CN113939601A

CN113939601A - Multivalent binding compositions for nucleic acid analysis

Info

Publication number: CN113939601A
Application number: CN202080042516.6A
Authority: CN
Inventors: 锡南·阿尔斯兰; 莫利·何; 马修·克林格; 杰克·勒维厄; 迈克尔·普雷维特; 赵军花; 张夙; 泰勒·洛佩兹
Original assignee: Element Bioscience Corp
Current assignee: Element Bioscience Corp
Priority date: 2019-05-24
Filing date: 2020-05-22
Publication date: 2022-01-14
Also published as: SG11202112049VA; IL287528B1; KR20230165871A; EP3947731A4; GB202115667D0; KR20210144929A; IL301380A; WO2020243017A1; EP3947731A1; DE112020002516T5; AU2020285657A1; KR102607124B1; AU2022291540A1; GB2597398A; IL287528A; AU2020285657B2; JP2022535187A; IL287528B2; CA3137120A1; GB2597398B

Abstract

Multivalent binding compositions are described that include particle-nucleotide conjugates having multiple copies of nucleotides attached to the particles. Multivalent binding compositions allow for the localization of detectable signals to active regions of biochemical interactions, such as sites of protein-protein interactions, protein-nucleic acid interactions, nucleic acid hybridization, or enzymatic reactions, and can be used to identify sites of base incorporation into an elongated nucleic acid strand during polymerase reactions and provide improved base discrimination for sequencing and array-based applications.

Description

Multivalent binding compositions for nucleic acid analysis

Cross-referencing

This application is a continuation-in-part application of U.S. patent application No. 16/579,794 filed on 23.9.2019 and claims the benefit of U.S. provisional application No. 62/897,172 filed on 6.9.2019 and U.S. provisional application No. 62/852,876 filed on 24.5.24.2019, each of which is incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates generally to multivalent binding compositions and their use in the analysis of nucleic acid molecules. In particular, the inventive concept relates to a multivalent binding composition having multiple copies of nucleotides attached to a particle or polymer core that effectively increases the local concentration of nucleotides and enhances the binding signal. Multivalent binding compositions find use, for example, in the field of sequencing and biosensor microarrays.

Background

Nucleic acid sequencing can be used to obtain information in a variety of biomedical contexts, including diagnosis, prognosis, biotechnology, and forensic biology. Various sequencing methods have been developed, including Maxam-Gilbert sequencing and chain termination methods, or de novo sequencing methods including shotgun sequencing and bridge PCR, or next generation methods including polymerase chain sequencing, 454 pyrosequencing, Illumina sequencing, SOLID sequencing, ion torrent semiconductor sequencing, HeliScope single molecule sequencing, PCR, or the like,

Sequencing, and the like. Despite advances in DNA sequencing, many challenges facing cost-effective, high-throughput sequencing remain unsolved. The present disclosure provides novel solutions and methods that address many of the shortcomings of the prior art.

Disclosure of Invention

Disclosed herein are methods of determining the identity of a nucleotide in a target nucleic acid sequence, comprising: a. providing a composition comprising: i. two or more copies of the target nucleic acid sequence; two or more primer nucleic acid molecules complementary to one or more regions of the target nucleic acid sequence; two or more polymerase molecules; b. contacting a polymer-nucleotide conjugate with the polymer-nucleotide conjugate under conditions sufficient to allow formation of a multivalent binding complex between the polymer-nucleotide conjugate and the two or more copies of the target nucleic acid sequence in the composition of (a), wherein the polymer-nucleotide conjugate comprises copies of two or more nucleotide moieties and optionally one or more detectable labels; detecting the multivalent binding complex, thereby determining the identity of the nucleotide in the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is DNA. In some embodiments, the detection of multivalent binding complexes is performed in the absence of unbound or solution-borne polymer-nucleotide conjugates. In some embodiments, the target nucleic acid sequence has been replicated or amplified or has been produced by replication or amplification. In some embodiments, the one or more detectable labels are fluorescent labels. In some embodiments, detecting the multivalent complex comprises fluorescence measurement. In some embodiments, contacting comprises the use of one type of polymer-nucleotide conjugate. In some embodiments, contacting comprises the use of two or more types of polymer-nucleotide conjugates. In some embodiments, each of the two or more types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety. In some embodiments, the contacting comprises the use of three types of polymer-nucleotide conjugates, and wherein each of the three types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety. In some embodiments, the polymer-nucleotide conjugate comprises a blocked nucleotide moiety. In some embodiments, the blocked nucleotide is a 3' -O-azidomethyl nucleotide, a 3' -O-methyl nucleotide, or a 3' -O-alkylhydroxylamine nucleotide. In some embodiments, the contacting occurs in the presence of an ion that stabilizes the multivalent binding complex. In some embodiments, the contacting is performed in the presence of strontium ions, magnesium ions, calcium ions, or any combination thereof. In some embodiments, the polymerase molecule is catalytically inactive. In some embodiments, the polymerase molecule has been rendered catalytically inactive by mutation or chemical modification. In some embodiments, the polymerase molecule has been rendered catalytically inactive by the absence of an essential ion or cofactor. In some embodiments, the polymerase molecule is catalytically active. In some embodiments, the polymer-nucleotide conjugate does not comprise a blocked nucleotide moiety. In some embodiments, the multivalent binding complex has a duration of greater than 2 seconds. In some embodiments, the process may be carried out at a temperature in the range of 25 ℃ to 62 ℃. In some embodiments, the polymer-nucleotide conjugate further comprises one or more fluorescent labels, and two or more copies of the target nucleic acid sequence are deposited, attached, or hybridized to a surface, wherein the contrast to noise ratio of the fluorescence image of the multivalent binding complex on the surface in the detecting step is greater than 20. In some embodiments, the composition of (a) is deposited onto a surface using a buffer incorporating a polar aprotic solvent. In some embodiments, the contacting is performed under the following conditions: stabilizing the multivalent binding complex when the nucleotide moiety is complementary to the next base of the target nucleic acid sequence, and destabilizing the multivalent binding complex when the nucleotide moiety is not complementary to the next base of the target nucleic acid sequence. In some embodiments, the polymer-nucleotide conjugate comprises a polymer having a plurality of branches and the two or more nucleotide moieties are attached to the branches. In some embodiments, the polymer has a star, comb, cross-linked, bottle brush, or dendrimer configuration. In some embodiments, the polymer-nucleotide conjugate comprises one or more binding groups selected from avidin, biotin, an affinity tag, and combinations thereof. In some embodiments, the method further comprises a dissociation step that destabilizes the multivalent binding complex formed between the composition of (a) and the polymer-nucleotide conjugate, the dissociation step being capable of removing the polymer-nucleotide conjugate. In some embodiments, the method further comprises an extension step of incorporating a nucleotide complementary to the next base of the target nucleic acid sequence into the two or more primer nucleic acid molecules. In some embodiments, the extending step occurs simultaneously with or after the dissociating step.

Disclosed herein are methods of determining the identity of a nucleotide in a target nucleic acid sequence, comprising: a. providing a composition comprising: i. two or more copies of the target nucleic acid sequence; two or more primer nucleic acid molecules complementary to one or more regions of the target nucleic acid sequence; two or more polymerase molecules; b. contacting a polymer-nucleotide conjugate with the composition of (a) under conditions sufficient to allow formation of a multivalent complex between the polymer-nucleotide conjugate and the two or more copies of the target nucleic acid sequence in the composition, wherein the polymer-nucleotide conjugate comprises two or more copies of a reversibly terminated nucleotide moiety and optionally one or more cleavable detectable labels; detecting the multivalent complex, thereby determining the identity of the nucleotide in the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is DNA. In some embodiments, the method further comprises contacting the composition of (a) with a reversibly terminated nucleotide or a polymer-nucleotide conjugate comprising two or more copies of a reversibly terminated nucleotide after detection of the multivalent binding complex. In some embodiments, the target nucleic acid sequence has been replicated or amplified or has been produced by replication or amplification. In some embodiments, the one or more detectable labels are fluorescent labels. In some embodiments, detecting the multivalent complex comprises fluorescence measurement. In some embodiments, contacting comprises the use of one type of polymer-nucleotide conjugate. In some embodiments, contacting comprises the use of two or more types of polymer-nucleotide conjugates. In some embodiments, each of the two or more types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety. In some embodiments, the contacting comprises the use of three types of polymer-nucleotide conjugates, and wherein each of the three types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety. In some embodiments, the polymer-nucleotide conjugate comprises a blocked nucleotide moiety. In some embodiments, the blocked nucleotide is 3' -O-azidomethyl, 3' -O-methyl or 3' -O-alkylhydroxylamine. In some embodiments, the contacting occurs in the presence of an ion that stabilizes the multivalent binding complex. In some embodiments, the polymerase molecule is catalytically inactive. In some embodiments, the polymerase molecule has been rendered catalytically inactive by mutation or chemical modification. In some embodiments, the polymerase molecule is catalytically active. In some embodiments, the polymer-nucleotide conjugate does not comprise a blocked nucleotide moiety. In some embodiments, the process may be carried out at a temperature in the range of from 25 ℃ to 80 ℃. In some embodiments, the polymer-nucleotide conjugate further comprises one or more fluorescent labels, and two or more copies of the target nucleic acid sequence are deposited, attached, or hybridized to a surface, wherein the contrast to noise ratio of the fluorescence image of the multivalent binding complex on the surface in the detecting step is greater than 20.

