Methods and systems for selecting aptamers from aptamer candidates

Abstract

This invention provides a method and system for selecting an aptamer from a plurality of candidate aptamers. A target is coupled to each of a plurality of wells disposed in a substrate. Each well is brought into contact with a fluid containing the plurality of candidate aptamers. In at least one well, any candidate aptamer selective for the target is coupled to the target. Any candidate aptamers not coupled to the target are removed. In the wells, any candidate aptamers remaining after removal are sequenced to identify any aptamer selective for the target.

Description

Technical Field

[0001] This invention relates to a method and system for selecting aptamers. Prior Technology

[0002] The detection of specific nucleic acid sequences present in biological samples has been used, among other things, as a method for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying cancer-related genetic changes, studying genetic susceptibility to diseases, and determining responses to various types of treatments. The nucleic acid sequence that binds to a specific target molecule is called an "aptamer" and can be synthetic or derived from a biological sample. A common technique used to detect specific nucleic acid sequences (whether synthetic or derived from a biological sample) is nucleic acid sequencing.

[0003] Nucleic acid sequencing methods have evolved from the chemical degradation methods used by Maxam and Gilbert and the strand elongation methods used by Sanger. Several sequencing methods are now in use that allow for the parallel processing of millions or even billions of nucleic acids on a single flow cell. Some platforms include bead-based and microarray-based forms, where silica beads are probe-functionalized, depending on the application (including sequencing, genotyping, or gene expression mapping). Some sequencing systems (whether for sequencing-by-synthesis or for genotyping) use substrates, including multiple different reservoirs carrying different reagents for sequencing operations. Summary of the Invention

[0004] The examples provided herein relate to the use of ordered selection of adaptors. Apparatus and methods for performing such selection are disclosed.

[0005] Some examples of this document provide a method for selecting an aptamer from a plurality of candidate aptamers. The method may include coupling a target to each of a plurality of wells disposed in a substrate. The method may include contacting each of the wells with a fluid containing the plurality of candidate aptamers. The method may include coupling any candidate aptamer selective to the target to the target in at least one of the wells. The method may include removing any candidate aptamers not coupled to the target. The method may include sequencing any candidate aptamers remaining in the wells after removal to identify any aptamer selective to the target.

[0006] In some instances, coupling a target to each of a plurality of traps includes coupling a first portion to each of those traps; coupling a plurality of second portions to each of the targets; and coupling the second portions to each of the traps of the first portion. In some instances, the first portion comprises streptavidin and the second portion comprises biotin. In some instances, the first portion is coupled to each of the traps by a capture primer. In some instances, the first portion is separated from the capture primer prior to sequencing.

[0007] In some instances, the substrate includes detection circuitry for sequencing candidate aptamers that remain after removal.

[0008] In some instances, the plurality of traps comprises flow pools through which fluid flows in parallel.

[0009] In some instances, any candidate aptamer that is selective for the target has a tertiary structure that changes when it becomes coupled to the target.

[0010] In some instances, the method further includes generating an amplicon of any candidate aptamer selective to the target. In some instances, generating an amplicon of any candidate aptamer selective to the target includes: uncoupling the candidate aptamer from the target; and using the candidate aptamer to generate an amplicon using polymerase chain reaction (PCR). In some instances, PCR is performed in an amplification chamber different from the traps. In some instances, the method further includes contacting each of the traps with a fluid containing amplicons; coupling any target-selective amplicon in each of the traps to the target; and removing any amplicon not coupled to the target. In some instances, any sequenced candidate aptamer includes any amplicon retained after removal of any amplicon not coupled to the target. In some instances, the method further includes generating additional amplicons of any target-selective amplicon.

[0011] In some instances, sequencing any candidate aptamer includes: coupling any candidate aptamer retained after removal to a capture primer configured in a trap; performing amplification in the traps to generate an amplicon coupled to the capture primer; and sequencing the amplicon coupled to the capture primer. In some instances, the target is coupled to the trap via individual capture primers in the capture primer. In some instances, the capture primer couples a first portion to the trap and a second portion to the first portion and then to the target in the trap. In some instances, the capture primer is coupled to the trap separately from the target. In some instances, each of the candidate aptamers includes a first and a second aptamer complementary to the respective capture primer. In some instances, each of the candidate aptamers further includes a first spacer configured between the first aptamer and a region of a candidate to be selective for the target, and a second spacer configured between the second aptamer and a region of a candidate to be selective for the target.

[0012] In some instances, each of the candidate aptamers contains an oligonucleotide.

[0013] Some examples of this document provide a system for selecting an aptamer from a plurality of candidate aptamers. The system may include a substrate comprising a plurality of wells. The system may include a target coupled to each of the wells. The system may include a fluid containing the plurality of candidate aptamers and contacting each of the wells, wherein any candidate aptamer selective to the target becomes coupled to the target. The system may include detection circuitry that orders any candidate aptamers in the wells to identify any aptamer selective to the target.

[0014] In some instances, the first portion is coupled to each of the traps. In some instances, a plurality of second portions are coupled to individual targets of a target. In some instances, the second portion is coupled to the first portion in each of the traps so that the target is coupled to each of the traps. In some instances, the first portion contains streptavidin and the second portion contains biotin. In some instances, the first portion is coupled to each of the traps by a capture primer. In some instances, the first portion is separable from the capture primer.

[0015] In some instances, the plurality of traps are configured on the detection circuit.

[0016] In some instances, the plurality of traps comprises flow pools through which fluid flows in parallel.

[0017] In some instances, any candidate aptamer that is selective for the target has a tertiary structure that changes when it becomes coupled to the target.

[0018] In some instances, amplicons of any candidate aptamer selective to the target are generated. In some instances, amplicons of any candidate aptamer selective to the target are generated by the following steps: uncoupling the candidate aptamer from the target; and using the candidate aptamer to generate amplicons via polymerase chain reaction (PCR). In some instances, the system includes an amplification chamber different from the trap for performing PCR.

[0019] In some instances, the system further includes a fluid containing amplicons and in contact with each of the traps, wherein any amplicon selective for the target becomes coupled to the target. In some instances, any sequenced candidate aptamer includes any amplicons retained after removal of any amplicons not coupled to the target. In some instances, the system further includes additional amplicons of any amplicon selective for the target.

[0020] In some instances, the system further includes a capture primer configured in a trap and coupled to any candidate aptamers retained after removal; and an amplicon coupled to the candidate aptamers of the capture primer. Detection circuitry can sequence the amplicon coupled to the capture primer. In some instances, the target is coupled to the trap via individual capture primers in the capture primer. In some instances, the capture primer couples a first portion to the traps and a second portion to the first portion and then to the target in the traps. In some instances, the capture primer is coupled to the trap separately from the target. In some instances, each of the candidate aptamers includes a first and a second aptamer complementary to the respective capture primer. In some instances, each of the candidate aptamers further includes a first spacer configured between the first aptamer and a region of a candidate to be selective for the target, and a second spacer configured between the second aptamer and a region of a candidate to be selective for the target.

