. Accordingly, the ABCCs 10 including inactive aptamers 410 may release relatively fewer protein-bound aptamers 38. As such, fewer proteins 32 may be analyzed for a given biological sample. In particular, it is presently recognized that by tuning the relative amount of the inactive aptamers 410, the signal related to the amount of proteins 32 may be reduced (e.g., into the dynamic range of an instrument). This may be advantageous in instances where the amount of proteins 32 that bind the aptamers 14 is relatively high, and would otherwise saturate the signal and make it difficult to obtain quantitative information.

[0061] In some embodiments, the aptamers 14 may be rendered inactive by eliminating the cleavage group of the aptamer. Thus, aptamers 14 that still have the cleavage group will still elute the protein-bound aptamer 38 from the substrate 12.

[0062] In another embodiment, the inactive aptamer may be an aptamer that lacks the ability to bind the surface substrate (e.g., is lacking the surface binding moiety). For example, additional inactive aptamers to the most abundant proteins which may saturate the signal is added to a mixture of the aptamers and sample comprising the proteins allowing for the most abundant proteins to bind both the inactive and active aptamers compressing the dynamic range. This mixture can then contact the solid surface (e.g., bead, well, flow cell) where the active aptamers can bind to said surface while the inactive aptamers (some of which are boundto proteins) can be removed. This allows for data compression and the ability to have a wider dynamic range of protein detection within a sample by lowering the most abundant proteins in order for the signals of less abundant proteins to be measured.

[0063] To illustrate this, FIG. 10 shows a method 500 for attenuating a signal using an ABCC 10 in a mixture with inactive aptamers. At block 502, the process 500 includes providing a mixture that includes the ABCC 10 which includes aptamers 14 that are bound to the solid substrate 12 via the detachable linker 16. Additionally, the mixture includes free aptamers 504 that lack a linker that may bind to the solid substrate 12. The free aptamers 504 may compete with the aptamers 14 of the ABCC 10 in the binding of proteins 32 or other cognate targets. At block 506, which illustrates the mixture after a time period, a first portion of the proteins 32 are bound to the ABCC 10, while a second portion of the proteins are bound to the free aptamers 54. As such, at least a portion of the aptamers 14 of the ABCC 10 may remain unbound. At block 508, the free aptamers 504 (e.g., bound to proteins 32) and / or unbound proteins 32 may be washed away or removed from the mixture, leaving the ABCC 10 bound to the first portion of proteins 32. At block 510, the bound proteins 32 may be detached from the ABCC 10 and utilized in a Next-generation sequencing (NGS) protocol or microarray for detection and relative quantification of the amount of protein. In this way, the amount of free aptamers 504 utilized in process 500 may be increased or decreased to tune the amount of proteins 32 that are bound to the ABCC 10 and, ultimately, measured. As such, process 500 provides an additional technique for using inactive aptamers (e.g., aptamers that do not include a linker that may bind to the substrate 12) to attenuate a signal. At least in some instances, the techniques of FIG. 9 and FIG. 10 may be combined. For example, the ABCC 10 with modified aptamers may be utilized in combination with free aptamers 504 to attenuate a signal. It is noted that the analyte / protein 32 may be first mixed with the active aptamers 14 that contain a surface binding moiety and inactive aptamers 504 (lacking surface binding moiety) before being combined with the solid substrate 12 to form the ABCC and is encompassed in this disclosure although not pictured in the figures.

[0064] Accordingly, the process 400 and / or the process 500 may be utilized for dynamic range compression. For example, the disclosed techniques may be utilized to compress a dynamic range of aptamers with positive binding results (e.g., that bind to target molecules in a sample) and that may occur before or in conjunction with an aptamer detection step. The techniques preserve the aptamer binding for low-abundancy proteins that are assessed together with high-abundancy proteins. Further, because low-abundancy proteins may correspond to biomarkers that can be used for diagnostic purposes, the disclosed techniques prevent noise or false negative results of an aptamer-based assay caused by high-abundancy proteins obscuring the results. In addition, reducing the dynamic range can also reduce the amount of total sequencing data required to detect aptamers in a detection assay by reducing the amounts of reads wasted on high-abundance aptamer sequences. In certain embodiments, the disclosed techniques may provide streamlined workflows with reduced equipment burden via reduction in a number of steps (e.g., single hybridization reactions or reduced number of wash steps). The disclosed techniques may include sample preparation steps and / or sample preparations that permit improved aptamer abundance measurement.