Also disclosed herein is a system comprising: a) one or more computer processors individually or collectively programmed to implement a method comprising: i) contacting a substrate comprising multiple copies of a target nucleic acid sequence tethered to a surface of the substrate with a reagent comprising a polymerase and one or more primer nucleic acid sequences complementary to one or more regions of the target nucleic acid sequence to form a primed target nucleic acid sequence; ii) contacting a substrate surface with an agent comprising a polymer-nucleotide conjugate under conditions sufficient to allow formation of a multivalent binding complex between the polymer-nucleotide conjugate and two or more copies of the primed nucleic acid target nucleic acid sequence, wherein the polymer-nucleotide conjugate comprises two or more copies of a known nucleotide moiety and a detectable label; iii) acquiring and processing an image of the substrate surface to detect the multivalent binding complex, thereby determining the identity of the nucleotide in the target nucleic acid sequence. In some embodiments, the system further comprises a fluidics module configured to deliver a series of reagents to the substrate surface in a specified order and at specified time intervals. In some embodiments, the system further comprises an imaging module configured to acquire an image of the surface of the substrate. In some embodiments, (ii) and (iii) are repeated two or more times, thereby determining the identity of a series of two or more nucleotides in the target nucleic acid sequence. In some embodiments, the series of steps further comprises a dissociation step that destabilizes the multivalent binding complex, the dissociation step being capable of removing the polymer-nucleotide conjugate. In some embodiments, the series of steps further comprises an extension step of incorporating a nucleotide complementary to the next base of the target nucleic acid sequence into the two or more primer nucleic acid molecules. In some embodiments, the extending step occurs simultaneously with or after the dissociating step. In some embodiments, the detectable label comprises a fluorophore and the image comprises a fluorescent image. In some embodiments, when the fluorophore is cyanine dye 3(Cy3) and images were taken using an inverted fluorescence microscope equipped with a 20X objective, NA ═ 0.75, dichroic mirror optimized for 532nm light, band pass filter optimized for cyanine dye 3 emission, and camera under non-signal saturation conditions while the surface was immersed in 25mM ACES, pH 7.4 buffer, the contrast to noise ratio of the fluorescence image of the multivalent binding complex on the substrate surface was greater than 20. In some embodiments, the series of steps is completed in less than 60 minutes. In some embodiments, the series of steps is completed in less than 30 minutes. In some embodiments, the series of steps is completed in less than 10 minutes. In some embodiments, the accuracy of base detection is characterized by a Q score greater than 25 for at least 80% of the nucleotide identities determined. In some embodiments, the accuracy of base detection is characterized by a Q score greater than 30 for at least 80% of the nucleotide identities determined. In some embodiments, the accuracy of base detection is characterized by a Q score greater than 40 for at least 80% of the nucleotide identities determined.

Disclosed herein are compositions comprising: a) a polymeric core; and b) two or more nucleotides, nucleotide analogs, nucleoside or nucleoside analog moieties attached to the polymer core; wherein the length of the linker is dependent on the nucleotide, nucleotide analogue, nucleoside or nucleoside analogue moiety attached to the polymer core. Also disclosed herein are compositions comprising: a) a mixture of polymer-nucleotide conjugates, wherein each polymer-nucleotide conjugate comprises: i) a polymeric core; and ii) two or more nucleotides, nucleotide analogs, nucleosides, or nucleoside analog moieties attached to the polymer core, wherein the length of the linker depends on the nucleotide, nucleotide analog, nucleoside, or nucleoside analog moieties attached to the polymer core; and wherein the mixture comprises polymer-nucleotide conjugates having at least two different types of attached nucleotide, nucleotide analog, nucleoside, or nucleoside analog moieties. In some embodiments, the polymeric core comprises a polymer having a plurality of branches and two or more nucleotide, nucleotide analog, nucleoside, or nucleoside analog moieties are attached to the branches. In some embodiments, the polymer has a star, comb, cross-linked, bottle brush, or dendrimer configuration. In some embodiments, the polymer-nucleotide conjugate comprises one or more binding groups selected from avidin, biotin, an affinity tag, and combinations thereof. In some embodiments, the polymeric core comprises branched polyethylene glycol (PEG) molecules. In some embodiments, the polymer-nucleotide conjugate comprises a blocked nucleotide moiety. In some embodiments, the blocked nucleotide is a 3' -O-azidomethyl nucleotide, a 3' -O-methyl nucleotide, or a 3' -O-alkylhydroxylamine nucleotide. In some embodiments, the polymer-nucleotide conjugate further comprises one or more fluorescent labels.

In some embodiments, the present disclosure provides a method of determining the identity of a nucleotide in a target nucleic acid, comprising the following steps, regardless of any particular order of operation, 1) providing a composition comprising: a target nucleic acid comprising two or more repeats of the same sequence; two or more primer nucleic acids complementary to one or more regions of the target nucleic acid; and two or more polymerase molecules; 2) contacting the composition with a multivalent binding or incorporation composition comprising a polymer-nucleotide conjugate under conditions sufficient to allow formation of a binding or incorporation complex between the polymer-nucleotide conjugate and the composition of step (a), wherein the polymer-nucleotide conjugate comprises two or more copies of a nucleotide and optionally one or more detectable labels; and 3) detecting the binding or incorporation complex, thereby determining the identity of the nucleotide in the target nucleic acid polymer. In some further embodiments, the disclosure provides the methods, wherein the target nucleic acid is DNA, and/or wherein the target nucleic acid has been replicated, e.g., by any commonly practiced method of DNA replication or amplification, such as rolling circle amplification, bridge amplification, helicase-dependent amplification, isothermal bridge amplification, rolling circle multiple displacement amplification (RCA/MDA), and/or recombinase-based replication or amplification methods. In some further embodiments, the present disclosure provides the method, wherein the detectable label is a fluorescent label and/or wherein detecting the complex comprises a fluorescence measurement. In some further embodiments, the present disclosure provides the method, wherein the multivalent binding composition comprises one type of polymer-nucleotide conjugate, wherein the multivalent binding composition comprises two or more types of polymer-nucleotide conjugates, and/or wherein each type of the two or more types of polymer-nucleotide conjugates comprises a different type of nucleotide. In some embodiments, the present disclosure provides the method, wherein the binding complex or incorporation complex further comprises a blocked nucleotide, particularly wherein the blocked nucleotide is a 3' -O-azidomethyl nucleotide, a 3' -O-alkylhydroxylamine nucleotide, or a 3' -O-methyl nucleotide. In some further embodiments, the present disclosure provides the method, wherein the contacting is performed in the presence of strontium ions, barium, magnesium ions, and/or calcium ions. In some embodiments, the present disclosure provides the methods wherein the polymerase molecule is non-catalytically active, e.g., wherein the polymerase molecule is rendered non-catalytically active by mutation, by chemical modification, or by the absence of an essential ion or cofactor. In some embodiments, the present disclosure also provides the method, wherein the polymerase molecule is catalytically active, and/or wherein the binding complex does not comprise a blocked nucleotide. In some embodiments, the present disclosure provides the method wherein the binding complex has a duration of greater than 2 seconds and/or wherein the method is or can be performed at a temperature equal to or greater than 15 ℃, equal to or greater than 20 ℃, equal to or greater than 25 ℃, equal to or greater than 35 ℃, equal to or greater than 37 ℃, equal to or greater than 42 ℃, equal to or greater than 55 ℃, equal to or greater than 60 ℃ or equal to or greater than 72 ℃ or within a range as defined in any of the foregoing. In some embodiments, the present disclosure provides the method wherein the binding complex is deposited, attached or hybridized onto a surface that exhibits a contrast to noise ratio of greater than 20 in the detecting step. In some embodiments, the present disclosure provides the method wherein the composition is deposited under buffer conditions incorporating a polar aprotic solvent. In some embodiments, the present disclosure provides the method, wherein the contacting is performed under the following conditions: stabilizing the binding complex when the nucleotide is complementary to the next base of the target nucleic acid and destabilizing the binding complex when the nucleotide is not complementary to the next base of the target nucleic acid. In some embodiments, the present disclosure provides the method, wherein the polymer-nucleotide conjugate comprises a polymer having a plurality of branches and the plurality of copies of the first nucleotide are attached to the branches, particularly wherein the first polymer has a star, comb, cross-link, bottle brush, or dendrimer configuration. In some embodiments, the present disclosure provides the method, wherein the polymer-nucleotide conjugate comprises one or more binding groups selected from avidin, biotin, an affinity tag, and combinations thereof. In some embodiments, the present disclosure provides the method further comprising a dissociation step to destabilize the binding complex formed between the composition of (a) and the polymer-nucleotide conjugate to remove the polymer-nucleotide conjugate. In some embodiments, the present disclosure provides the method further comprising an extension step of incorporating a nucleotide complementary to the next base of the target nucleic acid into the primer nucleic acid, and optionally wherein the extension step occurs simultaneously with or after the dissociation step.