[0021] In some instances, each of the candidate aptamers contains an oligonucleotide.

[0022] It should be understood that any individual feature / instance of the various forms disclosed herein may be implemented together in any suitable combination, and any feature / instance from any one or more of these forms may be implemented together with any feature of other forms as described herein in any suitable combination to achieve the benefits described herein. Simple Explanation of the Diagram

[0023] Figures 1A to 1I schematically illustrate example equipment and operations for using a sequenced selection process for adaptors.

[0024] Figures 2A to 2F schematically illustrate example equipment and operations for an alternative process of selecting adaptors using a sequence.

[0025] Figure 3 schematically illustrates example candidate fits for devices or processes described with reference to Figures 1A-1I or 2A-2F.

[0026] Figure 4 schematically illustrates an example operation in the process of selecting a fitness model using a sequence. Implementation

[0027] Cross-reference to related applications

[0028] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 128,669, filed December 21, 2020, entitled “Selecting Aptamers Using Sequencing,” the entire contents of which are incorporated herein by reference.

[0029] The examples provided herein relate to the use of ordered selection of adaptors. Apparatus and methods for performing such selection are disclosed.

[0030] Some aptamers are oligonucleotide strands that bind selectively to target molecules such as enzymes, antibodies, single cells, or any other molecular target of interest. During this selective binding, the aptamer can achieve tertiary structure. SELEX (or Systematic Evolution of Ligands by Exponential Enrichment) is a procedure for selecting aptamers. Previously known SELEX procedures were typically performed on a conventional solid substrate (such as a column containing a target-coupled medium). A fluid containing candidate aptamers was passed through the substrate. Any aptamer selective for the target became coupled to the target, while other aptamers did not become coupled and thus flowed out of the substrate. The target-selective aptamer was then uncoupled from the target and amplified, for example, in a 96-well plate using polymerase chain reaction (PCR). The PCR product could again flow through the substrate and be amplified to provide further enrichment. The amplified aptamers were then sequenced to identify the aptamer that became coupled to the target and was therefore the most "successful". These procedures can be time- and labor-intensive.

[0031] In comparison, as provided herein, this apparatus and method can streamline and simplify the SELEX procedure by utilizing sequencing. More specifically, in some instances, the operations for selecting and sequencing aptamers can be performed in the same traps, such as traps on a substrate within a sequencing system. The substrate on which the selection procedure is performed may include detection circuitry for sequencing aptamers. Inventively, a target can be coupled to each of these traps, and multiple aptamers can flow into each of these traps. Any aptamer selective to the target can become coupled to the target, and any other aptamer can be removed. Target-selective aptamers can be amplified and then sequenced in the traps, for example, using capture initiators also coupled to each of the traps. Therefore, this apparatus and method can provide a significantly streamlined and simplified procedure with a substantial reduction in material use and waste compared to previously known SELEX procedures.

[0032] First, some of the terminology used in this paper will be briefly explained. Then, some example methods and related devices for utilizing ordered selection of aptamers will be described. [, the term , ] [, , ]

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The use of the term "including" and other forms (such as "include," "includes," and "included") is non-limiting. The use of the term "having" and other forms (such as "have," "has," and "had") is non-limiting. As used herein, whether in transitional phrases or in the body of a technical solution, the terms "comprise(s)" and "comprising" should be interpreted as having an open meaning. That is, these terms should be interpreted synonymously with the phrases "having at least" or "including at least." For example, when used in the context of a procedure, the term "comprising" means that the procedure includes at least the cited 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 cited features or components, but may also include additional features or components.

[0034] The terms “substantially,” “approximately,” and “about” used throughout this specification are used to describe and indicate small fluctuations, such as those due to variations in the processing. For example, they may refer to less than or equal to ±10%, such as less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%.

[0035] As used herein, "hybridization" refers to the non-covalent binding of a first polynucleotide to a second polynucleotide along the length of the two polymers to form a double-stranded "duplex." For example, two DNA polynucleotide strands can bind through complementary base pairing. The strength of the binding between the first and second polynucleotides increases with the complementarity of the nucleotide sequences in the polynucleotides. The strength of the hybridization between polynucleotides can be characterized by the melting temperature (Tm) at which 50% of the duplexes dissociate from each other.

[0036] As used herein, the term "nucleotide" is intended to refer to a molecule that contains a sugar and at least one phosphate ester group, and in some instances also contains a nucleoside base. A nucleotide lacking a nucleoside base may be called "base-free." Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate ester 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), and deoxyadenosine monophosphate (dAMP). Deoxyadenosine 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).

[0037] As used herein, the term "nucleotide" is also intended to encompass any nucleotide analogues, which are a class of nucleotides that, compared to naturally occurring nucleotides, contain modified nucleoside bases, sugars, and / or phosphate esters. Examples of modified nucleoside bases include inosine, xanthine, hypoxanthine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymidine, 2-thiocytosine, 15-halogenuridine, 5-propynyluridine, 6-azouracil, and 6-azocytosine. 6-Azothymidine, 5-uracil, 4-thiouracil, 8-halogenated adenine or guanine, 8-aminoadenine or guanine, 8-thiol-adenine or guanine, 8-thioalkyladenine or guanine, 8-hydroxyadenine or guanine, 5-halogenated uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, or similar compounds. As known in the art, certain nucleotide analogs cannot be incorporated into polynucleotides (e.g., nucleotide analogs, such as adenosine 5'-phosphate sulfate). Nucleotides may contain any suitable number of phosphate groups, such as three, four, five, six, or more than six phosphate groups.

[0038] As used herein, the term "polynucleotide" refers to a molecule containing a sequence of nucleotides bound together. A polynucleotide is a non-limiting example of a polymer. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and analogues. Polynucleotides can be single-stranded sequences of nucleotides (such as RNA or single-stranded DNA), double-stranded sequences of nucleotides (such as double-stranded DNA), or mixtures of single-stranded and double-stranded sequences of nucleotides. Double-stranded DNA (dsDNA) contains genomic DNA and PCR and amplification products. Single-stranded DNA (ssDNA) can be converted to dsDNA and vice versa. Polynucleotides can contain DNA that is not naturally occurring, such as enantiomeric DNA. The exact sequence of nucleotides in a polynucleotide can be known or unknown. Examples of polynucleotides include: genes or gene fragments (e.g., probes, primers, expressed sequence tags (ESTs), or serial analysis of gene expression (SAGE) tags), genomic DNA, genomic DNA fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, synthetic polynucleotides, branched-chain polynucleotides, plastids, vectors, any sequence of isolated DNA, any sequence of isolated RNA, nucleic acid probes, primers, or any of the aforementioned amplified copies.

