Aptamer selection using sequencing

Abstract

A method and system are provided for selecting an aptamer from a plurality of aptamer candidates. A target is bound in each well of a plurality of wells disposed in a substrate. Each well is contacted with a fluid containing the plurality of aptamer candidates. In at least one well, any aptamer candidates that are selective for the target are bound to the target. Any aptamer candidates that are not bound to the target are removed. Any aptamer candidates remaining after removal are sequenced to identify any aptamers in the well that are selective for the target.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 128,669, entitled "Selecting Aptamers Using Sequencing," filed December 21, 2020, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] The detection of specific nucleic acid sequences that may be present in biological samples has been used as a method to identify and classify microorganisms, diagnose infectious diseases, detect and characterize genetic abnormalities, identify genetic changes associated with cancer, examine genetic susceptibility to disease, and measure response to various types of treatment, as some examples. Nucleic acid sequences that bind to specific target molecules may be referred to as "aptamers" and may be synthetic or derived from biological samples. A common technique for detecting specific nucleic acid sequences, whether synthetic or from biological samples, is nucleic acid sequencing.

[0003] Nucleic acid sequencing methods have evolved from the chemical degradation method used by Maxam and Gilbert and the strand extension method used by Sanger. Currently, several sequencing methods are in use that allow parallel processing of millions or even billions of nucleic acids on a single flow cell. Some platforms include bead-based and microarray formats, where silica beads are functionalized with probes depending on the application of such formats in applications including sequencing, genotyping, or gene expression profiling. Some sequencing systems utilize a substrate that includes multiple different reservoirs carrying different reagents for use in the sequencing operation, whether it is "sequencing by synthesis" or genotyping. Summary of the Invention

[0004] Examples provided herein relate to the selection of aptamers using sequencing. Apparatus and methods for carrying out such selection are disclosed.

[0005] Some examples herein provide a method for selecting an aptamer from a plurality of aptamer candidates. The method may include binding a target in each well of a plurality of wells arranged in a substrate. The method may include contacting each of the wells with a fluid containing a plurality of aptamer candidates. The method may include binding any aptamer candidate that is selective for the target to the target in at least one of the wells. The method may include removing any aptamer candidate that is not bound to the target. The method may include sequencing any aptamer candidate remaining after removal in the well to identify any aptamer that is selective for the target.

[0006] In some examples, binding the targets in each well of the plurality of wells includes binding a first portion in each of the wells, binding a plurality of second portions to respective ones of the targets, and binding the second portion to the first portion in each of the wells. In some examples, the first portion comprises streptavidin and the second portion comprises biotin. In some examples, the first portion is bound in each of the wells by a capture primer. In some examples, the first portion is detached from the capture primer before performing sequencing.

[0007] In some instances, the substrate includes detection circuitry that is used to sequence the aptamer candidates that remain after removal.

[0008] In some examples, the multiple wells comprise flow cells through which fluids flow in parallel.

[0009] In some instances, any aptamer candidate that is selective for a target has a tertiary structure that is altered upon binding to the target.

[0010] In some examples, the method further comprises generating an amplicon of any aptamer candidate that is selective for the target. In some examples, generating an amplicon of any aptamer candidate that is selective for the target comprises separating the aptamer candidate from the target and using polymerase chain reaction (PCR) to generate an amplicon with the aptamer candidate. In some examples, the PCR is performed in an amplification chamber separate from the well. In some examples, the method further comprises contacting each of the wells with a fluid containing an amplicon, allowing any amplicon that is selective for the target in each of the wells to bind to the target, and removing any amplicon that is not bound to the target. In some examples, any aptamer candidate that is sequenced comprises any amplicon that remains after removing any amplicon that is not bound to the target. In some examples, the method further comprises generating an additional amplicon of any amplicon that is selective for the target.

[0011] In some examples, sequencing any aptamer candidate comprises binding any aptamer candidate remaining after removal to a capture primer disposed in the well, performing amplification in the well to generate amplicons bound to the capture primer, and sequencing the amplicons bound to the capture primer. In some examples, the target is bound in the well via each of the capture primers. In some examples, the capture primer binds to a first portion in the well, and a second portion binds to the first portion and the target in the well. In some examples, the capture primer is bound to the well separately from the target. In some examples, each of the aptamer candidates comprises a first adaptor and a second adaptor that are complementary to the respective capture primer. In some examples, each of the aptamer candidates further comprises a first spacer disposed between the first adaptor and the region that is a candidate selective for the target, and a second spacer disposed between the second adaptor and the region that is a candidate selective for the target.

[0012] In some examples, each of the aptamer candidates comprises an oligonucleotide.

[0013] Some examples herein provide a system for selecting an aptamer from a plurality of aptamer candidates. The system may include a substrate that includes a plurality of wells. The system may include a target bound in each of the wells. The system may include a fluid that includes a plurality of aptamer candidates and contacts each of the wells, where any aptamer candidate that is selective for the target is bound to the target. The system may include a detection circuit for sequencing any aptamer candidate in the well to identify any aptamer that is selective for the target.

[0014] In some examples, a first portion is bound in each well. In some examples, a plurality of second portions are bound to each of the targets. In some examples, the second portion is bound to the first portion in each of the wells so as to bind the target in each of the wells. In some examples, the first portion comprises streptavidin and the second portion comprises biotin. In some examples, the first portion is bound in each of the wells by a capture primer. In some examples, the first portion is detachable from the capture primer.

[0015] In some examples, the multiple wells are disposed on a detection circuit.

[0016] In some examples, the multiple wells comprise flow cells through which fluids flow in parallel.

[0017] In some instances, any aptamer candidate that is selective for a target has a tertiary structure that is altered upon binding to the target.

[0018] In some examples, amplicons are generated from any aptamer candidate that is selective for the target. In some examples, amplicons of any aptamer candidate that is selective for the target are generated using a process that includes separating the aptamer candidate from the target and using polymerase chain reaction (PCR) to generate amplicons with the aptamer candidate. In some examples, the system includes an amplification chamber separate from the well to perform PCR.

[0019] In some examples, the system further comprises a fluid containing the amplicons and contacting each of the wells, where any amplicons selective for the target are bound to the target. In some examples, any aptamer candidate to be sequenced comprises any amplicons remaining after removing any amplicons not bound to the target. In some examples, the system further comprises additional amplicons of any amplicons selective for the target.

[0020] In some examples, the system further comprises a capture primer disposed in the well and bound to any aptamer candidate remaining after removal, and an amplicon of the aptamer candidate bound to the capture primer. The detection circuit can be for sequencing the amplicon bound to the capture primer. In some examples, the target is bound in the well via each of the capture primers. In some examples, the capture primer binds to a first portion in the well, and a second portion binds to the first portion and the target in the well. In some examples, the capture primer is bound to the well separately from the target. In some examples, each of the aptamer candidates comprises a first adaptor and a second adaptor that are complementary to the respective capture primer. In some examples, each of the aptamer candidates further comprises a first spacer disposed between the first adaptor and the region that is a candidate selective for the target, and a second spacer disposed between the second adaptor and the region that is a candidate selective for the target.