In some embodiments, the present disclosure provides a composition comprising a branched polymer having two or more branches and two or more copies of nucleotides, wherein the nucleotides are attached to a first plurality of the branches or arms, and optionally, wherein one or more interacting moieties are attached to a second plurality of the branches or arms. In some embodiments, the composition may further comprise one or more markers on the polymer. In some embodiments, the present disclosure provides the composition, wherein the nucleoside has a surface density of at least 4 nucleotides per polymer. In some embodiments, the present disclosure provides the compositions comprising or incorporating a nucleotide or nucleotide analog modified to prevent its incorporation into an extended nucleic acid strand during a polymerase reaction. In some embodiments, the composition may comprise or incorporate nucleotides or nucleotide analogs that are reversibly modified to prevent their incorporation into an extended nucleic acid strand during a polymerase reaction. In some embodiments, the present disclosure provides the composition, wherein the one or more labels comprise a fluorescent label, a FRET donor, and/or a FRET acceptor. In some embodiments, the composition may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more branches or arms, or 2, 4, 8, 16, 32, 64, or more branches or arms. In some embodiments, the branches or arms may radiate from the central portion. In some embodiments, the composition may comprise one or more interacting moieties, which may comprise avidin or streptavidin; a biotin moiety; an affinity tag; enzymes, antibodies, minibodies (minibodies), receptors or other proteins; a non-protein tag; a metal affinity tag, or any combination thereof. In some embodiments, the present disclosure provides the composition, wherein the polymer comprises polyethylene glycol, polypropylene glycol, polyvinyl acetate, polylactic acid, or polyglycolic acid. In some embodiments, the present disclosure provides the composition, wherein the nucleotide or nucleotide analog is attached to the branch or arm by a linker; in particular wherein the linker comprises PEG, and wherein the PEG linker moiety has an average molecular weight of about 1K Da, about 2K Da, about 3K Da, about 4K Da, about 5K Da, about 10K Da, about 15K Da, about 20K Da, about 50K Da, about 100K Da, about 150K Da, or about 200K Da, or greater than about 200K Da. In some embodiments, the present disclosure provides the composition, wherein the linker comprises PEG, and wherein the PEG linker moiety has an average molecular weight of about 5K Da to about 20K Da. In some embodiments, the present disclosure provides the composition, wherein the at least one nucleotide or nucleotide analog comprises a deoxyribonucleotide, a ribonucleotide, a deoxyribonucleoside, or a ribonucleoside; and/or wherein the nucleotide or nucleotide analogue is conjugated to the linker via the 5' end of the nucleotide or nucleotide analogue. In some embodiments, the present disclosure provides the composition, wherein one of the nucleotides or nucleotide analogs comprises deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, deoxycytidine, adenosine, guanosine, 5-methyl-uridine, and/or cytidine; and wherein the length of the linker is 1nm to 1,000 nm. In some embodiments, the present disclosure provides the composition, wherein the at least one nucleotide or nucleotide analog is a nucleotide modified to inhibit extension during a polymerase reaction or a sequencing reaction, for example wherein the at least one nucleotide or nucleotide analog is a nucleotide lacking a 3' hydroxyl group; a nucleotide modified to include a blocking group at the 3' position; and/or a nucleotide that has been modified with a 3 '-O-azido group, a 3' -O-azidomethyl group, a 3 '-O-alkylhydroxylamine group, a 3' -phosphorothioate group, a 3 '-O-malonyl group, or a 3' -O-benzyl group. In some embodiments, the present disclosure provides the composition, wherein the at least one nucleotide or nucleotide analog is a nucleotide that is unmodified at the 3' position.

In some embodiments, the present disclosure provides a method of determining the sequence of a nucleic acid molecule, comprising the steps, regardless of any particular order, of 1) providing a nucleic acid molecule comprising a template strand and a complementary strand at least partially complementary to the template strand; 2) contacting a nucleic acid molecule with one or more nucleic acid binding compositions according to any embodiment disclosed herein; 3) detecting binding of the nucleic acid binding composition to a nucleic acid molecule, and 4) determining the identity of the terminal nucleotide to be incorporated into the complementary strand of the nucleic acid molecule. In some embodiments, the present disclosure provides a method of determining the sequence of a nucleic acid molecule, comprising the steps, regardless of any particular order, of 1) providing a nucleic acid molecule comprising a template strand and a complementary strand at least partially complementary to the template strand; 2) contacting a nucleic acid molecule with one or more nucleic acid binding compositions according to any embodiment disclosed herein; 3) detecting partial or complete incorporation of the nucleic acid binding composition with a nucleic acid molecule, and 4) determining the identity of the terminal nucleotide to be incorporated into the complementary strand of the nucleic acid molecule from the partial or complete incorporation of the embodiments described herein. In some embodiments, the present disclosure provides the method, further comprising incorporating the terminal nucleotide into the complementary strand, and repeating the contacting, detecting, and incorporating steps for one or more additional iterations, thereby determining the sequence of the template strand of the nucleic acid molecule. In some embodiments, the present disclosure provides the method, wherein the nucleic acid molecule is coated withTethered to a solid support; and particularly wherein the solid support comprises a glass or polymeric substrate, at least one hydrophilic polymeric coating, and a plurality of oligonucleotide molecules attached to the at least one hydrophilic polymeric coating. In some embodiments, the present disclosure provides the method, further including embodiments wherein at least one hydrophilic polymer coating comprises PEG; and/or wherein at least one hydrophilic polymer layer comprises a branched hydrophilic polymer having at least 8 branches. In some embodiments, the present disclosure provides the method, wherein the plurality of oligonucleotide molecules are at least 500 molecules/mm²At least 1,000 molecules/mm²At least 5,000 molecules/mm²At least 10,000 molecules/mm²At least 20,000 molecules/mm²At least 50,000 molecules/mm²At least 100,000 molecules/mm²Or at least 500,000 molecules/mm²The surface density of (a) exists. In some embodiments, the present disclosure provides the method, wherein the nucleic acid molecule has been clonally amplified on a solid support. In some embodiments, the present disclosure provides the method, wherein clonal amplification comprises using Polymerase Chain Reaction (PCR), Multiple Displacement Amplification (MDA), Transcription Mediated Amplification (TMA), Nucleic Acid Sequence Based Amplification (NASBA), Strand Displacement Amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, Rolling Circle Amplification (RCA), loop-to-loop amplification, helicase dependent amplification, recombinase dependent amplification, Single Strand Binding (SSB) protein dependent amplification, or any combination thereof. In some embodiments, the present disclosure provides the method, wherein the one or more nucleic acid binding compositions are labeled with a fluorophore and the detecting step comprises using fluorescence imaging; particularly wherein fluorescence imaging comprises two-wavelength excitation/four-wavelength emission fluorescence imaging. In some embodiments, the present disclosure provides the method, wherein the identity of the terminal nucleotide is determined using four different nucleic acid binding compositions each comprising a different nucleotide or nucleotide analog, wherein the four different nucleic acid binding compositions are labeled with separate respective fluorophores, and wherein the detecting step comprises simultaneous excitation at a wavelength sufficient to excite all four fluorophores, and detection at a wavelength sufficient to detect eachThe fluorescence emission is imaged at the wavelength of the corresponding fluorophore. In some embodiments, the present disclosure provides the method, wherein the identity of the terminal nucleotide is determined using four different nucleic acid binding compositions each comprising a different nucleotide or nucleotide analog, wherein the four different nucleic acid binding compositions are labeled with cyanine dye 3(Cy3), cyanine dye 3.5(Cy3.5), cyanine dye 5(Cy5), and cyanine dye 5.5(Cy5.5), and wherein the detecting step comprises simultaneous excitation at any two of 532nm, 568nm, and 633nm, and imaging of fluorescence emissions at about 570nm, 592nm, 670nm, and 702nm, respectively; and/or wherein the fluorescence imaging comprises dual wavelength excitation/dual wavelength emission fluorescence imaging. In some embodiments, the disclosure provides the method wherein the identity of the terminal nucleotide is determined using four different nucleic acid binding compositions each comprising a different nucleotide or nucleotide analog, wherein one, two, three, or four different nucleic acid binding compositions are each separately labeled with a different fluorophore or set of fluorophores, and wherein the detecting step comprises simultaneous excitation at a wavelength sufficient to excite one, two, three, or four fluorophores or sets of fluorophores, and imaging of fluorescence emission at a wavelength sufficient to detect each respective fluorophore. In some embodiments, the disclosure provides the method wherein the identity of the terminal nucleotide is determined using three different nucleic acid binding or incorporation compositions each comprising a different nucleotide or nucleotide analog, wherein one, two, or three different nucleic acid binding or incorporation compositions are each separately labeled with a different fluorophore or set of fluorophores, and wherein the detecting step comprises simultaneous excitation at a wavelength sufficient to excite one, two, or three fluorophores or sets of fluorophores, and imaging of fluorescence emission at a wavelength sufficient to detect each respective fluorophore, and wherein detection of the fourth nucleotide is determined or determinable with reference to the location of "dark" or unlabeled spots or target nucleotides. In some embodiments, the present disclosure provides the methods, wherein the multivalent binding or incorporation composition can comprise three types of polymer-nucleotide conjugates, and wherein the three types of polymer-nucleosidesEach type of acid conjugate comprises a different type of nucleotide. In some embodiments, the present disclosure provides the methods wherein the detection of bound or incorporated complexes is performed in the absence of unbound or solution-borne polymer-nucleotide conjugates.

In some embodiments, the present disclosure provides the method, wherein the identity of the terminal nucleotide is determined using four different nucleic acid binding compositions or three different nucleic acid binding or incorporation compositions each comprising a different nucleotide or nucleotide analog, wherein one of the four or three different nucleic acid binding or incorporation compositions is labeled with a first fluorophore, one is labeled with a second fluorophore, one is labeled with first and second fluorophores, and one is unlabeled or absent, and wherein the detecting step comprises simultaneous excitation at a first excitation wavelength and a second excitation wavelength, and images are acquired at the first fluorescence emission wavelength and the second fluorescence emission wavelength. In some embodiments, the invention provides the method, wherein the first fluorophore is Cy3, the second fluorophore is Cy5, the first excitation wavelength is 532nm or 568nm, the second excitation wavelength is 633nm, the first fluorescence emission wavelength is about 570nm, and the second fluorescence emission wavelength is about 670 nm. In some embodiments, the present disclosure provides the methods wherein the detection label may comprise one or more portions of a Fluorescence Resonance Energy Transfer (FRET) pair such that multiple classifications may be made under a single excitation and imaging step. In some embodiments, the present disclosure provides the methods wherein the cycle of the sequencing reaction including the contacting, detecting, and incorporating/extending steps is performed in less than 30 minutes, less than 20 minutes, or less than 10 minutes. In some embodiments, the present disclosure provides the method, wherein the average Q score for base detection accuracy in a sequencing run is greater than or equal to 30, and/or greater than or equal to 40. In some embodiments, the present disclosure provides the method, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the identified terminal nucleotides have a Q score greater than 30 and/or greater than or equal to 40. In some embodiments, the present disclosure provides the methods, wherein at least 95% of the identified terminal nucleotides have a Q score greater than 30.