[0039] As used herein, "polymerase" refers to an enzyme with an active site that assembles polynucleotides by polymerizing nucleotides into polynucleotides. A polymerase can bind to an initiated single-stranded target polynucleotide and sequentially add nucleotides to a growth primer to form a "complementary copy" polynucleotide with a sequence complementary to the target polynucleotide. Another polymerase (or the same polymerase) can then form a copy of the target nucleotide by forming a complementary copy of that complementary copy polynucleotide. Any such copy may be referred to herein as an "amplifier". DNA polymerases bind to a target polynucleotide and then sequentially move the target polynucleotide down to add nucleotides to the free hydroxyl group at the 3' end of the growth polynucleotide strand (growth amplicon). DNA polymerases synthesize complementary DNA molecules from a DNA template, and RNA polymerases synthesize RNA molecules from a DNA template (transcription). Polymerases can use short RNA or DNA strands (primers) to initiate strand growth. Some polymerases can replace the strand upstream of the site where they add bases to the chain. This type of polymerase can be called a strand substitution polymerase, meaning it has the activity of removing a complementary strand from the template strand read by the polymerase. Examples of polymerases with strand substitution activity include (but are not limited to) large fragments of Bst (Bacillus stearothermophilus) polymerase, exokinetic Klenow polymerase, or sequencing-level T7 exokinetic polymerase. Some polymerases degrade the preceding strand, effectively replacing it with the following growth strand (5' exonuclease activity). Some polymerases have the activity of degrading the following strand (3' exonuclease activity). Some useful polymerases have been modified by mutation or other means to reduce or eliminate 3' and / or 5' exonuclease activity.

[0040] As used herein, the term "introducer" refers to a polynucleotide to which a nucleotide can be added via a free 3' OH group. Introducers can be of any suitable number of bases in length and can contain any suitable combination of natural and non-natural nucleotides. Target polynucleotides (such as (but not limited to) aptamers) may contain "aptamers" that hybridize with the introducer (having a sequence complementary to the introducer) and can be amplified to produce complementary copy polynucleotides by adding a nucleotide to the free 3' OH group of the introducer. Introducers can be coupled to a substrate. "Complementary" introducers hybridize with each other along their substantially full length, while "orthogonal" introducers do not substantially hybridize with each other, and their amplicon do not hybridize with each other. "Capture introducer" is intended to refer to an introducer coupled to a substrate and capable of hybridizing with the first aptamer of the target polynucleotide, while "orthogonal capture introducer" is intended to refer to an introducer coupled to a substrate and capable of hybridizing with the second aptamer of the target polynucleotide. The first aptamer may have a sequence complementary to the sequence of the capture primer, and the second aptamer may have a sequence complementary to the sequence of the orthogonal capture primer. The capture primer and the orthogonal capture primer may have different and independent sequences.

[0041] In some instances, the capturing primers are P5 or P7 primers available from Illumina, Inc. P5 and P7 primers are non-limiting examples of mutually orthogonal primers. In some instances, the P5 and P7 primer sequences may have the following sequences: [Paired read group:] [] P5:5'-AATGATACGGCGACCACCGAGAUCTACAC-3' (SEQ ID NO:1) P7:5'-CAAGCAGAAGACGGCATACGAG*AT-3' (SEQ ID NO:2) [one] [one] [Reading group:] [] P5:5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO:3) P7:5'-CAAGCAGAAGACGGCATACGA3' (SEQ ID NO:4) Where G* is G or 8-side-oxyguanine.

[0042] In some instances, the attached oligonucleotide (such as a primer or a P5 or P7 primer) contains 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 other desired properties, such as enabling covalent attachment to a polymer or support, or to act as a spacer to position the cleavage site at the optimal distance from the support. In some cases, 10 spacer nucleotides may be positioned between the attachment sites of the P5 or P7 primer to the polymer or support. In some instances, polyT spacers are used; however, other nucleotides and combinations thereof may also be used. In one instance, the spacer is a 6T to 10T spacer. In some instances, the linker contains a cleavable nucleotide containing a chemically cleavable functional group, such as vicinal diol or allyl T.

[0043] As used herein, the term "amplifier," when referring to a polynucleotide, is intended to mean the product of replicating the polynucleotide, wherein the product has a nucleotide sequence that is substantially identical or substantially complementary to at least a portion of the nucleotide sequence of the polynucleotide. "Amplification" and "amplifying" refer to the process of preparing an amplifier of a polynucleotide. The first amplifier of the target polynucleotide may be a complementary copy. Subsequent amplifiers are copies created from the target polynucleotide or from the first amplifier after its generation. Subsequent amplifiers may have a sequence substantially complementary to or substantially identical to the target polynucleotide. It should be understood that a few mutations in the polynucleotide (e.g., due to the amplification of an artifact) may occur during the generation of the amplifier of that polynucleotide.

[0044] As used herein, the term "substrate" refers to the material used as a support for the compositions described herein. Examples of substrate materials may include glass, silicon dioxide, plastic, quartz, metal, metal oxide, organosilicone (e.g., polyhedral organosilicon silsesquioxane (POSS)), polyacrylate, tantalum oxide, complementary metal-oxide-semiconductor (CMOS), or combinations thereof. An example of a POSS may be the one described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, which is incorporated herein by reference in its entirety. In some instances, the substrate used in this application comprises a silicon dioxide-based substrate, such as glass, fused silicon dioxide, or other silicon dioxide-containing materials. In some instances, the substrate may comprise silicon, silicon nitride, or silicon hydride. In some instances, the substrate used in this application comprises 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 instances, the substrate is or comprises a silicon dioxide-based material or a plastic material or a combination thereof. In certain instances, the substrate has at least one surface comprising a glass or silicon-based polymer. In some instances, the substrate may comprise a metal. In some such instances, the metal is gold. In some instances, the substrate has at least one surface comprising a metal oxide. In one instance, the surface comprises tantalum oxide or tin oxide. Acrylamide, ketones, or acrylates may also be used as substrate materials or components. Other substrate materials may include, but are not limited to, gallium arsenide, indium phosphide, aluminum, ceramics, polyimide, quartz, resins, polymers, and copolymers. In some instances, the substrate and / or the substrate surface may be or comprise quartz. In some other instances, the substrate and / or substrate surface may be or contain semiconductors, such as GaAs or ITO. The foregoing list is intended to illustrate, but is not limited to, the present application. The substrate may contain a single material or a plurality of different materials. The substrate may be a composite or laminate. In some instances, the substrate contains an organosilicone material. The substrate may be flat, circular, spherical, rod-shaped, or any other suitable shape. The substrate may be rigid or flexible. In some instances, the substrate is a bead or a flow cell.

[0045] In some instances, the substrate includes a patterned surface. A "patterned surface" refers to an arrangement of different regions in or above an exposed layer of the substrate. For example, one or more of these regions may be features containing one or more trapping leads. These features can be separated by gap regions where trapping leads are absent. In some instances, the pattern may be an xy-form of features in columns and rows. In some instances, the pattern may be a repeating arrangement of features and / or gap regions. In some instances, the pattern may be a random arrangement of features and / or gap regions. In some instances, the substrate includes an array of wells (recesses) in the surface. These wells may be provided by substantially vertical sidewalls. The wells can be manufactured using various techniques generally known in the art, including but not limited to lithography, stamping, molding, and micro-etching. As will be understood by those skilled in the art, the technique used will depend on the composition and shape of the array substrate.