[0021] In some examples, each of the aptamer candidates comprises an oligonucleotide.

[0022] It should be understood that any individual feature / example of each of the aspects of the disclosure described herein may be implemented together in any suitable combination, and any feature / example from any one or more of these aspects may be implemented together in any suitable combination with any of the features of the other aspects described herein, in order to achieve the benefits described herein. [Brief description of the drawings]

[0023] [Figure 1A] 1 shows a schematic of an exemplary apparatus and operations used in a process flow for selecting aptamers using sequencing. [Figure 1B] 1 shows a schematic of an exemplary apparatus and operations used in a process flow for selecting aptamers using sequencing. [Figure 1C] 1 shows a schematic of an exemplary apparatus and operations used in a process flow for selecting aptamers using sequencing. [Figure 1D] 1 shows a schematic of an exemplary apparatus and operations used in a process flow for selecting aptamers using sequencing. [Figure 1E] 1 shows a schematic of an exemplary apparatus and operations used in a process flow for selecting aptamers using sequencing. [Figure 1F] 1 shows a schematic of an exemplary apparatus and operations used in a process flow for selecting aptamers using sequencing. [Figure 1G] 1 shows a schematic of an exemplary apparatus and operations used in a process flow for selecting aptamers using sequencing. [Figure 1H] 1 shows a schematic of an exemplary apparatus and operations used in a process flow for selecting aptamers using sequencing. [Figure 1I] 1 shows a schematic of an exemplary apparatus and operations used in a process flow for selecting aptamers using sequencing. [Figure 2A] 1 shows a schematic of an exemplary apparatus and operations used in an alternative process flow for selecting aptamers using sequencing. [Figure 2B] 1 shows a schematic of an exemplary apparatus and operations used in an alternative process flow for selecting aptamers using sequencing. [Figure 2C] 1 shows a schematic of an exemplary apparatus and operations used in an alternative process flow for selecting aptamers using sequencing. [Figure 2D] 1 shows a schematic of an exemplary apparatus and operations used in an alternative process flow for selecting aptamers using sequencing. [Figure 2E] 1 shows a schematic of an exemplary apparatus and operations used in an alternative process flow for selecting aptamers using sequencing. [Figure 2F] 1 shows a schematic of an exemplary apparatus and operations used in an alternative process flow for selecting aptamers using sequencing. [Diagram 3] 1A-1I or 2A-2F show schematic diagrams of exemplary aptamer candidates for use in an apparatus or process flow as described with reference to FIGS. 1A-1I or 2A-2F. [Figure 4] 1 shows a schematic of exemplary operations in a process flow for selecting aptamers using sequencing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Examples provided herein relate to the selection of aptamers using sequencing. Apparatus and methods for carrying out such selection are disclosed.

[0025] Some aptamers are oligonucleotide chains that bind with high selectivity to target molecules, such as enzymes, antibodies, single cells, or any other molecular target of interest. During such selective binding, aptamers can acquire tertiary structures. SELEX, or Systematic Evolution of Ligands by EXponential Enrichment, is a process for selecting aptamers. Conventionally known SELEX processes are typically carried out on a conventional solid-phase substrate, such as a column containing a medium to which the target is bound. A fluid containing aptamer candidates is run over the substrate. Any aptamers that are selective for the target are bound to the target, while other aptamers are not bound and therefore flow out of the substrate. The aptamers selective for the target are then separated from the target and amplified, for example in a 96-well plate, using polymerase chain reaction (PCR). The PCR products may be run over the substrate again to be amplified and further enriched. The amplified aptamers are then sequenced to identify the aptamers that bind to the target and thus are the most "successful". These processes can be time and labor intensive.

[0026] In contrast, as provided herein, the apparatus and method can streamline and simplify the SELEX process through the use of sequencing. More specifically, in some examples, the operations for both aptamer selection and sequencing can be performed in the same well, for example, in a well of a substrate in a sequencing system. The substrate on which the selection process is performed can include detection circuitry for use in sequencing the aptamers. Illustratively, a target can be bound in each of the wells, and multiple aptamers flowed into each of the wells. Any aptamers selective for the target can be bound to the target, and any other aptamers can be removed. Aptamers selective for the target can be amplified and then sequenced in the wells, for example, using capture primers that are also bound in each well. In this way, the apparatus and method can provide a significantly streamlined and simplified process with significantly less material usage and waste compared to previously known SELEX processes.

[0027] We first provide a brief overview of some of the terms used herein, then we describe some exemplary methods for selecting aptamers using sequencing and related instrumentation.

[0028] term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The use of the term "including" and other forms such as "include", "includes" and "included" is not limiting. The use of the term "having" and other forms such as "have", "has" and "had" is not limiting. As used herein, whether in a transitional phrase or in the body of a claim, the terms "comprise" and "comprising" should be interpreted as having an open-ended meaning. That is, the above terms should be interpreted as synonymous with the phrase "having at least" or "comprising at least". For example, when used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may include additional features or components.

[0029] As used throughout this specification, the terms "substantially," "approximately," and "about" are used to describe and take into account small variations due to processing variations, etc. For example, they can refer to ±10% or less, such as ±5% or less, such as ±2% or less, such as ±1% or less, such as ±0.5% or less, such as ±0.2% or less, such as ±0.1% or less, such as ±0.05% or less.

[0030] As used herein, "hybridize" refers to the non-covalent binding of a first polynucleotide to a second polynucleotide along the length of the polymer to form a double stranded "duplex". For example, two DNA polynucleotide strands can associate through complementary base pairing. The strength of association between a first and a second polynucleotide increases with the complementarity between the nucleotide sequences within the polynucleotides. The strength of hybridization between polynucleotides can be characterized by the melting temperature (Tm) at which 50% of the duplex dissociates from each other.

[0031] As used herein, the term "nucleotide" is intended to mean a molecule that includes a sugar and at least one phosphate group, and in some instances also includes a nucleobase. A nucleotide that lacks a nucleobase may be referred to as "abasic." Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine triphosphate (DTPA ... These include adenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP) and deoxyuridine triphosphate (dUTP).

[0032] As used herein, the term "nucleotide" is also intended to encompass any nucleotide analog, which is a type of nucleotide that contains a modified nucleobase, sugar and / or phosphate moiety as compared to naturally occurring nucleotides. Exemplary modified nucleobases include inosine, xanthate, hypoxanthate, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil ... These include cytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, and the like. As is known in the art, certain nucleotide analogs cannot become incorporated into polynucleotides, such as nucleotide analogs such as adenosine 5'-phosphosulfate. A nucleotide can include any suitable number of phosphates, such as 3, 4, 5, 6, or more than 6 phosphates.