In some embodiments, the present disclosure provides reagents comprising one or more nucleic acid binding compositions as disclosed herein and a buffer. For example, in some embodiments, the present disclosure provides an agent, wherein the agent comprises 1,2, 3, 4, or more nucleic acid binding or incorporation compositions, wherein each nucleic acid binding or incorporation composition comprises a single type of nucleotide. In some embodiments, the agents of the present disclosure comprise 1,2, 3, 4, or more nucleic acid binding or incorporation compositions, wherein each nucleic acid binding or incorporation composition comprises a single type of nucleotide or nucleotide analog, and wherein the nucleotide or nucleotide analog may correspond to one or more of: adenosine Triphosphate (ATP), Adenosine Diphosphate (ADP), Adenosine Monophosphate (AMP), deoxyadenosine triphosphate (dATP), deoxyadenosine diphosphate (dADP), and deoxyadenosine monophosphate (dAMP); one or more of the following: thymidine Triphosphate (TTP), Thymidine Diphosphate (TDP), Thymidine Monophosphate (TMP), deoxythymidine triphosphate (dTTP), deoxythymidine diphosphate (dTDP), deoxythymidine monophosphate (dTMP), Uridine Triphosphate (UTP), Uridine Diphosphate (UDP), Uridine Monophosphate (UMP), deoxyuridine triphosphate (dUTP), deoxyuridine diphosphate (dupg), and deoxyuridine monophosphate (dUMP); one or more of the following: cytidine Triphosphate (CTP), Cytidine Diphosphate (CDP), Cytidine Monophosphate (CMP), deoxycytidine triphosphate (dCTP), deoxycytidine diphosphate (dCDP), and deoxycytidine monophosphate (dCMP); and one or more of the following: guanosine Triphosphate (GTP), Guanosine Diphosphate (GDP), Guanosine Monophosphate (GMP), deoxyguanosine triphosphate (dGTP), deoxyguanosine diphosphate (dGDP) and deoxyguanosine monophosphate (dGMP). In some other or some further examples, the present disclosure provides an agent comprising or further comprising 1,2, 3, 4 or more nucleic acid binding or incorporation compositions, wherein each nucleic acid binding or incorporation composition comprises a single type of nucleotide or nucleotide analog, and wherein the nucleotide or nucleotide analog may correspond to one or more of: ATP, ADP, AMP, dATP, dADP, dAMP, TTP, TDP, TMP, dTTP, dTDDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP, CDP, CMP, dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, and dGMP.

Disclosed herein are kits comprising a nucleic acid binding or incorporation composition of any embodiment disclosed herein and/or a reagent of any embodiment disclosed herein and/or one or more buffers; and instructions for their use.

Disclosed herein is a system for performing the methods of any of the embodiments disclosed herein, comprising the nucleic acid binding or incorporation composition of any of the embodiments disclosed herein, and/or the reagent of any of the embodiments disclosed herein. In some embodiments, the system is configured to iteratively perform sequential contacting of the tethered, primed nucleic acid molecule with the nucleic acid binding or incorporation composition and/or the agent; and for detecting binding or incorporation of the disclosed nucleic acid binding or incorporation compositions with one or more primed nucleic acid molecules.

In some embodiments, the present disclosure provides a composition comprising a particle (e.g., a nanoparticle or a polymeric core) comprising a plurality of enzymes or proteins bound or incorporated into a substrate, wherein the enzymes or proteins bound or incorporated into the substrate are bound to one or more enzymes or proteins to form one or more bound or incorporated complexes (e.g., multivalent bound or incorporated complexes), and wherein the binding or incorporation can be monitored or identified by observing the location, presence, or persistence of the one or more bound or incorporated complexes. In some embodiments, the particle may comprise a polymer, a branched polymer, a dendrimer, a liposome, a micelle, a nanoparticle, or a quantum dot. In some embodiments, the substrate may comprise a nucleotide, nucleoside, nucleotide analog, or nucleoside analog. In some embodiments, the enzyme or protein binding or incorporation into the substrate may comprise a reagent that can bind to a polymerase. In some embodiments, the enzyme or protein may comprise a polymerase. In some embodiments, for one or more of binding to or incorporating into the complexSaid observation of location, presence or persistence may comprise fluorescence detection. In some embodiments, the present disclosure provides a composition comprising a plurality of different particles as disclosed herein, wherein each particle comprises a single type of nucleoside or nucleoside analog, and wherein each nucleoside or nucleoside analog is associated with a fluorescent label that detectably differs in emission or excitation wavelength. In some embodiments, the present disclosure provides the composition further comprising one or more labels, e.g., fluorescent labels, on the particle. In some embodiments, the present disclosure provides the composition, wherein the composition comprises at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or more than 20 tethered nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs tethered to the particle. In some embodiments, the present disclosure provides the composition, wherein the nucleoside or nucleoside analog is present at 0.001 to 1,000,000/μm²0.01 to 1,000,000/. mu.m²0.1 to 1,000,000/μm ²1 to 1,000,000/. mu.m²10 to 1,000,000/. mu.m²100 to 1,000,000/μm²1,000 to 1,000,000/. mu.m²1,000 to 100,000/. mu.m²10,000 to 100,000/μm²Or 50,000 to 100,000/μm²Or a surface density within a range defined by any two of the above values. In some embodiments, the present disclosure provides the composition, wherein the nucleoside or nucleoside analog is present in a nucleotide or nucleotide analog. In some embodiments, the present disclosure provides the composition, wherein the composition comprises or incorporates a nucleotide or nucleotide analog that is modified to prevent its incorporation into an extended nucleic acid strand during a polymerase reaction. In some embodiments, the present disclosure provides the composition, wherein the composition comprises or incorporates a nucleotide or nucleotide analog that is reversibly modified to prevent its incorporation into an extended nucleic acid strand during a polymerase reaction. In some embodiments, the present disclosure provides the composition, wherein the one or more labels comprise a fluorescent label, a FRET donor, and/or a FRET acceptor. In some embodiments, the present disclosure provides the composition, wherein the substrate (e.g., core)A nucleotide, nucleotide analog, nucleoside, or nucleoside analog) is attached to the particle via a linker. In some embodiments, the present disclosure provides the composition, wherein the at least one nucleotide or nucleotide analog is a nucleotide modified to inhibit extension during a polymerase reaction or a sequencing reaction, such as, for example, a nucleotide lacking a 3' hydroxyl group; a nucleotide modified to include a blocking group at the 3' position; nucleotides that have been modified with a 3 '-O-azido group, a 3' -O-azidomethyl group, a 3 '-O-alkylhydroxylamine group, a 3' -phosphorothioate group, a 3 '-O-malonyl group, or a 3' -O-benzyl group; and/or nucleotides that are unmodified at the 3' position.

In some embodiments, the present disclosure provides a method of determining the sequence of a nucleic acid molecule, comprising the steps, regardless of order, of 1) providing a nucleic acid molecule comprising a template strand and a complementary strand at least partially complementary to the template strand; 2) contacting a nucleic acid molecule with one or more nucleic acid binding or incorporation compositions according to any embodiment disclosed herein; 3) detecting binding or incorporation of the nucleic acid binding or incorporation composition to a nucleic acid molecule, and 4) determining the identity of the terminal nucleotide to be incorporated into said complementary strand of said nucleic acid molecule. In some embodiments, the method may further comprise incorporating the terminal nucleotide into the complementary strand, and repeating the contacting, detecting, and incorporating steps for one or more additional iterations, thereby determining the sequence of the template strand of the nucleic acid molecule. In some embodiments, the present disclosure provides the method, wherein the nucleic acid molecule has been clonally amplified on a solid support. In some embodiments, the present disclosure provides the method, wherein clonal amplification comprises the use of Polymerase Chain Reaction (PCR), Multiple Displacement Amplification (MDA), Transcription Mediated Amplification (TMA), Nucleic Acid Sequence Based Amplification (NASBA), Strand Displacement Amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification, loop-to-loop amplification, helicase dependent amplification, recombinase dependent amplification, Single Strand Binding (SSB) protein dependent amplification, or any combination thereof. In some embodiments, the present disclosure provides the methods wherein the cycle of the sequencing reaction including the contacting, detecting, and incorporating steps is performed in less than 30 minutes, less than 20 minutes, or less than 10 minutes. In some embodiments, the disclosure provides the method, wherein the average Q score for base detection accuracy in a sequencing run is greater than or equal to 30, or greater than or equal to 40. In some embodiments, the present disclosure provides the method, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the identified terminal nucleotides have a nucleotide sequence greater than 30; or a Q-score greater than 40. In some embodiments, the present disclosure provides the method, wherein at least 95% of the identified terminal nucleotides have a Q score greater than 30.