[0046] Features in the patterned surface of a substrate may include traps in an array of traps (e.g., micro-traps or nano-traps) on a patterned, covalently linked gel (such as poly(N-(5-azidoacetylammonopentyl)acrylamide-copolymer-acrylamide) (PAZAM)) on glass, silicone, plastic, or other suitable materials. This process establishes a gel pad for sequencing, which can be stabilized during sequencing operations with a large number of cycles. The covalent bonding of the polymer to the traps helps maintain the gel in the structured features throughout the entire lifetime of the structured substrate during various uses. However, in many instances, the gel does not need to be covalently bonded to the traps. For example, under certain conditions, silane-free acrylamide (SFA) not covalently attached to any part of the structured substrate can be used as the gel material.

[0047] In specific instances, structured substrates can be fabricated by patterning suitable materials with traps (e.g., micro-traps or nano-traps), coating the patterned material with a gel material (e.g., PAZAM, SFA, or chemically modified variants thereof, such as an azide-based variant of SFA (azido-SFA)), and polishing the surface of the gel-coated material, for example, by chemical or mechanical polishing. This retains the gel within the traps but removes or deactivates substantially all of the gel from the interstitial regions on the surface of the structured substrate between the traps. Primers can be attached to the gel material. A solution containing multiple target polynucleotides (e.g., fragments of the human genome or portions thereof) can then be contacted with the polished substrate, such that individual target polynucleotides are seeded into individual traps via interaction with primers attached to the gel material; however, the target polynucleotides will not occupy interstitial regions due to the absence or inactivity of the gel material. The amplification of the target polynucleotides can be confined to the traps because the absence or inactivity of the gel in the interstitial regions inhibits the outward migration of growth clusters. The procedure is easy to manufacture, scalable, and utilizes known microfabrication or nanofabrication methods.

[0048] Patterned substrates can include, for example, wells etched into a glass slide or wafer. The patterning of the etched geometry of the wells can take various shapes and sizes, and this feature can be physically or functionally separable. Particularly useful substrates with this structural feature include patterned substrates with selectable sizes of solid particles (such as microspheres). One example of a patterned substrate with these characteristics is an etched substrate used in conjunction with BEAD ARRAY technology (Illumina, Inc., San Diego, Calif.).

[0049] In some instances, a substrate forms at least a portion of a flow cell, is positioned within the flow cell, or is coupled to the flow cell. The flow cell may comprise flow chambers divided into a plurality of lanes or sectors. Examples of flow cells and substrates used in the methods and systems described herein, including but not limited to those purchased from Illumina, Inc. (San Diego, CA), may also be used in the fabrication of the flow cells.

[0050] As used herein, the term "complex" is intended to refer to a group of two or more distinct members. Complexity can range in size from small, medium, large to extremely large. Small complexities can range in size from, for example, a few members to tens of members. Medium-sized complexities can range in size from, for example, tens of members to about 100 members or hundreds of members. Large complexities can range in size from, for example, about hundreds of members to about 1000 members, to thousands of members, and up to tens of thousands of members. Extremely large complexities can range in size from, for example, tens of thousands of members to hundreds of thousands, millions, millions, tens of millions, and up to or more than hundreds of millions of members. Therefore, complexity can range in size from two to over one hundred million members, and all sizes (as determined by the number of members) are between and greater than the ranges in the examples above. Example polynucleotide complexities include, for example, groups of about 1 × 10⁵ or more, 5 × 10⁵ or more, or 1 × 10⁶ or more distinct polynucleotides. Therefore, the definition of this term is intended to include all integer values ​​greater than 2. The upper limit of the complex number can be set, for example, by the theoretical diversity of polynucleotide sequences in the sample.

[0051] The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein. Unless otherwise specifically indicated, the different terms are not intended to indicate any particular difference in size, sequence, or other properties. For clarity of description, the terms may be used to distinguish one polynucleotide species from another when describing a particular method or composition involving several polynucleotide species.

[0052] As used herein, the term "sequencing system" refers to a system structurally designed to determine the sequence of polynucleotides. A variety of sequencing systems are commercially available. Exemplarily, a sequencing system may be or include the iSEQ™ 100 sequencing system, available from Illumina, Inc. (San Diego, CA). The iSEQ™ 100 sequencing system is a mesa-type system for synthetic sequencing using a pre-filled cartridge containing reservoirs of various 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.

[0053] As used herein, "aptamer" refers to an oligonucleotide having a tertiary structure that causes the oligonucleotide to be selective for a target, while "candidate aptamer" refers to an oligonucleotide that is potentially selective for a target. "Selectivity" for a target means coupling to that target rather than coupling to a different target. Therefore, among a given plurality of candidate aptamers, one or more candidate aptamers are potentially selective for a given target and are therefore aptamers for that target. However, it should be understood that any given plurality of candidate aptamers does not necessarily include an aptamer for a given target. A candidate aptamer can be defined as an aptamer that is selective for a target using operations involving coupling the candidate aptamer to the target (indicating selectivity) and sequencing the candidate aptamer. The amplicon of a candidate aptamer can be itself a candidate aptamer, whether the amplicon is a copy of the candidate aptamer or a complementary copy. The amplicon of an aptamer may be an aptamer itself, for example, in which the amplicon is a copy of the aptamer (as opposed to a complementary copy of the aptamer, which may be a candidate aptamer but not necessarily an aptamer). The aptamer may contain any suitable type of oligonucleotide, such as DNA, RNA, and / or nucleic acid analogs as exemplified elsewhere herein. The aptamer may become coupled to a target through any suitable combination of interactions, such as electrostatic interactions, hydrophobic interactions, and any suitable combination of tertiary structure formation.

[0054] As used herein, "target" is intended to refer to a chemical element for which an aptamer is desired. A target may include chemical entities that are not nucleotides. One example of a target is a protein target. A protein comprises a series of polypeptides folded into a structure. Another example of a target is a metabolite target. A metabolite target is a chemical element that is formed or used during metabolism. Other examples of targets include, but are not limited to, carbohydrates, fatty acids, sugars (such as glucose), amino acids, nucleosides, neurotransmitters, phospholipids, and heavy metals. In this disclosure, analytes may be selected against targets for any suitable application, such as analyzing disease states, analyzing metabolic health, analyzing the microbiome, analyzing drug interactions, analyzing drug responses, analyzing toxicity, or analyzing infectious diseases. By way of example, metabolites may contain chemical elements that are upregulated or downregulated in response to disease. Non-limiting examples of targets include kinases, serine hydrolases, metalloproteinases, disease-specific biomarkers (such as antigens of specific diseases), and glucose.