[0033] As used herein, the term "polynucleotide" refers to a molecule that comprises a sequence of nucleotides that are linked together. A polynucleotide is a non-limiting example of a polymer. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and their analogs. A polynucleotide can be a single-stranded sequence of nucleotides, such as RNA or single-stranded DNA, a double-stranded sequence of nucleotides, such as double-stranded DNA, or a mixture of single-stranded and double-stranded sequences of nucleotides. Double-stranded DNA (dsDNA) includes genomic DNA, and PCR and amplification products. Single-stranded DNA (ssDNA) can be converted to dsDNA and vice versa. A polynucleotide can include non-naturally occurring DNA, such as enantiomeric DNA. The exact sequence of nucleotides in a polynucleotide can be known or unknown. The following are examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, expressed sequence tag (EST), or serial analysis of gene expression (SAGE) tag), genomic DNA, genomic DNA fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, synthetic polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers, or amplified copies of any of the foregoing.

[0034] As used herein, "polymerase" is intended to mean an enzyme having an active site that assembles a polynucleotide by polymerizing nucleotides into a polynucleotide. A polymerase can bind to a primed single-stranded target polynucleotide and grow it by successively adding nucleotides to the primer to form a "complementary copy" polynucleotide having a sequence complementary to that of the target polynucleotide. Another polymerase, or the same polymerase, can then form a copy of the target nucleotide by forming a complementary copy of the complementary copy polynucleotide. Any such copy may be referred to herein as an "amplicon." A DNA polymerase can bind to a target polynucleotide and then move downstream while growing the target polynucleotide by successively adding nucleotides to the free hydroxyl group at the 3' end of the polynucleotide chain (amplicon growth). A DNA polymerase can synthesize a complementary DNA molecule from a DNA template, and an RNA polymerase can synthesize an RNA molecule from a DNA template (transcription). Polymerases may use short RNA or DNA strands (primers) to initiate strand growth. Some polymerases may displace the strand upstream of the site where they add a base to the strand. Such polymerases may also be said to be strand displacing, that is, to have the activity of removing a complementary strand from the template strand that is read by the polymerase. Exemplary polymerases with strand displacement activity include, but are not limited to, Bst (Bacillus stearothermophilus) polymerase, Exo-Klenow polymerase, or a large fragment of sequencing grade T7 exo-polymerase. Some polymerases cleave the strand in front of them and effectively replace it with the growing strand (5' exonuclease activity). Some polymerases have the activity of degrading the strand behind them (3' exonuclease activity). Some useful polymerases have been mutated or otherwise modified to reduce or eliminate 3' and / or 5' exonuclease activity.

[0035] As used herein, the term "primer" is defined as a polynucleotide to which nucleotides can be added via a free 3'OH group. The length of the primer can be any suitable number of bases long and can include a suitable combination of natural and / or non-natural nucleotides. A target polynucleotide (such as, but not limited to, an aptamer) can include an "adapter" that hybridizes to the primer (having a sequence complementary thereto) and can be amplified to generate a complementary copy polynucleotide by adding a nucleotide to the free 3'OH group of the primer. The primer can be bound to a substrate. Primers that are "complementary" to each other can hybridize to each other substantially along their entire length, while primers that are "orthogonal" to each other do not substantially hybridize to each other and do not hybridize to their amplicons. "Capture primer" is intended to mean a primer that can be bound to a substrate and hybridize to a first adapter of a target polynucleotide, and "orthogonal capture primer" is intended to mean a primer that can be bound to a substrate and hybridize to a second adapter of the target polynucleotide. The first adaptor may have a sequence complementary to that of the capture primer and the second adaptor may have a sequence complementary to that of the orthogonal capture primer. The capture primer and the orthogonal capture primer may have independent sequences that are different from each other.

[0036] In some examples, the capture primer is a P5 or P7 primer, commercially available from Illumina, Inc. P5 and P7 primers are non-limiting examples of primers that are orthogonal to each other. The P5 and P7 primer sequences, in some examples, can have the following sequences: Paired lead set: P5:5'-AATGATACGGCGACCACCGAGAUCTACAC-3' P7: 5'-CAAGCAGAAGACGGCATACGAG * AT-3' Single Lead Set: P5: 5'-AATGATACGGCGACCACCGA-3' P7: 5'-CAAGCAGAAGACGGCATACGA3' In the formula, G * is G or 8-oxoguanine.

[0037] In some examples, the attached oligonucleotide (such as the primer, or the P5 or P7 primer) includes a linker or spacer at the 5' end. Such a linker or spacer may be included to allow chemical or enzymatic cleavage or to impart some other desired property, for example, to allow covalent attachment to the polymer or solid support, or to act as a spacer to position the cleavage site at an optimal distance from the solid support. In certain cases, 10 spacer nucleotides may be placed between the attachment points of the P5 or P7 primer to the polymer or solid support. In some examples, a poly-T spacer is used, although other nucleotides and combinations thereof may also be used. In one example, the spacer is a 6T-10T spacer. In some examples, the linker includes a cleavable nucleotide that includes a chemically cleavable functional group, such as a vicinal diol or an allyl T.

[0038] As used herein, the term "amplicon" when used in reference to a nucleic acid refers to a product of the replication of a nucleic acid, which product has a nucleotide sequence that is substantially the same as or complementary to at least a portion of the nucleotide sequence of the nucleic acid. "Amplification" and "amplifying" refer to the process of creating an amplicon of a polynucleotide. A first amplicon of a target polynucleotide can typically be a complementary copy. Additional amplicons are copies made from the target polynucleotide or the first amplicon after the generation of the first amplicon. Subsequent amplicons can have a sequence that is substantially complementary to or substantially identical to the target polynucleotide. It will be understood that a small number of mutations of a polynucleotide (e.g., due to amplification artifacts) may occur when generating an amplicon of that polynucleotide.