In some embodiments, the present disclosure provides an agent comprising one or more nucleic acid binding or incorporation compositions as disclosed herein and a buffer. In some embodiments, the present disclosure provides the agent, wherein the agent comprises 1,2, 3, 4 or more nucleic acid binding or incorporation compositions, wherein each nucleic acid binding or incorporation composition comprises a single type of nucleotide or nucleotide analog, and wherein the nucleotide or nucleotide analog comprises a nucleotide, nucleotide analog, nucleoside or nucleoside analog. In some embodiments, the present disclosure provides the method, wherein the agent comprises 1,2, 3, 4, or more nucleic acid binding or incorporation compositions, wherein each nucleic acid binding or incorporation composition comprises a single type of nucleotide or nucleotide analog, and wherein the nucleotide or nucleotide analog may correspond to one or more of: ATP, ADP, AMP, dATP, dADP, and dAMP; one or more of the following: TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP and dUMP; one or more of the following: CTP, CDP, CMP, dCTP, dCDP, and dCMP; and one or more of the following: GTP, GDP, GMP, dGTP, dGDP and dGMP. In some embodiments, the present disclosure provides the method, wherein the agent comprises 1,2, 3, 4, or more nucleic acid binding or incorporation compositions, wherein each nucleic acid binding or incorporation composition comprises a single type of nucleotide or nucleotide analog, and wherein the nucleotide or nucleotide analog may correspond to one or more of: ATP, ADP, AMP, dATP, dADP, dAMP, TTP, TDP, TMP, dTTP, dTDDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP, CDP, CMP, dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, and dGMP.

In some embodiments, the present disclosure provides a kit comprising any of the compositions disclosed herein; and/or any agent disclosed herein; one or more buffering agents; and instructions for their use.

In some embodiments, the present disclosure provides a system for performing any of the methods disclosed herein; wherein the method may comprise the use of any composition as disclosed herein; and/or any agent disclosed herein; one or more buffers, and one or more nucleic acid molecules optionally tethered or attached to a solid support, wherein the system is configured to iterate the sequential contacting of the nucleic acid molecules with the composition and/or the reagents; and for detecting binding or incorporation of the nucleic acid binding or incorporation composition to one or more nucleic acid molecules.

In some embodiments, the present disclosure provides a composition as disclosed herein for increasing the contrast to noise ratio (CNR) of a labeled nucleic acid complex bound or associated with a surface.

In some embodiments, the present disclosure provides compositions as disclosed herein for establishing or maintaining control of the duration of signal from a labeled nucleic acid complex bound or associated with a surface.

In some embodiments, the present disclosure provides compositions as disclosed herein for establishing or maintaining control over the duration of a fluorescent, luminescent, electrical, electrochemical, colorimetric, radioactive, magnetic, or electromagnetic signal from a labeled nucleic acid complex bound or associated with a surface.

In some embodiments, the present disclosure provides compositions as disclosed herein for increasing the specificity, accuracy or read length of nucleic acid sequencing and/or genotyping applications.

In some embodiments, the present disclosure provides compositions as disclosed herein for increasing specificity, accuracy or read length in sequencing by binding or incorporation, sequencing-by-synthesis, single molecule sequencing or integrated sequencing methods.

In some embodiments, the present disclosure provides an agent as disclosed herein for increasing the contrast to noise ratio (CNR) of a labeled nucleic acid complex bound or associated with a surface.

In some embodiments, the present disclosure provides an agent as disclosed herein for establishing or maintaining control of the duration of a signal from a labeled nucleic acid complex bound or associated with a surface.

In some embodiments, the present disclosure provides reagents as disclosed herein for establishing or maintaining control over the duration of a fluorescent, luminescent, electrical, electrochemical, colorimetric, radioactive, magnetic, or electromagnetic signal from a labeled nucleic acid complex bound or associated with a surface.

In some embodiments, the present disclosure provides reagents as disclosed herein for increasing the specificity, accuracy or read length of nucleic acid sequencing and/or genotyping applications.

In some embodiments, the present disclosure provides reagents as disclosed herein for increasing specificity, accuracy or read length in sequencing by binding or incorporation, sequencing-by-synthesis, single molecule sequencing or integrated sequencing methods.

Is incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated in its entirety by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein controls.

Drawings

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and at the expense of providing the necessary fee.

The novel features believed characteristic of the inventive concept disclosed herein are set forth with particularity in the appended claims. The features and advantages of the disclosed compositions, methods, and systems may be better understood by reference to the following detailed description of illustrative embodiments that utilize the principles of the present inventive concepts when taken in conjunction with the accompanying drawings, wherein:

FIGS. 1A-1H show the steps of sequencing a target nucleic acid using non-limiting examples of multivalent binding compositions: FIG. 1A shows a non-limiting example of attaching a target nucleic acid to a surface 4-; FIG. 1B shows a target nucleic acid in a clonal fashion to form clusters of amplified target nucleic acid molecules; FIG. 1C shows a non-limiting example of priming a target nucleic acid to produce a primed nucleic acid target nucleic acid; FIG. 1D shows a non-limiting example of contacting a primed target nucleic acid with a multivalent binding composition and a polymerase to form a binding complex; FIG. 1E shows a non-limiting example of an image of a binding complex captured on a surface; FIG. 1F shows a non-limiting example of extending a primer strand by one nucleotide; FIG. 1G shows a non-limiting example of another cycle of contacting a primed target nucleic acid with a multivalent binding composition and a polymerase to form a binding complex; FIG. 1H shows a non-limiting example of an image of a binding complex captured on a surface in a subsequent sequencing cycle.

FIG. 2 shows a flow chart summarizing the steps of sequencing a target nucleic acid and extending a primer strand by single base addition.

Figure 3 shows a flow chart summarizing the steps for sequencing a target nucleic acid and extending a primer strand by incorporating nucleotides on a particle-nucleotide conjugate.

FIGS. 4A-4B show non-limiting examples of the detection of a target nucleic acid using polymer-nucleotide conjugates. FIG. 4A shows the step of contacting a polymerase and a polymer-nucleotide conjugate with a number of nucleic acid molecules; FIG. 4B shows a binding complex formed between a polymerase, a polymer-nucleotide conjugate, and a target nucleic acid molecule.

Fig. 5A-5C show schematic diagrams of non-limiting examples of different configurations of polymer-nucleotide conjugates: figure 5A shows polymer-nucleotide conjugates having a variety of multi-arm configurations; figure 5B shows a polymer-nucleotide conjugate with a polymer branch radiating from the center; and figure 5C shows a polymer-nucleotide conjugate with a binding moiety biotin.

FIG. 6 shows a generalized illustration of the increase in signal intensity observed during binding, persistence, washing and removal of multivalent substrates.

Fig. 7A-7J show fluorescence images of steps in a sequencing reaction using a multivalent PEG-substrate composition. FIG. 7A shows the PCR amplification in a medium containing 20nM Klenow polymerase and 2.5mM Sr⁺²In the exposure buffer of (1), red and green fluorescence images of the DNA RCA template (G and A first base) after exposure to 500nM base-labeled nucleotides (A-Cy3 and G-Cy 5). Images were collected after washing with imaging buffer, which had the same composition as the exposure buffer but did not contain nucleotides or polymerase. The contrast was scaled to maximize visualization of the darkest signal, but no signal persisted after washing with imaging buffer (fig. 7A, inset). Fig. 7B-7E: fluorescence images of multivalent PEG-nucleotide (base-labeled) ligands PB1 (fig. 7B), PB2 (fig. 7C), PB3 (fig. 7D), and PB5 (fig. 7E) with an effective nucleotide concentration of 500nM after mixing in exposure buffer and imaging in imaging buffer as described above are shown. FIG. 7F: fluorescence images of multivalent PEG-nucleotide (base-labeled) ligand PB5 at 2.5uM after mixing in exposure buffer and imaging in imaging buffer as described above are shown. Fig. 7G-fig. 7I: fluorescence images showing further base discrimination by exposure of multivalent binding compositions to inactive Klenow polymerase mutants (fig. 7 g.882h; fig. 7 h.882e; fig. 7 i.882a) and wild-type Klenow (control) enzyme (fig. 7J) are shown.

Fig. 8A-8B show the efficacy of multivalent reporter compositions in determining the base sequence of a DNA sequence over 5 sequencing cycles: FIG. 8A shows images and expected sequences of templates obtained after each sequencing cycle; and fig. 8B shows the results of alignment sequencing using the images captured in fig. 8A.

FIGS. 9A-9J show the polymerase activity in a sample containing 20nM Klenow polymerase and 2.5mM Sr⁺²In an exposure buffer ofFluorescence images of multivalent polyethylene glycol (PEG) polymer-nucleotide (base-labeled) conjugates with an effective nucleotide concentration of 500nM and varying PEG branch lengths after contact with a support surface containing a DNA template containing G or A as the first base; prepared using Rolling Circle Amplification (RCA). The images were obtained after washing with imaging buffer, which had the same composition as the exposure buffer but lacked nucleotides and polymerase. The panels show images obtained using multivalent PEG-nucleotide ligands with arm lengths as follows. FIG. 9A: 1K PEG. FIG. 9B: 2K PEG. FIG. 9C: 3K PEG. FIG. 9D: 5K PEG. FIG. 9E: 10K PEG. FIG. 9F: 20K PEG. Figure 9G shows the use of 10K PEG and contains mutation D882H inactive klenow polymerase obtained image. Figure 7H shows the image obtained using 10K PEG and inactive klenow polymerase containing mutation D882E. Figure 7I shows an image obtained using 10K PEG and inactive klenow polymerase containing mutation D882A. Figure 7J shows the use of 10K PEG and activity of wild type klenow polymerase obtained image.

FIG. 10 shows a quantitative representation of fluorescence intensity in the images shown in FIGS. 9A-9F, separated by color values, with an orange trace corresponding to the red label (Cy3 label; A bases) and a blue trace corresponding to the green label (Cy5 label; G bases).

Figure 11 shows normalized fluorescence from multivalent substrates bound to DNA clusters as described for figures 7A-7J, substrate complexes formed in the presence (condition B) and absence (condition a) of Triton-X100 (0.016%).

Fig. 12A-12B show graphs of normalized fluorescence intensity measured for multivalent polymer-nucleotide conjugates and free nucleotides. FIG. 12A: a binding complex of two repeats of a multivalent polymer-nucleotide conjugate bound to a DNA cluster (conditions a and B) with a labeled free nucleotide (condition C) after 1 minute; FIG. 12B: time course of fluorescence from multivalent substrate complexes over the course of 60 minutes.