[0055] As used herein, an oligonucleotide with a “tertiary structure” is intended to refer to an oligonucleotide that has folded into a three-dimensional tertiary structure with internal crosslinks that hold the fold in place. In contrast, oligonucleotides with a primary structure (e.g., a specific sequence of linked nucleic acids) and a secondary structure (e.g., a local structure) but without internal crosslinks that hold the fold in place will not be considered to have a tertiary structure as used herein. The tertiary structure of an aptamer can change when it becomes coupled to a target.

[0056] As used herein, the term "dissimilar" means that one element has at least one variation relative to another, making the elements distinguishable from each other. For example, dissimilar oligonucleotides may have nucleic acid sequences that vary by at least one nucleic acid relative to each other. As another example, dissimilar proteins may have peptide sequences that vary by at least one peptide relative to each other. As another example, metabolites may vary by at least one chemical group relative to each other. As provided herein, this device and method can be used to select different aptamers for different targets.

[0057] As used herein, the term "luciferoid" is intended to refer to a molecule that emits light of the first wavelength when excited by a second wavelength of light, which is different from the first wavelength. The light emitted by a luciferoid can be called "fluorescence" and can be detected by appropriate optical and electrical detection methods.

[0058] Fluorescence can be detected using any suitable optical detection circuit, which may include an optical detector that generates an electrical signal based on light received from the phosphor, and electronic circuitry that uses this electrical signal to determine the light received from the phosphor. As an example, the optical detector may include an active-pixel sensor (APS), comprising an array of amplified photodetectors structurally designed to generate an electrical signal based on light received by the photodetector. APSs may be based on complementary metal-oxide-semiconductor (CMOS) technology known in this art. CMOS-based detectors may include field-effect transistors (FETs), such as metal-oxide-semiconductor field-effect transistors (MOSFETs). In a particular instance, a CMOS imager with a single-photon breakdown diode (CMOS-SPAD) may be used, for example, for fluorescence lifetime imaging (FLIM). In other instances, the optical detector may include photodiodes, such as collapsible photodiodes, charge-coupled devices (CCDs), cryogenic photon detectors, reverse-biased light-emitting diodes (LEDs), photoresistors, photocells, photovoltaic cells, photomultiplier tubes (PMTs), quantum dot photoconductors, or the like. The optical detection circuitry may further include any suitable combination of hardware and software operatively connected to the optical detector to receive electrical signals from it and be structurally designed to detect fluorescence based on these signals, for example, based on an optical detector detecting light from a phosphorescent cluster. For example, the electronic circuitry may include memory and a processor coupled to the memory. The memory may store instructions for inducing the processor to receive signals from the optical detector and use these signals to detect a phosphorescent cluster. For example, the instructions may cause the processor to use the signals from the optical detector to determine that fluorescence is emitted within the field of view of the optical detector and use this determination to confirm the presence of a phosphorescent cluster. The substrate may include optical detection circuitry, for example, it may include one or more optical detectors configured thereon for using a trap for sequentially selecting aptamers. [, Using ordinal selection of aptamers , ] [, , ]

[0059] As described above and in more detail below, this device and method can be used to select aptamers using sequencing. A target can be coupled to each of a plurality of traps, where aptamer selection and sequencing will be performed. A plurality of candidate aptamers (e.g., a SELEX aptamer library) can be introduced into the traps to bring the target into contact with the candidate aptamers, and any candidate aptamer that is selective to the target becomes coupled to the target while remaining uncoupled and washable. The candidate aptamers that become coupled to the target can be amplified and sequenced in the traps to identify aptamers selective to the target.

[0060] For example, Figures 1A to 1I schematically illustrate example apparatus and operations for a process of selecting adaptors using a sequence. The apparatus 100 illustrated in Figure 1A may include a substrate 110 comprising a plurality of reservoirs. In a non-limiting example illustrated in Figure 1A, the reservoirs may comprise wells 121, 122, 123, and 124, which are provided together in a common, integrally formed substrate 110. However, it should be understood that one or more of wells 121, 122, 123, and 124, and indeed all wells 121, 122, 123, and 124, may be physically separable from each other and do not need to be formed together in a common substrate. Any suitable number of wells with any suitable size and arrangement can be provided. For example, substrate 110 may contain thousands, tens of thousands, hundreds of thousands, or even millions of wells. Each of traps 121, 122, 123, and 124 may contain a plurality of first and second capture primers 111, 112, which can be used to amplify and sequence candidate aptamers in a manner further described below with reference to Figures 1H to 1I. Device 100 may further include detection circuitry 190 to sequence any candidate aptamers in the traps and identify any aptamer selective for a target, for example, in a manner described with reference to Figure 1I. Trapes 121, 122, 123, and 124 may be configured on or otherwise coupled to detection circuitry 190. In a non-limiting example, detection circuitry 190 includes a CMOS optical detector, traps 121, 122, 123, and 124 include flow cells through which fluid can flow in parallel, and the CMOS detects fluorescence during the sequencing of oligonucleotides in the flow cells.

[0061] Examplely, device 100 can be used in a sequencing system to utilize sequencing to select aptamers. Examplely, the sequencing system may be or include the iSEQ™ 100 sequencing system, available from Illumina, Inc. (San Diego, CA). The iSEQ™ 100 sequencing system is a benchtop system that uses a pre-filled cartridge containing reservoirs storing different sequencing reagents to perform synthetic sequencing in a manner similar to device 100 illustrated in Figures 1A-1B. However, it should be understood that device 100 is suitably compatible with any other sequencing system, such as the cBot 2, NovaSeq 6000, or MiniSeq systems available from Illumina, Inc., or sequencing systems from another source.

[0062] Targets (which may require finding aptamers) may be coupled to each of the wells 121, 122, 123, and 124. Non-limiting examples of targets are provided elsewhere herein. Targets may be directly coupled via any suitable connection, such as via a trapping initiator, as further described below with reference to Figures 2A to 2F. Alternatively, targets may be indirectly coupled via interacting portions. For example, as illustrated in Figure 1A, a first portion 130 may be coupled to each of the wells 121, 122, 123, and 124, for example, via any suitable connection. Wells 121, 122, 123, and 124 may be contacted with a fluid containing a plurality of molecules 140, each of which may contain a second portion 150 and a target 160 coupled to each other via any suitable connection, which may be cleaved as needed. The individual connections between the first part 130 and traps 121, 122, 123, and 124, and between the second part 150 and target 160, may be formed via any suitable interaction (such as NTA-His-Tag, Spytag-Spycatcher, oligonucleotide hybridization to complementary oligonucleotides, copper(I)-catalyzed click reactions, or strain-promoted azide-alkyne cycloaddition). Any suitable number of such connections may contain cleavable moieties, such as 8-side oxygen-G, or proteins that can be cleaved using a protease, which may be used to decouple one or more elements from the substrate in a manner as described in more detail below.