[0039] As used herein, the term "substrate" refers to a material used as a support for the compositions described herein. Exemplary substrate materials can include glass, silica, plastic, quartz, metal, metal oxide, organo-silicates (e.g., polyhedral organic silsesquioxanes (POSS)), polyacrylates, tantalum oxide, complementary metal oxide semiconductor (CMOS), or combinations thereof. An example of a POSS can be that described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, which is incorporated herein by reference in its entirety. In some examples, the substrate used in this application includes a silica-based substrate, such as glass, fused silica, or other silica-containing materials. In some examples, the substrate can include silicon, silicon nitride, or hydrogenated silicone. In some examples, the substrates used in the present application include plastic materials or components such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylon, polyester, polycarbonate, and poly(methyl methacrylate). Examples of plastic materials include poly(methyl methacrylate), polystyrene, and cyclic olefin polymer substrates. In some examples, the substrate is or includes a silica-based material or a plastic material, or a combination thereof. In certain examples, the substrate has at least one surface that includes glass or a silicon-based polymer. In some examples, the substrate may include a metal. In some such examples, the metal is gold. In some examples, the substrate has at least one surface that includes a metal oxide. In one example, the surface includes tantalum oxide or tin oxide. Acrylamides, enones, or acrylates can also be utilized as substrate materials or components. Other substrate materials can include, but are not limited to, gallium arsenide, indium phosphide, aluminum, ceramic, polyimide, quartz, resin, polymer, quartz, resin, polymer, and copolymer.In some examples, the substrate and / or substrate surface may be or may include quartz. In some other examples, the substrate and / or substrate surface may be or may include a semiconductor, such as GaAs or ITO. The above list is intended to illustrate, but not limit, the present application. The substrate may include a single material or multiple different materials. The substrate may be a composite or laminate. In some examples, the substrate includes an organosilicate material. The substrate may be flat, round, spherical, rod-like, or any other suitable shape. The substrate may be rigid or flexible. In some examples, the substrate is a bead or a flow cell.

[0040] In some examples, the surface is a patterned surface. A "patterned surface" refers to an arrangement of different regions in or on an exposed layer of a substrate. For example, one or more regions can be features in which one or more capture primers are present. The features can be separated by gap regions in which no capture primers are present. In some examples, the pattern can be an xy format of features in rows and columns. In some examples, the pattern can be a repeating sequence of features and / or gap regions. In some examples, the pattern can be a random sequence of features and / or gap regions. In some examples, the substrate includes an array of wells (recesses) on the surface. The wells can be provided with substantially vertical sidewalls. The wells can be manufactured as commonly known in the art using a variety of techniques, including but not limited to photolithography, stamping techniques, molding techniques, and microetching techniques. As is understood in the art, the techniques used will depend on the composition and shape of the array substrate.

[0041] The features within the patterned surface of the substrate may include wells in which a covalently bonded gel, such as poly(N-(5-azidoacetamylpentyl)acrylamide-co-acrylamide) (PAZAM), is patterned and arrayed on glass, silicon, plastic, or other suitable material. This process creates a gel pad used for sequencing, which may be stable over many cycles of sequencing operations. Covalently bonding the polymer to the wells may be useful to maintain the gel in the structured features throughout the life of the structured substrate during various applications. However, in many instances, the gel does not need to be covalently bonded to the wells. For example, in some conditions, silane-free acrylamide (SFA) that is not covalently bonded to any part of the structured substrate may be used as the gel material.

[0042] In a particular example, a structured substrate can be created by patterning a suitable material with wells (e.g., microwells or nanowells), coating the patterned material with a gel material (e.g., PAZAM, SFA, or chemically modified variants thereof, such as the azido form of SFA (azido-SFA)), and polishing the gel-coated material, for example, by chemical or mechanical polishing, thereby retaining the gel in the wells but removing or inactivating substantially all of the gel from the interstitial regions of the surface of the structured substrate between the wells. Primers can be bound to the gel material. A solution containing a plurality of target polynucleotides (e.g., a fragmented human genome or a portion thereof) can then be contacted with the polished substrate such that individual target nucleotides are seeded into individual wells through interaction with primers attached to the gel material, but the target polynucleotides do not occupy the interstitial regions due to the absence or inactivity of the gel material. Amplification of the target polynucleotides can be confined to the wells due to the absence or inactivity of the gel in the interstitial regions, preventing outward migration of clusters. The process is easily manufacturable, scalable, and utilizes conventional micro- or nano-fabrication methods.

[0043] Patterned substrates may include, for example, wells etched into a slide or chip. The pattern of wells etched and geometric shapes may be of a variety of different shapes and sizes, and such features may be physically or functionally separable from one another. Particularly useful substrates with such structural features include patterned substrates that allow selection of the size of solid particles, such as microspheres. An example of a patterned substrate with these characteristics is the etched substrate used in conjunction with BEAD ARRAY technology (Illumina, Inc., San Diego, Calif.).

[0044] In some examples, the substrate forms at least a portion of a flow cell, or is located within or coupled to a flow cell. A flow cell may include a flow chamber that is divided into multiple lanes or multiple sectors. Examples of flow cells and substrates for the manufacture of flow cells that may be used in the methods and systems described herein include, but are not limited to, those commercially available from Illumina, Inc. (San Diego, Calif.).

[0045] As used herein, the term "plurality" is intended to mean a population of two or more distinct members. A plurality can range in size from small, medium, large, to very large. A small size plurality can range, for example, from a few members to tens of members. A medium size plurality can range, for example, from tens of members to about 100 members or hundreds of members. A large plurality can range, for example, from about hundreds of members to about 1000 members, thousands of members, and tens of thousands of members. A very large plurality can range, for example, from tens of thousands of members to about hundreds of thousands, millions, tens of millions, or hundreds of millions or more members. Thus, a plurality can range in size from 2 to well over 100 million members, as well as between all sizes measured by number of members and larger than the exemplary ranges listed above. Exemplary polynucleotides include, for example, about 1×105 That's it, 5 x 10 5 or more or 1×10 6 The term includes a population of more than one different polynucleotides. Thus, the definition of this term is intended to include all integer values ​​greater than 2. The upper limit of the plurality of values ​​can be set, for example, by the theoretical diversity of polynucleotide sequences in a sample.

[0046] The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein. The difference in the terms is not intended to indicate any particular difference in size, sequence, or other properties, unless otherwise specified. For clarity of explanation, when describing a particular method or composition that includes several polynucleotide species, different terms may be used to distinguish one type of polynucleotide from another.

[0047] As used herein, the term "sequencing system" refers to a system configured to determine the sequence of a polynucleotide. Various sequencing systems are commercially available. Illustratively, the sequencing system can be or include the iSEQ™ 100 sequencing system available from Illumina, Inc. (San Diego, CA). The iSEQ™ 100 sequencing system is a bench-top system that performs sequencing by synthesis using a pre-filled cartridge that includes reservoirs that store different sequencing reagents. Other non-limiting examples of sequencing systems include the cBot 2, NovaSeq 6000, and MiniSeq systems available from Illumina, Inc., as well as sequencing systems from other sources.