Detailed Description

I. Definition of

As used herein, a "nucleic acid" (also referred to as a "polynucleotide", "oligonucleotide", ribonucleic acid (RNA), or deoxyribonucleic acid (DNA)) is a linear polymer of two or more nucleotides connected by covalent internucleoside linkages, or variants or functional fragments thereof. In the natural example of nucleic acids, the internucleoside linkage is typically a phosphodiester linkage. However, other examples optionally include other internucleoside linkages, such as phosphorothioate linkages, and may or may not include a phosphate group. Nucleic acids include double-and single-stranded DNA, as well as double-and single-stranded RNA, DNA/RNA hybrids, Peptide Nucleic Acids (PNA), hybrids between PNA and DNA or RNA, and may also include other types of nucleic acid modifications.

As used herein, "nucleotide" refers to a nucleotide, nucleoside, or analog thereof. Nucleotides refer to naturally occurring and chemically modified nucleotides and may include, but are not limited to, nucleosides, ribonucleotides, deoxyribonucleotides, protein-nucleic acid residues, or derivatives. Examples of nucleotides include adenine, thymine, uracil, cytosine, guanine or residues thereof; deoxyadenine, deoxythymine, deoxyuracil, deoxycytidine, deoxyguanine or residues thereof; adenine PNA, thymine PNA, uracil PNA, cytosine PNA, guanine PNA or residues or equivalents thereof, N-or C-glycosides of purine or pyrimidine bases (e.g., 2-deoxy-D-ribose containing deoxyribonucleosides or D-ribose containing ribonucleosides).

As used herein, "complementary" refers to the topological compatibility or matching together of the interacting surfaces of a ligand molecule and its receptor. Thus, a receptor and its ligand can be described as complementary, and in addition, the contact surface features are complementary to each other.

As used herein, "branched polymer" refers to a polymer having multiple functional groups that facilitate conjugation to a biologically active molecule such as a nucleotide, and which may be located on a side chain of the polymer or attached directly to the central core or central backbone of the polymer. The branched polymer may have a linear backbone in which one or more functional groups are detached from the backbone for conjugation. The branched polymer may also be a polymer having one or more side chains, wherein the side chains have sites suitable for conjugation. Examples of functional groups include, but are not limited to, hydroxyl, ester, amine, carbonate, acetal, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, hydrazide, thiol, alkanoic acid, acid halide, isocyanate, isothiocyanate, maleimide, vinyl sulfone, dithiopyridine, vinyl pyridine, iodoacetamide, epoxide, glyoxal, diketone, mesylate, tosylate, and triflate.

As used herein, "polymerase" refers to an enzyme that contains a nucleotide binding moiety and facilitates the formation of a binding complex between a target nucleic acid and a complementary nucleotide. The polymerase may have one or more activities including, but not limited to, base analog detection activity, DNA polymerization activity, reverse transcriptase activity, DNA binding or incorporation, strand displacement activity, and nucleotide binding or incorporation and recognition. Polymerases can include non-catalytically active polymerases, reverse transcriptases, and other enzymes that contain nucleotide binding or incorporation moieties.

As used herein, "duration" refers to the length of time that a binding complex formed between a target nucleic acid, polymerase, conjugated or unconjugated nucleotides remains stable without any binding components dissociating from the binding complex. The duration of time indicates the stability of the binding complex and the strength of the binding interaction. The duration may be measured by observing the onset and/or duration of the binding complex, for example by observing a signal from the label component of the binding complex. For example, a labeled nucleotide or a labeling reagent comprising one or more nucleotides may be present in the binding complex, allowing detection of a signal from the label during the duration of the binding complex. One non-limiting example of a label is a fluorescent label.

Method for analyzing target nucleic acid

Disclosed herein are multivalent binding or incorporation compositions and their use in the analysis of nucleic acid molecules, including in sequencing or other bioassay applications. An increase in binding or incorporation of a nucleotide to an enzyme (e.g., polymerase) or enzyme complex can be affected by increasing the effective concentration of the nucleotide. The increase can be achieved by increasing the concentration of nucleotides in free solution or by increasing the amount of nucleotides in the vicinity of the relevant binding or incorporation site. An increase may also be achieved by physically confining many nucleotides within a limited volume, resulting in a local increase in concentration, and thus such a structure may bind or incorporate binding or incorporation sites with higher apparent affinity than observed with unconjugated, untethered, or otherwise unconstrained individual nucleotides. One non-limiting means of achieving this limitation is by providing multivalent binding or incorporation compositions in which a plurality of nucleotides are bound to particles such as polymers, branched polymers, dendrimers, micelles, liposomes, microparticles, nanoparticles, quantum dots, or other suitable particles known in the art.

Multivalent binding or incorporation compositions disclosed herein can include at least one particle-nucleotide conjugate, and the particle-nucleotide conjugate has multiple copies of the same nucleotide attached to the particle. When the nucleotide is complementary to the target nucleic acid, the particle-nucleotide conjugate forms a bound or incorporated complex with the polymerase and the target nucleic acid, and the bound or incorporated complex exhibits increased stability and longer duration than bound or incorporated complexes formed using a single unconjugated or untethered nucleotide. Each nucleotide moiety of the multivalent binding composition can bind to a complementary N +1 nucleotide of a primed target nucleic acid molecule, thereby forming a multivalent binding complex comprising two or more target nucleic acid molecules, two or more polymerase (or other enzyme) molecules, and a multivalent binding composition (e.g., a polymer-nucleotide conjugate). Each nucleotide moiety of the multivalent binding composition can bind to a complementary N nucleotide of a primed target nucleic acid molecule, thereby forming a multivalent binding complex comprising two or more target nucleic acid molecules, two or more polymerase (or other enzyme) molecules, and a multivalent binding composition (e.g., a polymer-nucleotide conjugate). From this bound complex, the nucleotide can interrogate the complementary base prior to incorporation of a modified reversibly blocked nucleotide that extends the replicative strand by 1 base. Furthermore, it is conceivable to interrogate the N nucleotide with the bound complex, step forward with a reversibly terminated nucleotide, and then probe the N +1 base before and after deblocking. In this way, error checking can be performed by reading two interrogations and the overall accuracy of base detection is improved. An important discrimination factor of the traditional method is the binding of the bases used for interrogating matches, whereas the stepping or incorporation step is only used to move forward on the extended strand.

Multivalent binding or incorporation compositions can be used to localize a detectable signal to an active region of a biochemical interaction, such as a site of a protein-nucleic acid interaction, a nucleic acid hybridization reaction, or an enzymatic reaction, such as a polymerase reaction. For example, multivalent binding or incorporation compositions described herein can be used to identify sites of base incorporation in an extended nucleic acid strand during a polymerase reaction and provide base discrimination for sequencing and array-based applications. When the nucleotide is complementary to the target nucleic acid, increased binding or incorporation between the target nucleic acid and the nucleotide in the multivalent binding or incorporation composition provides an enhanced signal, thereby greatly improving base detection accuracy and reducing imaging time.

Furthermore, the use of multivalent binding compositions allows the sequencing signal from a given sequence to originate within a cluster region that contains multiple copies of the target sequence. An advantage of a sequencing method incorporating multiple copies of the target sequence is that the signal can be amplified since there are multiple simultaneous sequencing reactions within a defined region, each providing its own signal. The presence of multiple signals within a defined region also reduces the effect of any single skipping cycle, since a large number of correctly base detected signals may overwhelm a small number of skipped or incorrectly base detected signals, thus providing a means to reduce phase separation errors and/or increase read length in a sequencing reaction.

The multivalent binding compositions and uses thereof disclosed herein result in one or more of: (i) compared with the traditional nucleic acid amplification and sequencing method, the method has stronger signal and higher detection accuracy of the basic group; ii) allows better discrimination of sequence specific signals from background signals; (iii) (iii) reduced requirement for required amount of starting material, (iv) increased sequencing rate and reduced sequencing time; (v) (vii) reduced phase separation errors, and (vi) increased read length in sequencing reactions.

In some embodiments, a target nucleic acid can refer to a target nucleic acid sample having one or more nucleic acid molecules. In some embodiments, a target nucleic acid can include a plurality of nucleic acid molecules. In some embodiments, a target nucleic acid can include two or more nucleic acid molecules. In some embodiments, a target nucleic acid can include two or more nucleic acid molecules having the same sequence.

A. Sequencing a target nucleic acid

FIGS. 1A-1H illustrate an exemplary method in which a target nucleic acid is sequenced using a multivalent binding composition. As shown in FIG. 1A, a target nucleic acid 102 can be tethered to a solid support surface 101. The target nucleic acid can be attached to the surface directly or indirectly. Although not shown in FIG. 1A, the target nucleic acid 102 can be hybridized to an adapter that is attached to the surface by covalent or non-covalent bonds. When one or more adapters are used to attach the target nucleic acid to the surface, the target surface may comprise fragments that are complementary to, and thus hybridize to, the adapters. In some cases, an adapter sequence may be tethered to the surface. In some cases, multiple adapter sequences can be tethered to a surface. In some cases, the target nucleic acid 102 can also be attached directly to the solid support surface without the use of adapters. The solid support may be a low non-specific binding surface.

In fig. 1B, after the initial step of attaching the target nucleic acid to the surface of the solid support surface (e.g., by hybridization with an adapter), the target nucleic acid is then clonally amplified to form an amplified nucleic acid cluster. When the target nucleic acid is attached to the surface by an adapter, the surface density of the clonally amplified nucleic acid sequence hybridized to the adapter on the surface of the vector can span the same range as the surface density of the tethered adapters (or primers). Clonal amplification can be performed using Polymerase Chain Reaction (PCR), Multiple Displacement Amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), Strand Displacement Amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification, loop-to-loop amplification, helicase-dependent amplification, recombinase-dependent amplification, Single Strand Binding (SSB) protein-dependent amplification, or any combination thereof.