[0063] In a manner as illustrated in FIG1B, the second portion 150 may be coupled to the first portion 130 to couple the target 160 to each of the traps 121, 122, 123, and 124. Any suitable portion may be provided for the first and second portions 130 and 150 to interact covalently or non-covalently with each other. In one non-limiting example, the first portion 130 comprises streptavidin and the second portion 150 comprises biotin. In another non-limiting example, the first portion 130 comprises biotin and the second portion 150 comprises streptavidin. Alternatively, target 160 may be coupled to each of traps 121, 122, 123, and 124 via any suitable interaction, such as NTA-His-Tag, Spytag-Spycatcher, oligonucleotide hybridization to complementary oligonucleotide, copper(I)-catalyzed click reaction, or strain-promoted azide-alkyne cycloaddition, including, as needed, a cleavable moiety, such as 8-side-oxy-G, or a protein that can be cleaved by a protease. Although for simplicity, a single first portion 130, a single second portion 150, and a single target 160 are illustrated in each of these traps, it should be understood that any suitable number of first and second portions and targets may be coupled to each of these traps, and each trap may have the same or different numbers of portions or targets coupled thereto. In an example such as that shown in Figure 1B, the trapping initiators 111 and 112 can be coupled from the target 160 to the traps 121, 122, 123, and 124, respectively.

[0064] As illustrated in FIG1C, system 100 may include a fluid comprising a plurality of candidate aptamers 171, 172, 173, 174 and contacting traps 121, 122, 123, 124. Each of candidate aptamers 171, 172, 173, 174 may include aptamers 181, 182, via which the candidate aptamers may be coupled to capture primers 111 or 112 for amplification as described below with reference to FIG1H. Any candidate aptamer that is selective to the target becomes coupled to target 160. For example, as illustrated in Figure 1D, candidate aptamer 174 is coupled to target 160 in trap 124, while candidate aptamers 171, 172, and 173 remain uncoupled to target 160 in traps 121, 122, and 123 and can be removed, for example, by allowing buffer solution to flow through traps 121, 122, 123, and 124, as illustrated in Figure 1E. In some instances, any candidate aptamer selective for target 160 has a tertiary structure that changes when becoming coupled to the target. As shown in Figure 1D, at least some of candidate aptamers 171, 172, 173, and 174 may include a hairpin structure comprising a single-stranded loop and a single-stranded stem, which can couple to target 160 when the candidate aptamer is selective for that target.

[0065] Amplicons of any candidate aptamer that are selective for a target can be generated. For example, generating amplicons of any candidate aptamer that are selective for a target may include uncoupling the candidate aptamer from the target and using the candidate aptamer to generate amplicons via PCR. For example, candidate aptamer 174, illustrated in Figure 1E, may be uncoupled from target 160 and subjected to PCR to generate amplicons of the candidate aptamer. Such uncoupling between candidate aptamer 174 and target 160 may be performed using any suitable reaction conditions that disrupt the interaction between the target and the aptamer (such as heat or buffer exchange where the salt concentration or pH becomes unfavorable to the tertiary structure of the aptamer or forces stabilizing the aptamer-target interface). In some instances, device 100 may include an amplification chamber remote from traps 121, 122, 123, and 124 for PCR. Then, in a manner as illustrated in Figure 1F, the target 160 in each of traps 121, 122, 123, and 124 is brought into fluid contact with an amplicon 174 containing a candidate aptamer 174. Note that the same reference numerals are used herein to refer to amplicon 174 and candidate aptamer 174 because they are equivalent structures; for example, amplicon 174 is also a candidate aptamer for target 160 and is expected to become coupled to it in a similar manner to candidate aptamer 174. In a manner similar to that described with reference to Figure 1D, any amplicon 174 selective for the target becomes coupled to a target, such as that shown in Figure 1G, where multiple amplicon 174 are coupled to individual targets 160. However, note that additional steps for decoupling the candidate aptamer from the target and using the candidate aptamer for PCR to generate amplicon can be omitted.

[0066] Then, device 100 can be used to sequence candidate aptamers 174 (including any combination thereof). For example, after removing candidate aptamers not coupled to target 160, any candidate aptamers that remain coupled to target 150 can be sequenced. Candidate aptamers 174 (including any amplicon thereof) illustrated in FIG. 1G can be decoupled from target 160 in a manner as described with reference to FIG. 1E, and cluster amplification can be used to generate amplicons of candidate aptamers 174, including additional amplicons that generate any amplicon selective to the target. For example, as described above with reference to FIG. 1A, capture primers 111, 112 can be configured in traps 121, 122, 123, 124. Capture primers 111 and 112 can be orthogonal to each other; for example, capture primer 111 can contain a P5 primer and capture primer 112 can contain a P7 primer. Capture primers 111 and 112 can be coupled to any candidate aptamer (including any of its amplicones) remaining after removal of any candidate aptamers (and amplicones) that have not become coupled to target 160. For example, as described above with reference to FIG1A, candidate aptamers 171, 172, 173, and 174 may include aptamers 181 and 182, which can be coupled to capture primers 111 or 112, respectively, for amplification.

[0067] As illustrated in FIG1I, aptamer 181 of candidate aptamer 174 (or its amplicons) can be coupled to capture primer 111, and aptamer 182 of candidate aptamer can be coupled to capture primer 112. Clusters of amplicons 174, 174' of candidate aptamer (or amplicons) 174 can be generated using bridge amplification or other suitable surface-based amplification procedures as illustrated in FIG1I. Note that for simplicity, the second portion 150 and target 160 are omitted in FIG1I, and either can be retained in the trap or can be removed, for example by cleaving the linker-linked target 160 to portion 130 or cleaving the linker-cleaved portion 130 to the surface. Detection circuitry 190 can be used, for example, to sequence the amplicons 174, 174' coupled to the capture primer in a manner known in this art. Examplely, polymerase can be used to extend primers 111 or 112 using labeled (e.g., fluorescently labeled) nucleotides based on the sequences of individual amplicons 174, 174', and detection circuitry 190 can identify sequences in which such nucleotides are added using signals generated by labeling those nucleotides. Sequentially ordered candidate aptamers (or their amplicons 174) can be considered aptamers for target 160, for example, because such candidate aptamers have been coupled to target 160 through multiple manipulations, indicating selectivity for the target.