[0048] As used herein, "aptamer" is intended to mean an oligonucleotide having a tertiary structure that results in an oligonucleotide that is selective for a target, and "aptamer candidate" is intended to mean an oligonucleotide that may potentially be selective for a target. Being "selective" for a target is intended to mean binding to that target and not binding to a different target. Thus, among a given plurality of aptamer candidates, one or more aptamer candidates may potentially be selective for a given target and therefore may be aptamers for that target. However, it will be understood that any given plurality of aptamer candidates does not necessarily have to include an aptamer for a given target. Aptamer candidates can be identified as aptamers selective for a target using a procedure that includes binding the aptamer candidate to a target (indicating selectivity) and sequencing the aptamer candidate. An amplicon of an aptamer candidate can itself be an aptamer candidate, regardless of whether the amplicon is a copy or a complementary copy of the aptamer candidate. An aptamer amplicon may itself be an aptamer, for example, if the amplicon is a copy of an aptamer (as opposed to a complementary copy of an aptamer, which may be an aptamer candidate, but is not necessarily an aptamer). Aptamers may include any suitable type of oligonucleotide, such as DNA, RNA, and / or nucleic acid analogs, as exemplified elsewhere herein. Aptamers may bind to targets through any suitable combination of interactions, for example, any suitable combination of electrostatic interactions, hydrophobic interactions, and formation of tertiary structures.

[0049] As used herein, "target" is intended to mean a chemical element for which it is desired to select an aptamer. Targets may include chemical entities that are not nucleotides. An example of a target is a protein target. A protein includes a sequence of a polypeptide folded into a structure. Another exemplary target is a metabolite target. A metabolite target is a chemical element that is formed or used during metabolism. Additional exemplary targets include, but are not limited to, carbohydrates, fatty acids, sugars (such as glucose), amino acids, nucleosides, neurotransmitters, phospholipids, and heavy metals. In the present disclosure, an analyte may be selected for a target in the context of any suitable application(s), such as analysis of a disease state, analysis of metabolic health, analysis of the microbiome, analysis of drug interactions, analysis of drug responses, analysis of toxicity, or analysis of infectious diseases. Illustratively, metabolites may include chemical elements that are upregulated or downregulated in response to a disease. Non-limiting examples of targets include kinases, serine hydrolases, metalloproteases, disease-specific biomarkers such as antigens for a particular disease, and glucose.

[0050] As used herein, the oligonucleotide having "tertiary structure" is intended to mean that the oligonucleotide that is folded into a three-dimensional tertiary structure has internal bridges that hold the fold in place.In contrast, an oligonucleotide that has a primary structure (e.g., a specific sequence of nucleic acids linked together) and a secondary structure (e.g., a local structure) but does not have internal bridges that hold the fold in place is not considered to have a tertiary structure as that term is used herein.The tertiary structure of an aptamer can change when the aptamer binds to a target.

[0051] As used herein, "different" elements are intended to mean that one of the elements has at least one change with respect to the other element that makes the elements distinguishable from each other. For example, different oligonucleotides can have nucleic acid sequences that differ from each other by at least one nucleic acid. As another example, different proteins can have peptide sequences that differ from each other by at least one peptide. As another example, metabolites can vary from each other by at least one chemical group. As provided herein, the device and method can be used to select different aptamers for different targets.

[0052] As used herein, the term "fluorophore" is intended to mean a molecule that emits light at a first wavelength in response to excitation by light at a second wavelength, different from the first wavelength. The light emitted by a fluorophore may be referred to as "fluorescence" and may be detected by suitable optical detection circuitry.

[0053] The fluorescence may be detected using any suitable optical detection circuitry, which may include an optical detector for generating an electrical signal based on the light received from the fluorophore, and an electronic circuit for determining that light has been received from the fluorophore using the electrical signal. As an example, the photodetector may include an active pixel sensor (APS) including an array of amplified photodetectors configured to generate an electrical signal based on the light received by the photodetector. The APS may be based on complementary metal oxide semiconductor (CMOS) technology known in the art. The CMOS-based detector may include a field effect transistor (FET), e.g., a metal oxide semiconductor field effect transistor (MOSFET). In a particular example, a CMOS imager with a single photon avalanche diode (CMOS-SPAD) may be used, e.g., to perform fluorescence lifetime imaging (FLIM). In other examples, the photodetector may include a photodiode, such as an avalanche photodiode, a charge-coupled device (CCD), a cryogenic photon detector, a reverse-biased light-emitting diode (LED), a photoresistor, a phototransistor, a photovoltaic cell, a photomultiplier tube (PMT), a quantum dot photoconductor, or a photodiode. The optical detection circuit may further include any suitable combination of hardware and software configured to operatively communicate with the optical detector to receive an electrical signal from the optical detector and detect fluorescence based on such signal, for example, based on the optical detector detecting light from a fluorophore. For example, the electronic circuit may include a memory and a processor coupled to the memory. The memory may store instructions for causing the processor to receive a signal from the optical detector and use such signal to detect a fluorophore. For example, the instructions may cause the processor to determine, using the signal from the optical detector, that fluorescence is emitted within the field of view of the optical detector and use such determination to determine that a fluorophore is present. The substrate may include the optical detection circuit and may include an optical detector in which, for example, one or more wells used to select aptamers using sequencing may be placed.

[0054] Aptamer selection using sequencing As described above and in more detail below, the apparatus and method may be used to select aptamers using sequencing. A target may be bound in each of a plurality of wells where both aptamer selection and aptamer sequencing are performed. A plurality of aptamer candidates (e.g., a SELEX aptamer library) may be introduced into the wells to contact the target with the aptamer candidates, and any aptamer candidates that are selective for the target will be bound to the target, while the remaining aptamer candidates will remain unbound to the target and may be washed away. Aptamer candidates that become bound to the target may be amplified and sequenced in the well to identify the aptamer(s) that are selective for the target.

[0055] For example, Figures 1A-1I show schematic diagrams of exemplary apparatus and operations used in a process flow for selecting aptamers using sequencing. The apparatus 100 shown in Figure 1A may include a substrate 110 including a plurality of reservoirs. In the non-limiting example shown in Figure 1A, the reservoirs may include wells 121, 122, 123, 124 provided with each other in a common integrally formed substrate 110. However, it will be understood that one or more of the wells 121, 122, 123, 124, and indeed all of the wells 121, 122, 123, 124, may be physically separated from each other and need not be formed with each other in a common substrate. Any suitable number of wells of any suitable size and arrangement may be provided. For example, the substrate 110 may include thousands, tens of thousands, hundreds of thousands, or even millions of wells. Each of the wells 121, 122, 123, 124 may include a plurality of first and second capture primers 111, 112, respectively, that may be used to amplify and sequence candidate aptamers, in a manner as described further below with reference to Figures 1H-1I. The device 100 may further include a detection circuit 190 for sequencing any aptamer candidates in the wells to identify any aptamers that are selective for the target, for example, in a manner as described with reference to Figure 1I. The wells 121, 122, 123, 124 may be disposed on or otherwise coupled to the detection circuit 190. In one non-limiting example, the detection circuit 190 includes a CMOS optical detector, and the wells 121, 122, 123, 124 include flow cells capable of flowing fluids in parallel, and the CMOS detects fluorescence during sequencing of oligonucleotides in the flow cells.