FIG. 1C shows a non-limiting step of annealing the primer 103 to the target nucleic acid 102 to form a primed target nucleic acid 104. FIG. 1B shows only one primer used in the annealing step, but more than one primer may be used depending on the type of target nucleic acid. In some cases, the adapter used to attach the target nucleic acid to the surface has the same sequence as the primer used to prepare the primed target nucleic acid. The primers can include forward amplification primers, reverse amplification primers, sequencing primers, and/or molecular barcode sequences, or any combination thereof. In some cases, one primer sequence may be used in the hybridization step. In some cases, multiple different primer sequences may be used in the hybridization step.

As shown in fig. 1D, the primed target nucleic acid 104 is combined with a multivalent binding or incorporation composition and a polymerase 106 to form a binding or incorporation complex. The non-limiting example of a multivalent binding or incorporation composition in fig. 10 comprises four particle-

nucleotide conjugates

105a, 105b, 105c, and 105 d. Each particle-nucleotide conjugate has multiple copies of nucleotides attached to the particle, and four particle-nucleotide conjugates encompass four types of nucleotides, respectively. A particle-nucleotide conjugate having a nucleotide complementary to the next base on the primed target nucleic acid will form a binding or incorporation complex with the polymerase and the target nucleic acid. In some cases, a multivalent binding or incorporation composition can include one, two, or three particle-nucleotide conjugates. In some embodiments, each different type of particle-nucleotide conjugate can be labeled with a separate label. In some embodiments, three of the four types of nucleotide conjugates can be labeled, with the fourth unlabeled or conjugated to an undetectable label. In some embodiments, 1,2, 3 or 4 particle-nucleotide conjugates may be labeled, either with the same label, or each with a label corresponding to the identity of its conjugated nucleotide, with 3, 2, 1 or no particle-nucleotide conjugates, respectively, that may be unlabeled or conjugated with an undetectable label. In some embodiments, detection of polymerase complexes incorporated into particle-nucleotide conjugates can be performed using four-color detection, such that conjugates corresponding to all four nucleotides, each conjugate having a separate label corresponding to the nucleotide conjugated thereto, are present in the sample. In some embodiments, four particle-nucleotide conjugates can be exposed to or contacted with a target nucleic acid simultaneously; in some other embodiments, the four particle-nucleotide conjugates can be exposed to or contacted with the target nucleic acid individually or sequentially in groups of two or three. In some embodiments, detection of polymerase complexes incorporating particle-nucleotide conjugates can be performed using a three-color detection, such that conjugates corresponding to three of the four nucleotides are present in the sample, where the three conjugates have a separate label corresponding to the nucleotide conjugated thereto and one conjugate that does not have a label or is conjugated to an undetectable label. In some embodiments, only three types of conjugates are provided, such that conjugates corresponding to three of the four nucleotides are present in the sample, wherein three conjugates have a separate label corresponding to the nucleotide conjugated thereto and one conjugate is absent. In some embodiments, the identity of the nucleotide corresponding to an unlabeled or absent nucleotide conjugate can be determined relative to the location of a "dark" spot and/or the identity or location of a known target nucleic acid that does not display a fluorescent signal. In some embodiments, the present disclosure provides the methods wherein the detection of bound or incorporated complexes is performed in the absence of unbound or solution-borne polymer-nucleotide conjugates.

In some embodiments in which three of the four particle-nucleotide conjugates are labeled, or in embodiments in which only three of the four particle-nucleotide conjugates are present, the identity of the nucleotide corresponding to the unlabeled or absent conjugate can be determined by the absence of a signal or by monitoring the presence of the unlabeled complex, for example by identifying a "dark" spot or unlabeled region in the sequencing reaction. In some embodiments, detection of polymerase complexes incorporating particle-nucleotide conjugates can be performed using a two-color detection, such that conjugates corresponding to two of the four nucleotides are present in the sample, where the two conjugates have a separate label corresponding to the nucleotide conjugated thereto and two conjugates that do not have a label or are conjugated to an undetectable label. In some embodiments, only two of the four particle-nucleotide conjugates are labeled. In some embodiments in which two of the four particle-nucleotide conjugates are labeled, the identity of the nucleotide corresponding to the unlabeled conjugate or conjugates can be determined by the absence of a signal or by monitoring the presence of the unlabeled complex, e.g., by identifying a "dark" spot or unlabeled region in the sequencing reaction. In some embodiments in which two of the four particle-nucleotide conjugates are labeled, the four particle-nucleotide conjugates can be exposed to or contacted with the target nucleic acid sequentially, individually, or in groups of two or three. In some embodiments, two of the four particle-nucleotide conjugates can share a common label, and the four particle-nucleotide conjugates can be exposed to or contacted with a target nucleic acid sequentially, individually, or in groups of two or three, wherein each contacting step shows a distinction between two or more different bases, such that after two, three, four, or more such contacting steps, the identity of all unknown bases has been determined.

Figure 1E shows an image captured on a surface after formation of a binding or incorporation complex between a polymerase, a target nucleic acid, and a particle-nucleotide conjugate with a nucleotide annotated to the next base of the primed target nucleic acid. The captured image includes four binding or

incorporation complexes

107a, 107b, 107c, and 107d formed on the surface, and each binding or incorporation complex has a different nucleotide that can be distinguished based on the label (e.g., fluorescence emission color) on the particle-nucleotide conjugate. Since the use of particle-nucleotide conjugates allows the binding or incorporation signal from a given sequence to originate within a cluster region containing multiple copies of the target sequence, the sequencing signal is greatly enhanced. Although fig. 1E involves four particle-nucleotide conjugates, each with a different type of nucleotide, some methods may use one, two, or three particle-nucleotide conjugates, each with a different type of nucleotide and label. In some embodiments, each different type of particle-nucleotide conjugate may be labeled with the same label, or each labeled with a label corresponding to the identity of the nucleotide to which it is conjugated. In some embodiments, three of the four types of nucleotide conjugates can be labeled, with the fourth unlabeled or conjugated to an undetectable label. In some embodiments, 1,2, 3, or 4 particle-nucleotide conjugates can be labeled with separate labels, with 3, 2, 1, or no particle-nucleotide conjugates, respectively, being unlabeled or conjugated to an undetectable label. In one embodiment, the detecting step may comprise simultaneous and/or sequential excitation of up to 4 different excitation wavelengths, for example where fluorescence imaging is performed by detecting single and/or multiple fluorescence emission bands that uniquely classify each possible base pairing (A, G, C or T). In some embodiments, the identity of the terminal nucleotide can be determined using four different nucleic acid binding or incorporation compositions each comprising a different nucleotide or nucleotide analog, wherein one of the four different nucleic acid binding or incorporation compositions is labeled with a first fluorophore, one is labeled with a second fluorophore, one is labeled with the first and second fluorophores, and one is unlabeled, and wherein the detecting step comprises simultaneous excitation at a first excitation wavelength and a second excitation wavelength, and images are acquired at the first fluorescence emission wavelength and the second fluorescence emission wavelength.

When a multivalent binding or incorporation composition is used in place of a single unconjugated or untethered nucleotide to form a binding or incorporation complex with the polymerase and primed target nucleic acid, the local concentration of nucleotides increases many-fold, which in turn enhances signal intensity. The formed bound or incorporated complexes also have a longer duration, which in turn helps to shorten the imaging step. The high signal intensity results from the high binding or incorporation affinity of the polymer-nucleotide conjugate (which may also contain multiple fluorophores or other labels), which thus forms a complex that remains stable throughout the binding or incorporation and imaging steps. Strong binding or incorporation between the polymerase, the primer target strand and the polymer-nucleotide or nucleotide analogue conjugate also means that the multivalent binding or incorporation complex formed thereby will remain stable during the washing step and the signal intensity will remain high when other reaction mixture components and mismatched nucleotide analogues are washed away. After the imaging step, the bound or incorporated complex can be destabilized (e.g., by changing the buffer composition), and the primed target nucleic acid can then be extended by one base.

The sequencing method may further comprise incorporating N +1 or terminal nucleotides into the primed strand, as shown in figure 1F. In FIG. 1F, the primed primer strand of the target nucleic acid 108 can be extended by one base to form an extended nucleic acid 109. The extension step may occur after or simultaneously with destabilization of the multivalent binding or incorporation complex. The primed target nucleic acid 108 can be extended using complementary nucleotides attached to the particles in the particle-nucleotide conjugate or using unconjugated or untethered free nucleotides provided after removal of the multivalent binding or incorporation composition.

After the extension step, a contact step as shown in fig. 1G. This can be done again to form bound or incorporated complexes and to simulate the next sequencing cycle. The contacting, detecting, and extending steps can be repeated for one or more cycles to determine the sequence of the target nucleic acid molecule. For example, FIG. 1H shows surface images obtained after performing multiple sequencing cycles, which can then be processed to determine the sequence of a target nucleic acid molecule.

Primed extension of the target nucleic acid can be prevented or inhibited due to blocked nucleotides on the strand or the use of a non-catalytically active polymerase. When a nucleotide in a polymer-nucleotide conjugate has a blocking group that prevents nucleic acid extension, incorporation of the nucleotide can be achieved by removing the blocking group from the nucleotide (e.g., by separating the nucleotide from its polymer, branched polymer, dendrimer, particle, etc.). When the primed extension of the target nucleic acid is inhibited due to the use of a non-catalytically active polymerase, incorporation of the nucleotide can be achieved by providing a cofactor or an activator, such as a metal ion.