[0068] As described above with reference to FIG. 1A, the target for the aptamer selection procedure can be coupled to the trap in any suitable manner, such as using a trapping initiator. For example, FIGS. 2A to 2F schematically illustrate example apparatus and operation for an alternative process of selecting aptamers using a sequence. In the example illustrated in FIG. 2A, the first portion is coupled to each of the traps by trapping initiators; for example, the first portion 230 is coupled to the substrate 210 via trapping initiator 211, and the first portion 230' is coupled to the substrate 210 via trapping initiator 212. The trapping initiators 211 and 212 can be orthogonal to each other; for example, trapping initiator 211 can include a P5 initiator and trapping initiator 212 can include a P7 initiator. The first portions 230, 230' can be contacted with the fluid containing molecule 140 (including the second portion 150 and the target 160) in a manner as described with reference to FIG. 1A. As illustrated in Figure 2B, the second portion 150 can be coupled to the first portion 230 to couple the target molecule 160 to the substrate 210 (e.g., in a trap, not specifically shown) via a trapping primer 211, while another second portion 150 can be coupled to the first portion 230' to couple the target molecule 160 to the substrate 210 (e.g., in the same trap or another trap, not specifically shown) via a trapping primer 212. As illustrated in Figure 2C, a candidate aptamer (or its amplicon) 174 selective for the target 160 can be coupled to it, for example, in a manner as described with reference to Figures 1D and 1G. As illustrated in Figure 2C, the candidate aptamer 174 can be decoupled from the target 160 for amplification in a manner as described with reference to Figure 1E.

[0069] In some instances, the first portions 230, 230' may be separable from the capture initiators 211, 212. Thus, in a manner as illustrated in FIG. 2E, the first portions 230, 230', the second portion 150, and the target 160 may be decoupled from the substrate 210 by separating the first portions 230, 230' from their respective capture initiators 211, 212. Inventively, the capture initiators 211, 212 may contain cleavable portions such as 8-side radical-G, which can be cleaved prior to sequencing to separate the first portions 230, 230' and any elements coupled thereto. For example, as illustrated in Figure 2F, candidate aptamer 174 and its amplicons 174' aptamers 181, 181' can be hybridized with individual capture primers 211, and candidate aptamers and their amplicons aptamers 182, 182' can be hybridized with individual capture primers 212 for amplification and sequencing as described with reference to Figures 1H to 1I. Alternatively, in examples such as those provided throughout this disclosure, any suitable component of target 160, linker, first portion, and second portion may contain a protein that can be digested by proteases and thus decoupled from the substrate and / or capture primer.

[0070] It should be understood that candidate aptamers that come into contact with the target can contain any suitable sequence and components. For example, Figure 3 schematically illustrates example candidate aptamers for use in devices or processes such as those described with reference to Figures 1A-1I or 2A-2F. In the non-limiting example shown in Figure 3, candidate aptamers 171, 172, 173, and 174 each contain oligonucleotide subsequences (regions) 171”, 172”, 173”, and 174” that are potentially selective for the target, and aptamers 181 and 182 coupled to the oligonucleotide subsequences. The oligonucleotide subsequences 171”, 172”, 173”, and 174” can be different from each other. Each aptamer 181 may contain an optional spacer 300, a capture primer aptamer 311 complementary to the capture primer 111, and an optional PCR aptamer 320 that, if suitable and if such an operation is included in the process, can be used to perform PCR amplification of the candidate aptamer. Similarly, each aptamer 182 may include an optional spacer 300, a capture primer aptamer 312 complementary to the capture primer 112, and an optional PCR aptamer 320, which, if suitable and if such an operation is included in the procedure, can be used for PCR amplification of the candidate aptamer. The spacer 300 may provide a suitable distance between the oligonucleotide subsequences 171”, 172”, 173”, 174” and the capture primer aptamer 311 or 312 such that the capture primer aptamer 311 can suitably couple to the target 160 without inhibiting the oligonucleotide subsequences. Inventively, the spacer 300 may contain 5 or more nucleotides, 10 or more nucleotides, or 15 or more nucleotides.

[0071] It should be understood that any suitable system, method, and composition can be used to select aptamers based on this teaching. For example, Figure 4 schematically illustrates an example operation in a process of sequential aptamer selection. Method 400 illustrated in Figure 4 includes coupling a target to each of a plurality of wells disposed in a substrate (operation 410). For example, the target 160 described with reference to Figure 1A can be directly coupled to the substrate in each well via suitable connectors (such as capture leads) in a manner as described with reference to Figure 2A. Alternatively, for example, the target 160 can be indirectly coupled to the substrate in each well via a first portion coupled to the substrate and a second portion coupled to the target, and thus coupled to the first portion in a manner as described with reference to Figures 1A to 1B. Method 400 illustrated in Figure 4 also includes contacting each of the wells with a fluid containing a plurality of candidate aptamers (operation 420). For example, these traps may contact aptamer libraries 171, 172, 173, and 174 in a manner as described with reference to FIG. 1C, or they may contact the amplicon of aptamer 174 in a manner as described with reference to FIG. 1F. Method 400 illustrated in FIG. 4 includes, in at least one of these traps, coupling any candidate aptamer selective for the target to the target (operation 430). For example, candidate aptamer 174 (or any amplicon thereof) may be coupled to target 160 in a manner as described with reference to FIG. 1D, 1G, or 2C. Method 400 illustrated in FIG. 4 includes removing any candidate aptamers not coupled to the target (operation 440). For example, candidate aptamers 171, 172, and 173 may be removed by flowing buffer through the trap, while candidate aptamer 174 remains coupled to target 160 despite such flow. Method 400, illustrated in Figure 4, includes sequencing any candidate aptamers retained in the trap after removal to identify any aptamer that is selective for the target (operation 450). For example, an amplicon of candidate aptamer 174 (or its amplicons) may be generated and such amplicons may be sequenced. [, Other reviews , ]

[0072] It should be understood that any individual feature / instance of the various forms disclosed herein may be implemented together in any suitable combination, and any feature / instance from any one or more of these forms may be implemented together with any feature of other forms as described herein in any suitable combination to achieve the benefits described herein.

[0073] While various illustrative examples have been described above, those skilled in the art will understand that various changes and modifications can be made without departing from the invention. The appended claims are intended to cover all such changes and modifications that fall within the true spirit and scope of the invention.

[0074] 100: Equipment 110:Substrate 111: The First Capture Prologue 112: The Second Capture Prologue 121: Trap 122: Trap 123: Trap 124: Trap 130: Part One 140: Molecules 150: Part Two 160: Target 171: Candidate Fitti 171'': Oligonucleotide subsequence 172: Candidate Fitti 172'': Oligonucleotide subsequence 173: Candidate Fitti 173": Oligonucleotide sequence 174: Candidate Fitti 174': Amplicon 174": Oligonucleotide subsequence 181: Fittest 182: Fittest 181': Fit 182': Fit 190: Detection circuit 210:Substrate 211: Capturing the Prologue 212: Capturing the Prologue 230: Part One 230': Part 1 300: Spacer 311: Capturing the Primitive Aptamer 312: Capturing the Primitive Aptamer 320:PCR Aptamer 400: Method 410: Operation 420: Operation 430: Operation 440: Operation 450: Operation