[0056] Illustratively, the device 100 may be for use in a sequencing system for selecting aptamers using sequencing. Illustratively, the sequencing system may be or include an iSEQ™ 100 sequencing system available from Illumina, Inc. (San Diego, Calif.). The iSEQ™ 100 sequencing system is a bench-top system that performs sequencing by synthesis using a pre-filled cartridge that includes reservoirs that store different sequencing reagents in a manner similar to that of the device 100 shown in FIG. 1A-1B. However, it will be understood that the device 100 may be suitably adapted for use in any other sequencing system, such as the cBot 2, NovaSeq 6000, or MiniSeq systems available from Illumina, Inc., or a sequencing system from another source.

[0057] A target for which it may be desired to find an aptamer may be bound in each of the wells 121, 122, 123, 124. Non-limiting examples of targets are provided elsewhere herein. The target may be directly bound via a capture primer via any suitable linkage, for example in a manner as further described below with reference to Figures 2A-2F. Alternatively, the target may be indirectly bound via moieties that interact with each other. For example, in a manner as shown in Figure 1A, a first portion 130 may be bound in each of the wells 121, 122, 123, 124, for example, via any suitable linkage. The wells 121, 122, 123, 124 may be contacted with a fluid that includes a plurality of molecules 140, each of which may include a second portion 150 and a target 160 bound to each other via any suitable linkage, which linkage may optionally be cleavable. The respective linkages between the first portion 130 and the wells 121, 122, 123, 124, and between the second portion 150 and the target 160 may be formed by any suitable interaction, such as NTA-His-Tag, Spytag-Spycatcher, hybridization of an oligonucleotide to a complementary oligonucleotide, copper(I)-catalyzed click reaction, or strain-promoted azide-alkyne cycloaddition. Any suitable number of such linkages may include a cleavable moiety, such as 8-oxo-G, or a protein that can be cleaved using a proteinase, and may be used to separate one or more elements from the substrate in a manner as described in more detail below.

[0058] 1B, the second portion 150 can be attached to the first portion 130 so as to bind a target 160 in each of the wells 121, 122, 123, 124. Any suitable moieties that interact with each other covalently or non-covalently may be provided on the first and second portions 130, 150. In one non-limiting example, the first portion 130 includes streptavidin and the second portion 150 includes biotin. In another non-limiting example, the first portion 130 includes biotin and the second portion 150 includes streptavidin. Alternatively, the target 160 may be bound in each of the wells 121, 122, 123, 124 via any suitable interaction, such as NTA-His-Tag, Spytag-Spycatcher, hybridization of an oligonucleotide to a complementary oligonucleotide, copper(I)-catalyzed click reaction, or strain-promoted azide-alkyne cycloaddition, optionally including a cleavable moiety such as 8-oxo-G, or a protein that can be cleaved using a proteinase. For simplicity, a single first moiety 130, a single second moiety 150, and a single target 160 are shown in each well, but it will be understood that any suitable number of first and second moieties and targets may be bound in each well, and each well may have the same or different number of moieties or targets bound therein. In the example as shown in FIG. 1B, the capture primers 111, 112 may be bound to the wells 121, 122, 123, 124 separately from the target 160.

[0059] In a manner as shown in Figure 1C, system 100 may include a plurality of aptamer candidates 171, 172, 173, 174 and may include fluid in contact with each of wells 121, 122, 123, 124. Each of aptamer candidates 171, 172, 173, 174 may include an adaptor 181, 182 capable of binding that aptamer candidate to capture primers 111 or 112, respectively, for amplification, in a manner as described below with reference to Figure 1H. Any aptamer candidates that are selective for the target are bound to target 160. For example, as shown in FIG. 1D, aptamer candidate 174 is bound to target 160 in well 124, while aptamer candidates 171, 172, 173 remain unbound to target 160 in wells 121, 122, 123 and can be removed in a manner as shown in FIG. 1E, for example, by flushing wells 121, 122, 123, 124 with a buffer. In some examples, any aptamer candidate that is selective for target 160 may have a tertiary structure that changes when bound to the target. As suggested by FIG. 1D, at least some of aptamer candidates 171, 172, 173, 174 may include a hairpin structure that includes a single-stranded loop and a single-stranded stem that may bind to target 160 if the aptamer candidate is selective for that target.

[0060] An amplicon may be generated from any aptamer candidate that is selective for a target. For example, generating an amplicon of any aptamer candidate that is selective for a target may include separating the aptamer candidate from the target and using PCR to generate an amplicon with the aptamer candidate. For example, the aptamer candidate 174 shown in FIG. 1E may be separated from the target 160 and PCR may be performed to generate an amplicon of the aptamer candidate. Such separation between the aptamer candidate 174 and the target 160 may be performed using any suitable reaction condition(s) that disrupts the interaction between the target and the aptamer, such as heat or buffer exchange, where the salt concentration or pH is unfavorable to the tertiary structure of the aptamer or to forces that stabilize the aptamer-target interface. In some examples, the device 100 may include an amplification chamber separate from the wells 121, 122, 123, 124 to perform PCR. Next, the target 160 in each well 121, 122, 123, 124 is contacted with a fluid containing an amplicon 174 of an aptamer candidate 174 in a manner as shown in FIG. 1F. It should be noted that the amplicon 174 and the aptamer candidate 174 are referred to herein using the same reference numbers since they are equivalent structures. For example, the amplicon 174 is also an aptamer candidate for the target 160 and is expected to bind to it in the same manner as the aptamer candidate 174. Any amplicon 174 that is selective for the target will be bound to the target, such as, for example, multiple amplicons 174 are bound to each target 160 as shown in FIG. 1G in a manner similar to that described with reference to FIG. 1D. However, it should be noted that the additional operation of separating the aptamer candidate from the target and using the aptamer candidate to generate an amplicon using PCR may be omitted.

[0061] The device 100 may then be used to sequence the aptamer candidates 174 (including any amplicons thereof). For example, after removing aptamer candidates that are not bound to the target 160, any aptamer candidates that remain bound to the target 150 may be sequenced. The aptamer candidates 174 (including any amplicons thereof) shown in FIG. 1G may be separated from the target 160 in a manner as described with reference to FIG. 1E, and cluster amplification may be used to generate amplicons of the aptamer candidates 174, including generating additional amplicons of any amplicons that are selective for the target. For example, the capture primers 111, 112 may be positioned in the wells 121, 122, 123, 124 as described above with reference to FIG. 1A. The capture primers 111 and 112 may be orthogonal to each other, for example, the capture primer 111 may include a P5 primer and the capture primer 112 may include a P7 primer. The capture primers 111, 112 may be attached to any aptamer candidates (including any amplicons thereof) 174 that remain after any aptamer candidates (and amplicons) that were not bound to the target 160 have been removed. For example, as described above with reference to Figure 1A, the aptamer candidates 171, 172, 173, 174 may include adaptor(s) 181, 182 that can attach the aptamer candidates to the capture primers 111 or 112, respectively, for amplification.