[0065] The techniques presented and claimed herein are referenced and applied to material objects and concrete examples of a practical nature that demonstrably improve the present technical field and, as such, are not abstract, intangible or purely theoretical. Further, if any claims appended to the end of this specification contain one or more elements designated as “means for (perform)ing (a function)...” or “step for (perform)ing (a function)...”, it is intended that such elements are to be interpreted under 35 U.S.C. 112(f). However, for any claims containing elements designated in any other manner, it is intended that such elements are not to be interpreted under 35 U.S.C. 112(f).

[0066] This written description uses examples to enable any person skilled in the art to practice the disclosed embodiments, including making and using any devices or systems and performing any incorporated methods. The patentable scope is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from theliteral language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

[0067] A set of aspects are provided in the claims below. Another set of aspects is described below herein.

[0068] An aptamer-based capture complex comprising a plurality of aptamers. The aptamer-based capture complex also comprises a plurality linkers coupled to a first portion of the plurality of aptamers, wherein a second portion of the plurality of aptamers is configured to bind cognate targets.

[0069] The aptamer-based capture complex of the aspect of paragraph

[0063] , comprising the cognate targets.

[0070] The aptamer-based capture complex of the aspect of paragraph

[0064] , comprising an additional plurality of linkers coupled to the cognate targets.

[0071] The aptamer-based capture complex of the aspect of paragraph

[0065] , comprising a solid support coupled to the plurality of linkers at a first location of the solid support, and wherein the solid support is coupled to the additional plurality of linkers at a second location of the solid support.

[0072] An aptamer-based capture complex comprising: a solid surface; a plurality of aptamers capable of being coupled to a first portion the solid surface via a first plurality of linkers, wherein a first portion of the plurality of aptamers is configured to bind cognate targets; and a second plurality of linkers coupled to a second portion of the solid surface, wherein the first plurality of linkers comprises different functional groups than the second plurality of linkers.

[0073] The aptamer-based capture complex of the aspect of paragraph

[0067] , wherein the first plurality of linkers comprises biotin.

[0074] The aptamer-based capture complex of the aspect of paragraph

[0068] or

[0069] , wherein the aptamers are bound to cognate targets prior to being coupled to the solid surface via the first plurality of linkers.

[0075] The aptamer-based capture complex of any one of the aspects of paragraphs

[0067] -

[0069] , wherein the first plurality of linkers comprises a biotin-amino bond.

[0076] The aptamer-based capture complex of any one of the aspects of paragraphs

[0067] -

[0070] , wherein the second plurality of linkers comprise an alkyne linker. The aptamerbased capture complex further comprises an additional portion of the plurality of aptamers comprise inactive aptamers.

[0077] The aptamer-based capture complex of any one of the aspects of paragraphs

[0067] -

[0070] , wherein the second plurality of linkers is configured to bind a tag coupled to the proteins while the plurality of aptamers is coupled to the first plurality of linkers.

[0078] Another aspect provides a method of preparing an aptamer-based capture complex, comprising: providing a solution comprising a plurality of aptamers; providing a sample comprising cognate targets to the solution including the aptamers to form a first plurality of aptamer-target complexes; capturing the aptamers via the plurality of adaptors that bind to the aptamers on a solid surface to form a first plurality of aptamer-target capture complexes and a second plurality of aptamer-unbound capture complexes on the surface; separating the surface bound capture complexes from the surface first surface by cleaving the first plurality of linkers into a second solution; and separating the aptamer-target capture complexes from the second solution by binding the aptamer-target capture complexes to a second solid support via a second plurality of linkers which bind the second surface to the cognate targets in the aptamertarget capture complexes.