[0075] <![CDATA[ <110> Illumina, Inc. (USA) <![CDATA[ <120> Using ordered selection of aptamers (APTAMERS) <![CDATA[ <140> TW 110147785]]> <![CDATA[ <141> 2021-12-20 <![CDATA[ <150> US 63 / 128,669 <![CDATA[ <151> 2020-12-21 <![CDATA[ <160> 4 ]]> <![CDATA[ <170> PatentIn version 3.5]]> <![CDATA[ <210> 1]]> <![CDATA[ <211> 29]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polynucleotides <![CDATA[ <220> ]]> <![CDATA[ <221> misc_feature]]> <![CDATA[ <222> (23)..(23)]]> <![CDATA[ <223> n is U]]> <![CDATA[ <400> 1]]> aatgatacgg cgaccaccgaganctacac 29 <![CDATA[ <210> 2]]> <![CDATA[ <211> 24]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polynucleotides <![CDATA[ <220> ]]> <![CDATA[ <221> misc_feature]]> <![CDATA[ <222> (22)..(22)]]> <![CDATA[ <223> G* represents G or 8-side-oxyguanine. <![CDATA[ <400> 2]]> caagcagaag acggcatacg anat 24 <![CDATA[ <210> 3]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polynucleotides <![CDATA[ <400> 3]]> aatgatacgg cgaccaccga 20 <![CDATA[ <210> 4]]> <![CDATA[ <211> 21]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polynucleotides <![CDATA[ <400> 4]]> caaccagaag acggcatacg a 21

[0076]

Claims

1. A method for selecting an aptamer from a plurality of candidate aptamers, the method comprising: The target is coupled to each of the plurality of wells disposed in the substrate; Each trap is brought into contact with a fluid containing a plurality of candidate aptamers; wherein each candidate aptamer includes a first and a second aptamer complementary to a respective capture primer; in at least one trap, any candidate aptamer selective to the target is coupled to the target; any candidate aptamer not coupled to the target is removed; and in the traps, any candidate aptamers retained after removal are sequenced to identify any aptamer selective to the target; wherein sequenced any candidate aptamer includes: coupling any candidate aptamer retained after removal to a capture primer disposed in the traps; amplifying in the traps to generate an amplicon coupled to the capture primers; and sequenced the amplicon coupled to the capture primers.

2. The method of claim 1, wherein coupling the target to each of the plurality of wells comprises: Couple the first part to each trap; couple the plurality of second parts to each individual part of the target; And to couple the second portion to the first portion in each trap; wherein, whereby, the first portion contains streptavidin and the second portion contains biotin.

3. The method of claim 2, wherein the first portion is coupled to each trap by a capture primer; wherein, where applicable, the method includes separating the first portion from the capture primer prior to the sequencing.

4. The method of any one of requests 1 to 3, wherein: (i) The substrate includes a detection circuit for sequencing candidate aptamers retained after removal; or (ii) The plurality of traps includes a flow cell through which fluid flows in parallel; or (iii) Any candidate aptamer selective for the target has a tertiary structure that changes when coupled to the target; or (iv) Any combination of two or more of (i), (ii) and (iii).

5. The method of any one of claims 1 to 3, further comprising generating an amplicon of any candidate aptamer that is selective for the target.

6. The method of claim 5, wherein generating an amplicon that is selective for any candidate aptamer of the target includes: The candidate aptamer is decoupled from the target; and the candidate aptamer is used to generate the amplicon using polymerase chain reaction (PCR); wherein, if applicable, the PCR is performed in an amplification chamber different from the traps.

7. The method of claim 5, further comprising: Contact each trap with the fluid containing the amplicon; In each trap, any amplicon selective to the target is coupled to the target; and any amplicon not coupled to the target is removed; wherein: (i) any sequenced candidate aptamer is included in any amplicon retained after removal of any amplicon not coupled to the target; and / or (ii) the method further includes generating additional amplicons selective to the target.

8. As in request item 1, where: (i) the target is coupled to the traps via individual trapping initiators, wherein the trapping initiators couple a first portion to the traps and a second portion to the first portion and the target coupled to the traps; or (ii) the trapping initiators are coupled to the traps separately from the target.

9. The method of claim 1, wherein each candidate aptamer further comprises a first spacer disposed between the first aptamer and a candidate region selective to the target, and a second spacer disposed between the second aptamer and a candidate region selective to the target.

10. The method of any one of claims 1 to 3, wherein each candidate aptamer comprises an oligonucleotide.

11. A system for selecting an aptamer from a plurality of candidate aptamers, the system comprising: A substrate containing a plurality of wells; A target coupled to each trap; a fluid containing a plurality of candidate aptamers and in contact with each trap, wherein any candidate aptamer selectively coupled to the target; wherein each candidate aptamer contains a first and a second aptamer complementary to a respective trapping primer; a trapping primer disposed in the traps and coupled to any candidate aptamer remaining after removal of any candidate aptamer not coupled to the target; an amplicon coupled to the candidate aptamers of the trapping primers; and a detection circuit that sequenced any candidate aptamers in the traps to identify any aptamer selectively coupled to the target, wherein the detection circuit is amplicon sequenced to the trapping primers.

12. The system as described in request item 11, wherein: The first part is coupled to each trap; a plurality of second parts are coupled to each individual target; and the second part is coupled to the first part in each trap to couple the target to each trap.

13. The system as described in request item 12, wherein: (i) the first portion contains streptavidin and the second portion contains biotin; and / or (ii) the first portion is coupled to each trap by a capture primer, wherein the first portion may be detached from the capture primer.

14. A system as described in any of requests 11 to 13, wherein: (i) the plurality of traps are configured on the detection circuit; or (ii) the plurality of traps comprise a flow cell through which fluid flows in parallel; or (iii) any candidate aptamer selective for the target has a three-level structure that changes when coupled to the target; or (iv) any combination of two or more of (i), (ii) and (iii).

15. A system as claimed in any of claims 11 to 13, wherein an amplicon of any candidate aptamer that is selective for the target is generated.

16. The system of claim 15, wherein an amplicon of any candidate aptamer selective for the target is generated by the following steps: decoupling the candidate aptamer from the target; and using the candidate aptamer to generate an amplicon by polymerase chain reaction (PCR), wherein, where applicable, the system includes an amplification chamber different from the traps for performing the PCR.

17. The system of claim 15, further comprising: a fluid containing the amplicons and contacting each trap, wherein any amplicon selectively coupled to the target is coupled to the target.

18. The system of claim 17, wherein any sequenced candidate aptamer comprises any amplicon retained after removal of any amplicon not coupled to the target; where applicable, the method further comprises additional amplicons of any amplicon selective to the target.

19. The system as described in request item 11, wherein: (i) the target is coupled to the traps via individual capture initiators, wherein the capture initiators couple a first portion to the traps and a second portion to the first portion and the target coupled to the traps; or (ii) the capture initiators are coupled to the traps separately from the target; or (iii) each candidate aptamer further includes a first spacer disposed between the first aptamer and a candidate region selective to the target, and a second spacer disposed between the second aptamer and a candidate region selective to the target.

20. A system as claimed in any of claims 11 to 13, wherein each candidate aptamer comprises an oligonucleotide.

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