[0062] In a manner as shown in FIG. 1I, an adaptor 181 of an aptamer candidate 174 (or its amplicon) may be attached to the capture primer 111, and an adaptor 182 of an aptamer candidate may be attached to the capture primer 112. Bridge amplification, or other suitable surface-based amplification process, may be used to generate clusters of amplicons 174, 174' of an aptamer candidate (or amplicon) 174 in a manner as shown in FIG. 1I. Note that the second portion 150 and the target 160 are simply omitted from FIG. 1I and may either remain in the well or may be removed, for example, by cleaving the linker-tethered target 160 to portions 130 or cleaving the linker cleavage portion 130 to the surface. A detection circuit 190 may sequence the amplicons 174, 174' attached to the capture primers, for example, using sequencing-by-synthesis in a manner as known in the art. Illustratively, a polymerase can be used to extend primers 111 or 112 with labeled (e.g., fluorescently labeled) nucleotides based on the sequence of the respective amplicon 174, 174', and detection circuitry 190 can identify the sequence to which such nucleotides are added using a signal generated by the labels of those nucleotides. The sequenced aptamer candidate (or its amplicon 174) can be considered to be an aptamer for target 160, for example, because such an aptamer candidate is bound to target 160 by multiple operations and exhibits selectivity for the target.

[0063] As described above with reference to FIG. 1A, the targets for the aptamer selection process can be bound in the wells in any suitable manner, such as by using a capture primer. For example, FIGS. 2A-2F show schematic diagrams of an exemplary apparatus and operation used in an alternative process flow for selecting aptamers using sequencing. In the example shown in FIG. 2A, a first portion is bound in each well by a capture primer, e.g., first portion 230 is bound to substrate 210 via capture primer 211, while first portion 230' is bound to substrate 210 via capture primer 212. Capture primer 211 and capture primer 212 may be orthogonal to each other, e.g., capture primer 211 may include a P5 primer and capture primer 212 may include a P7 primer. The first portions 230, 230' may be contacted with a fluid containing molecules 140, including second portion 150 and target 160, in a manner as described with reference to FIG. 1A. As shown in FIG. 2B, the second portion 150 can be bound to the first portion 230 to bind the target molecule 160 to the substrate 210 via a capture primer 211 (e.g., in a well, not specifically shown), while another second portion 150 can be bound to the first portion 230' to bind the target molecule 160 to the substrate 210 via a capture primer 212 (e.g., in the same well or another well, not specifically shown). As shown in FIG. 2C, an aptamer candidate (or its amplicon) 174 selective for the target 160 can be bound thereto, for example, in a manner as described with reference to FIG. 1D and FIG. 1G. As shown in FIG. 2D, the aptamer candidate 174 can be separated from the target 160 for amplification, in a manner as described with reference to FIG. 1E.

[0064] In some examples, the first portion 230, 230' may be detachable from the capture primer 211, 212. Thus, in the manner shown in FIG. 2E, the first portion 230, 230', the second portion 150, and the target 160 may be detached from the substrate 210 by separating the first portion 230, 230' from its respective capture primer 211, 212. Illustratively, the capture primer 211, 212 may include a cleavable portion, such as 8-oxo-G, that may be cleaved to detach the first portion 230, 230' and any elements bound thereto prior to performing sequencing. For example, as shown in Figure 2F, the adaptors 181, 181' of the aptamer candidate 174 and its amplicon 174' may be hybridized to respective capture primers 211, and the adaptors 182, 182' of the aptamer candidate and its amplicon may be hybridized to respective capture primers 212 for amplification and sequencing as described with reference to Figures 1H-I. Alternatively, in examples as provided throughout this disclosure, any suitable one of the target 160, linker(s), first portion, and second portion may comprise a protein that can be digested and thus separated from the substrate and / or capture primer using a proteinase.

[0065] It will be understood that the aptamer candidates that contact the target can include any suitable sequence and components. For example, FIG. 3 shows a schematic of exemplary aptamer candidates for use in an apparatus or process flow as described with reference to FIGS. 1A-1I or 2A-2F. In the non-limiting example shown in FIG. 3, aptamer candidates 171, 172, 173, 174 each include an oligonucleotide subsequence (region) 171", 172", 173", 174" that may potentially be selective for the target, and adaptors 181 and 182 attached to the oligonucleotide subsequence. The oligonucleotide subsequences 171", 172", 173", 174" may be different from each other. Each adaptor 181 may include an optional spacer 300, a capture primer adaptor 311 that is complementary to the capture primer 111, and an optional PCR adaptor 320 that may be used to perform PCR amplification of the aptamer candidates, if appropriate and if such an operation is included in the process flow. Similarly, each adaptor 182 may include an optional spacer 300, a capture primer adaptor 312 that is complementary to the capture primer 112, and an optional PCR adaptor 320 that may be used to perform PCR amplification of the aptamer candidates, if appropriate and if such an operation is included in the process flow. The spacer 300 may provide a suitable distance between the oligonucleotide subsequence 171", 172", 173", 174" and the capture primer adaptor 311 or 312 such that the capture primer adaptor 311 does not inhibit suitable binding of the oligonucleotide subsequence to the target 160. Illustratively, the spacer 300 may include 5 or more nucleotides, 10 or more nucleotides, or 15 or more nucleotides.

[0066] It will be understood that any suitable system, method, and composition can be used to select aptamers based on the present teachings. For example, FIG. 4 shows a schematic of an exemplary operation in a process flow for selecting aptamers using sequencing. The method 400 shown in FIG. 4 includes binding a target in each well of a plurality of wells disposed in a substrate (operation 410). For example, the target 160 described with reference to FIG. 1A can be directly bound to the substrate in each well via a suitable linker, such as a capture primer, in a manner such as described with reference to FIG. 2A. Or, for example, the target 160 can be indirectly bound to the substrate in each well via a first portion bound to the substrate and a second portion that is bound to the target and becomes bound to the first portion, in a manner such as described with reference to FIGS. 1A-1B. The method 400 shown in FIG. 4 also includes contacting each of the wells with a fluid containing a plurality of aptamer candidates (operation 420). For example, the wells may be contacted with aptamer libraries 171, 172, 173, 174 in a manner as described with reference to FIG. 1C, or with an amplicon of aptamer 174 in a manner as described with reference to FIG. 1F. The method 400 shown in FIG. 4 includes binding any aptamer candidate that is selective for the target to the target in at least one of the wells (operation 430). For example, the aptamer candidate 174 (or any amplicon thereof) may be bound to the target 160 in a manner as described with reference to FIG. 1D, FIG. 1G, or FIG. 2C. The method 400 shown in FIG. 4 includes removing any aptamer candidate that is not bound to the target (operation 440). For example, the aptamer candidates 171, 172, 173 may be removed by flowing a buffer through the wells in a manner as described with reference to FIG. 1E, while the aptamer candidate 174 remains bound to the target 160 despite such flow. 4 includes sequencing any aptamer candidates remaining after removal in the wells (operation 450) to identify any aptamers that are selective for the target. For example, amplicons (or amplicons thereof) of aptamer candidates 174 may be generated and such amplicons may be sequenced.