[0079] In another aspect, the disclosure provides a method of preparing an aptamer-based capture complex comprising: (a) mixing a plurality of aptamers to a sample comprising cognate targets, (b) capturing a plurality of aptamers bound to cognate targets on a solidsurface via a surface binding moiety on the aptamer to produce a target-aptamer-surface complexes; (c) mixing a plurality of different target-aptamers-surface complexes formed by step (a) and (b) to produce a plurality of aptamer-surface complexes bound to a plurality of cognate targets, wherein each solid surface comprises an adapter specific to a single cognate target; (d) releasing the aptamers bound to the solid surfaces to form a first plurality of cognatetarget bound aptamers and a second plurality of unbound aptamers; and (e) separating the first plurality of cognate-target bound aptamers from the second plurality of unbound aptamers to form a plurality of aptamer-based capture complexes bound to cognate targets.

[0080] In another aspect, this disclosure provides a method, comprising: providing a plurality of cognate targets; forming an aptamer-based capture complex comprising a plurality of aptamers that are bound to the plurality of cognate targets, releasing the aptamers bound to the solid surfaces to form a first plurality of cognate-target bound aptamers and a second plurality of unbound aptamers; and separating the first plurality of cognate-target bound aptamers from the second plurality of unbound aptamers to form a plurality of aptamer-based capture complexes bound to cognate targets.

Claims

1. CLAIMSWhat is claimed is:

1. An aptamer-based capture complex comprising: a solid surface; a plurality of aptamers coupled to a first portion the solid surface via a first plurality of linkers, wherein a portion the plurality of aptamers is configured to bind cognate targets; and a second plurality of linkers coupled to a second portion of the solid surface, wherein the first plurality of linkers comprises different functional groups than the second plurality of linkers.

2. The aptamer-based capture complex of claim 1, wherein the first plurality of linkers comprises biotin.

3. The aptamer-based capture complex of claim 2, wherein the first plurality of linkers comprises a biotin-amino bond.

4. The aptamer-based capture complex of claim 1, wherein the second plurality of linkers comprise an alkyne linker.

5. The aptamer-based capture complex of claim 1, wherein an additional portion of the plurality of aptamers comprise inactive aptamers.

6. The aptamer-based capture complex of claim 1, wherein the second plurality of linkers is configured to bind a tag coupled to the cognate targets while the plurality of aptamers is coupled to the first plurality of linkers.

7. A method of preparing an aptamer-based capture complex, comprising:providing a plurality of aptamer-based capture complexes into a solution, wherein each aptamer-based capture complex comprises a solid surface coupled to a first plurality of aptamers via a first plurality of linkers; providing sample comprising cognate targets to the solution including the aptamer-based capture complex to form a plurality of aptamer-target capture complexes; and separating unbound aptamer complexes from the plurality of aptamer-target capture complexes to obtain a plurality of bound aptamer complexes.

8. The method of claim 7, wherein step of separating comprises: cleaving the first plurality of linkers to release the aptamer-based capture complexes from the solid surface; and9. The method of claim 7, binding the plurality of aptamer-target capture complexes to a second surface via a second linker to isolate the plurality of aptamer-target complexes from the unbound aptamer complexes.

10. The method of claim 7, comprising cleaving the first plurality of linkers to release the unbound aptamer complexes using an enzyme.

11. The method of claim 10, wherein the enzyme comprises a base excision repair enzyme, a restriction endonuclease, or both.

12. The method of claim 7, wherein the plurality of aptamer-based capture complexes comprises a second plurality of linkers different than the first plurality of linkers.

13. The method of claim 12, wherein separating the unbound aptamer complexes to obtain the first plurality of bound aptamers comprises capturing the plurality of bound aptamer complexes using the second plurality of linkers before cleaving the first plurality of linkers.

14. The method of claim 7, further comprising: functionalizing the cognate target to include a tag; providing an additional aptamer-based capture complex comprising a second plurality of linkers different that the first plurality of linkers; and separating unbound aptamers to obtain a plurality of bound aptamers comprises capturing the aptamers including the tag based on a reaction between the tag and a second plurality of linkers.

15. The method of claim 14, comprising functionalizing the cognate target to include the tag after capturing the cognate target based on the plurality of aptamers.

16. The method of any one of claims 1-15, wherein the method further comprises: providing a plurality of inactive aptamers that cannot bind the solid surface when providing cognate targets.

17. The method of claim 16, wherein the plurality of inactive aptamers is directed toward the most abundant proteins found in a sample.