[0067] Further comments It should be understood that any individual feature / example of each of the aspects of the disclosure described herein may be implemented together in any suitable combination, and any feature / example from any one or more of these aspects may be implemented together in any suitable combination with any of the features of the other aspects described herein, in order to achieve the benefits described herein.

[0068] While various illustrative examples have been described above, it will be apparent to one skilled in the art that various changes and modifications can be made herein without departing from the present invention. It is intended that the appended claims cover all such changes and modifications that fall within the true spirit and scope of the present invention.

Claims

1. A method for selecting an aptamer from a plurality of aptamer candidates, comprising: binding a target within each well of a plurality of wells disposed within a substrate; contacting each of said wells with a fluid comprising a plurality of aptamer candidates; allowing any candidate aptamers selective for said target to bind to said target in at least one of said wells; removing any aptamer candidates that are not bound to the target; sequencing any aptamer candidates remaining in said wells after said removal to identify any aptamers that are selective for said target; A method comprising:

2. binding the target within each well of the plurality of wells; binding a first portion in each of said wells; binding a plurality of second moieties to respective ones of said targets; binding the second portion to the first portion in each of the wells; Including, 2. The method of claim 1, wherein optionally, the first portion comprises streptavidin and the second portion comprises biotin.

3. 3. The method of claim 2, wherein the first portion is bound in each of the wells by a capture primer, and optionally the method comprises detaching the first portion from the capture primer prior to performing the sequencing. (i) the substrate includes a detection circuit that is used to sequence the aptamer candidates remaining after the removal; or (ii) the plurality of wells comprises flow cells through which the fluids flow in parallel; or (iii) any candidate aptamer that is selective for said target has a tertiary structure that is altered upon binding to said target; or (iv) A method according to any one of claims 1 to 3, which is a combination of any two or three of (i), (ii) and (iii).

5. The method of any one of claims 1 to 4, further comprising generating amplicons of any aptamer candidates that are selective for said target.

6. generating amplicons of any candidate aptamers that are selective for said target; separating the aptamer candidates from the target; generating amplicons using the candidate aptamers using polymerase chain reaction (PCR); 6. The method of claim 5, comprising: optionally, the PCR is carried out in an amplification chamber separate from the wells.

7. contacting each of said wells with a fluid containing said amplicons; allowing any amplicons selective for said target to bind to said target in each of said wells; removing any amplicons that are not bound to the target; and and optionally further comprising: (i) any aptamer candidate that is sequenced includes any amplicon that remains after removing any amplicon that is not bound to the target; and / or The method of any one of claims 5 to 6, wherein (ii) the method further comprises generating additional amplicons of any amplicons that are selective for the target.

8. Sequencing of any aptamer candidate binding any aptamer candidates remaining after said removal to capture primers disposed in said wells; performing amplification in said wells to generate amplicons bound to said capture primers; sequencing the amplicons bound to the capture primers; The method according to any one of claims 1 to 7, comprising: (i) the target is bound in the well via a respective one of the capture primers, optionally wherein the capture primer binds to a first portion in the well and a second portion binds to the first portion and to the target in the well; or 9. The method of claim 8, wherein (ii) the capture primer is bound to the well separately from the target.

10. 10. The method of any one of claims 8 to 9, wherein each of the aptamer candidates comprises a first adaptor and a second adaptor complementary to a respective capture primer, and optionally each of the aptamer candidates further comprises a first spacer disposed between the first adaptor and a region that is a candidate selective for the target, and a second spacer disposed between the second adaptor and a region that is a candidate selective for the target.

11. The method of any one of claims 1 to 10, wherein each of the aptamer candidates comprises an oligonucleotide.

12. A system for selecting an aptamer from a plurality of aptamer candidates, comprising: a substrate including a plurality of wells; a target bound in each of said wells; a fluid comprising a plurality of aptamer candidates and in contact with each of said wells, wherein any aptamer candidate that is selective for said target is bound to said target; and a detection circuit for sequencing any aptamer candidates in the wells to identify any aptamers that are selective for the target; A system comprising:

13. a first portion is bound within each of said wells; a plurality of second moieties are bound to respective ones of said targets; the second portion is bound to the first portion in each of the wells so as to bind the target in each of the wells; The system of claim 12.

14. (i) the first portion comprises streptavidin and the second portion comprises biotin; and / or 14. The system of claim 13, wherein (ii) the first portion is bound in each of the wells by a capture primer, and optionally the first portion is detachable from the capture primer. (i) the plurality of wells are disposed on the detection circuit; or (ii) the plurality of wells comprises flow cells through which the fluids flow in parallel; or (iii) any candidate aptamer that is selective for said target has a tertiary structure that is altered upon binding to said target; or (iv) A system according to any one of claims 12 to 14, which is a combination of any two or three of (i), (ii) and (iii).

16. The system of any one of claims 12 to 15, wherein amplicons are generated from any aptamer candidate that is selective for said target.

17. The amplicons of any candidate aptamers selective for the target are separating the aptamer candidates from the target; generating amplicons using the candidate aptamers using polymerase chain reaction (PCR); and optionally, said system comprises an amplification chamber separate from said wells for performing said PCR.

18. a fluid containing said amplicons and contacting each of said wells, wherein any amplicons selective for said target are bound to said target; The system according to any one of claims 16 to 17, further comprising:

19. 20. The system of claim 18, wherein any aptamer candidates that are sequenced include any amplicons that remain after removing any amplicons that are not bound to the target, and optionally, the method further comprises additional amplicons of any amplicons that are selective for the target.

20. a capture primer disposed within the well and bound to any candidate aptamers remaining after the removal; an amplicon of the candidate aptamer bound to the capture primer; Further comprising: the detection circuit is for sequencing the amplicon bound to the capture primer; A system according to any one of claims 12 to 19. (i) the target is bound in the well via a respective one of the capture primers, optionally wherein the capture primer binds to a first portion in the well and a second portion binds to the first portion and to the target in the well; or (ii) the capture primer is bound to the well separately from the target; 21. The system of claim 20, wherein (iii) each of the aptamer candidates comprises a first adaptor and a second adaptor complementary to a respective capture primer, and optionally each of the aptamer candidates further comprises a first spacer disposed between the first adaptor and a region that is a candidate selective for the target, and a second spacer disposed between the second adaptor and a region that is a candidate selective for the target.

22. The system of any one of claims 12 to 21, wherein each of the aptamer candidates comprises an oligonucleotide.

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