18. The method of claim 16, wherein the solid surface is a bead, a well or a flow cell surface.

19. A method of preparing an aptamer-based capture complex comprising:(a) capturing an aptamer capable of binding a cognate target on a solid surface via a surface binding moiety to produce an aptamer- surface complex;(b) mixing a plurality of different aptamers-surface complexes formed by step (a) to produce a plurality of aptamer-surface complexes capable of binding a plurality of cognate targets, wherein each solid surface comprises an adapter specific to a single cognate target;(c) providing a sample comprising a plurality of cognate targets to the mixture;(d) capturing the cognate targets by binding to the plurality of aptamers on the aptamer-surface complexes;(e) releasing the aptamers bound to the solid surfaces to form a first plurality of cognate-target bound aptamers and a second plurality of unbound aptamers; and(f) separating the first plurality of cognate-target bound aptamers from the second plurality of unbound aptamers to form a plurality of aptamer-based capture complexes bound to cognate targets.

20. A method of preparing an aptamer-based capture complex comprising:(a) providing a plurality of aptamers capable of binding a plurality of cognate targets on a solid surface via a surface binding moiety to produce a plurality of aptamersurface complexes;(b) providing a sample comprising a plurality of cognate targets to the mixture;(c) capturing the cognate targets by binding to the plurality of aptamers on the aptamer-surface complexes;(d) releasing the aptamers bound to the solid surfaces via the surface binding moiety to form a first plurality of cognate-target bound aptamers and a second plurality of unbound aptamers; and(e) separating the first plurality of cognate-target bound aptamers from the second plurality of unbound aptamers to form a plurality of aptamer-based capture complexes bound to cognate targets.

21. The method of claim 19 or 20, wherein the cognate targets are proteins.

22. The method of any one of claims 19-21, wherein the surface binding moiety comprises a first plurality of linkers.

23. The method of claim 22, wherein the releasing the aptamer bound to the surface by the surface binding moiety comprises cleaving the first plurality of linkers using an enzyme or UV.

24. The method of claim 23, wherein the enzyme comprises a base excision repair enzyme, a restriction endonuclease, or both.

25. The method of any one of claims 19-24, wherein the plurality of aptamer-based capture complexes comprises a second plurality of linkers different than the first plurality of linkers.

26. The method of any one of claims 19-25, wherein separating the second plurality of unbound aptamers to obtain the first plurality of bound aptamers comprises capturing the first plurality of the bound aptamers using the second plurality of linkers before cleaving the first plurality of linkers.

27. The method of claim 26, further comprising: functionalizing the cognate target to include a tag; providing an additional aptamer-based capture complex comprising a second plurality of linkers different that the first plurality of linkers; and wherein separating the second plurality of unbound aptamers to obtain the first plurality of bound aptamers comprises capturing the aptamers including the tag based on a reaction between the tag and the second plurality of linkers.

28. The method of claim 26 or 27, comprising functionalizing the cognate target to include the tag after capturing the protein based on the plurality of aptamers.

29. A method of detecting a cognate target in a sample, the method comprising:detecting the cognate target bound to the aptamer-based capture complexes made by the method of any one of claims 6-28.

30. A method, comprising: providing a plurality of cognate targets; forming an aptamer-based capture complex comprising a plurality of aptamers that is bound to the plurality of cognate targets, wherein the plurality of aptamers is linked to a solid substrate of the aptamer-based capture complex via a detachable linker; releasing the aptamers bound to the solid surfaces to form a first plurality of cognate-target bound aptamers and a second plurality of unbound aptamers; and separating the first plurality of cognate-target bound aptamers from the second plurality of unbound aptamers to form a plurality of aptamer-based capture complexes bound to cognate targets.

31. The method of claim 30, wherein forming the aptamer-based capture complex comprises: providing the plurality of aptamers configured to bind the plurality of cognate targets; providing the solid substrate comprises a plurality of linkers; and forming the aptamer-based capture complex by capturing the plurality of aptamers bound to the plurality of cognate targets via the plurality of linkers.

32. The method of claim 30, wherein forming the aptamer-based capture complex comprises: providing the solid substrate comprising the plurality of aptamers bound to the substrate via a plurality of linkers; and forming the aptamer-based capture complex by capturing the plurality of cognate targets via the plurality of linkers.

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