Also disclosed herein are systems configured to perform any of the disclosed nucleic acid sequencing or nucleic acid analysis methods. The system can include a fluid flow controller and/or a fluid dispensing system configured to sequentially and repeatedly contact primed target nucleic acid molecules attached to a solid support with the disclosed polymerases and multivalent binding or incorporation compositions and/or reagents. The contacting may be performed in one or more flow cells. In some cases, the flow cell may be a stationary component of the system. In some cases, the flow cell may be a removable and/or disposable component of the system.

The sequencing system can include an imaging module, i.e., one or more light sources, one or more optical components, and one or more image sensors, for imaging and detecting the binding or incorporation of the disclosed nucleic acid binding or incorporation compositions to target nucleic acid molecules tethered to the interior of a solid support or flow cell. The disclosed compositions, reagents, and methods can be used in any of a variety of nucleic acid sequencing and analysis applications. Examples include, but are not limited to, DNA sequencing, RNA sequencing, whole genome sequencing, targeted sequencing, exome sequencing, genotyping, and the like.

The sequencing system may also include a computer control system programmed to implement the methods of the present disclosure. The computer system is programmed or otherwise configured to perform the methods of the present disclosure, including nucleic acid sequencing methods, interpretation of nucleic acid sequencing data and analysis of cellular nucleic acids, such as RNA (e.g., mRNA), and characterization of cells from the sequencing data. The computer system may be the user's electronic device or a computer system remotely located from the electronic device. The electronic device may be a mobile electronic device.

FIG. 2 is a flow chart summarizing the steps of sequencing a target nucleic acid. 201 describes the step of attaching target library sequences to a solid support surface by hybridizing target nucleic acid molecules to complementary adaptors on the substrate surface. The target nucleic acid molecule can be single-stranded or partially double-stranded. Prior to 201, the nucleic acid molecules in the target library may have been prepared by ligation or other methods to contain fragments complementary to the adapter sequences. 202 describes a clonal amplification step that produces clusters of target nucleic acid molecules on a surface. 203 describes hybridizing a sequencing primer to a complementary primer binding or incorporation sequence on a target nucleic acid to form a primed target nucleic acid. 204 describes a combination of a polymerase, a multivalent binding or incorporation composition comprising a labeled (e.g., fluorescently labeled) particle-nucleotide conjugate, and a primed target nucleic acid. 204 may also include the step of washing or removing unbound reagents including polymerase and particle-nucleotide conjugates.

Referring again to fig. 2, when the nucleotide on the particle-nucleotide conjugate is complementary to the next base of the primed target nucleic acid (205), the particle-nucleotide conjugate, polymerase, and primed target nucleic acid form a ternary binding or incorporation complex, which can be detected by a detection method (e.g., fluorescence imaging) compatible with the label on the particle-nucleotide conjugate. 205 may also include measuring the duration of ternary binding or incorporation into the complex. At 206, the binding or incorporation complex is destabilized to remove the binding or incorporation of the particle-nucleotide conjugate and the polymerase. Dissociation can be achieved by subjecting the bound or incorporated complex to conditions (e.g., addition of strontium ions) that alter the polymerase conformation and destabilize binding or incorporation. 206 may also include a step of washing or removing the dissociated particle-nucleotide conjugate and/or polymerase. 207 describes the step of extending the primed strand of the primed target nucleic acid by a single base addition reaction. After single base extension, steps 204, 205, 206, and 207 may be repeated in multiple cycles to determine the sequence of the target nucleic acid.

Figure 3 is another flowchart outlining the steps of sequencing a target nucleic acid, which includes cleaving nucleotides from particle-nucleotide conjugates and incorporating the cleaved nucleotides. 301 describes the step of attaching target library sequences to a solid support surface by hybridizing target nucleic acid molecules to complementary adaptors on the substrate surface. The target nucleic acid molecule can be single-stranded or partially double-stranded. Prior to 301, the nucleic acid molecules in the target library may have been prepared by ligation or other methods to contain fragments complementary to the adapter sequences. 302 describes a clonal amplification step that produces clusters of target nucleic acid molecules on a surface. 303, which describes hybridizing a sequencing primer to a complementary primer binding or incorporation sequence on a target nucleic acid to form a primed target nucleic acid. 304 describes a combination of a polymerase, a multivalent binding or incorporation composition comprising a labeled (e.g., fluorescently labeled) particle-nucleotide conjugate, and a primed target nucleic acid. In particle-nucleotide conjugates, nucleotides are attached to particles by chemical bonds or interactions that can be subsequently cleaved. 404 may also include a step of washing or removing unbound reagents including polymerase and particle-nucleotide conjugates.

Referring again to fig. 3, when the nucleotide on the particle-nucleotide conjugate is complementary to the next base of the primed target nucleic acid (305), the particle-nucleotide conjugate, polymerase, and primed target nucleic acid form a ternary binding or incorporation complex, which can be detected by a detection method (e.g., fluorescence imaging) compatible with the label on the particle-nucleotide conjugate. 305 may also include measuring the duration of ternary binding or incorporation into the complex. At 306, the polymerase is placed in conditions that render it catalytically active for incorporation of the nucleotide. The conditions may include exposing the polymerase to Mg or Mn ions in the reaction solution. The nucleotides bound by the polymerase and the primed target nucleic acid are then cleaved from the particle and then incorporated into the primed strand of the primed target nucleic acid. Destabilizes the bound or incorporated complex. 306 may also include a step of washing or removing the dissociated particle-nucleotide conjugate and/or polymerase. After extension, steps 304, 305, and 306 can be repeated in multiple cycles to determine the sequence of the target nucleic acid.

B. Detection of target nucleic acid molecules

FIGS. 4A-4B illustrate an exemplary method in which a target nucleic acid is sequenced using a multivalent binding or incorporation composition. As shown in fig. 4A, a polymer-nucleotide conjugate 401 is placed in contact with a polymerase 406, a first nucleic acid molecule 404, and a second nucleic acid molecule 405. The polymer-nucleotide conjugate 401 has multiple polymer branches radiating from the nucleus, with some branches attached to nucleotides or oligonucleotides 402 and some branches attached to labels 403. When the nucleotide or oligonucleotide 402 on the polymer-nucleotide conjugate 401 is complementary to at least a portion of the first nucleic acid 404, a multivalent binding or incorporation complex is formed as shown in fig. 4B, and a strong binding or incorporation signal can aid in the detection of a target nucleic acid having a sequence that is complementary or partially complementary to the nucleotide or oligonucleotide on the polymer-nucleotide conjugate. In some cases, at least one of the polymerase, the nucleic acid molecule, and the polymer-nucleotide conjugate is attached to a solid support.

The multivalent binding or incorporation compositions described herein can be used in methods of detecting a target nucleic acid in a sample. Also disclosed herein are systems configured to perform any of the disclosed nucleic acid analysis methods. The system can include a fluid flow controller and/or a fluid dispensing system configured to sequentially and repeatedly contact nucleic acid molecules with the disclosed polymerases and multivalent binding or incorporation compositions and/or reagents. The contacting may be performed in one or more flow cells. In some cases, the flow cell may be a stationary component of the system. In some cases, the flow cell may be a removable and/or disposable component of the system. The system can also include a cartridge comprising a sample collection unit and an assay component, wherein the sample collection unit is configured to collect a sample, and wherein the assay component comprises at least one reaction site comprising a multivalent binding or incorporation composition adapted to interact with the analyte, allow a predetermined portion of the sample to react with an assay reagent contained within the assay component to generate a signal indicative of the presence of the analyte in the sample, and detect the signal generated from the analyte.

Polyvalent binding or incorporation compositions

The present disclosure relates to multivalent binding or incorporation compositions having a plurality of nucleotides conjugated to a particle (e.g., a polymer, branched polymer, dendrimer, or equivalent structure). Contacting the multivalent binding or incorporation composition with the polymerase and multiple copies of the primed target nucleic acid can result in the formation of a ternary complex that can be detected and, in turn, more accurate determination of the target nucleic acid base.

When using multivalent binding or incorporation compositions instead of single unconjugated or untethered nucleotides to form complexes with one or more copies of the polymerase and target nucleic acid, the local concentration of nucleotides and the binding affinity of the complexes (in the case of complexes comprising two or more target nucleic acid molecules) is increased many-fold, which in turn enhances signal strength, particularly correct signals and mismatches. The multivalent binding or incorporation compositions described herein can include at least one particle-nucleotide conjugate (each particle-nucleotide conjugate comprising multiple copies of a single nucleotide moiety) for interacting with a target nucleic acid. The multivalent composition may also include two, three, or four different particle-nucleotide conjugates, each having a different nucleotide conjugated to the particle.

The multivalent binding or incorporation composition can comprise 1,2, 3, 4, or more types of particle-nucleotide conjugates, wherein each particle-nucleotide conjugate comprises a different type of nucleotide. The first type of particle-nucleotide conjugate can comprise a nucleotide selected from the group consisting of ATP, ADP, AMP, dATP, dADP, and dAMP. The second type of particle-nucleotide conjugate may comprise a nucleic acid sequence selected from the group consisting of: TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP and dUMP. The third type of particle-nucleotide conjugate can comprise a nucleotide selected from CTP, CDP, CMP, dCTP, dCDP, and dCMP. The fourth type of particle-nucleotide conjugate can comprise a nucleotide selected from GTP, GDP, GMP, dGTP, dGDP, and dGMP. In some embodiments, each particle-nucleotide conjugate comprises a single type of nucleotide corresponding to one or more nucleotides selected from ATP, ADP, AMP, dATP, dADP, dAMP TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP, CDP, CMP, dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, and dGMP, respectively. Each multivalent binding or incorporation composition can further comprise one or more labels corresponding to the specific nucleotides conjugated to each respective conjugate. Non-limiting examples of labels include fluorescent labels, colorimetric labels, electrochemical labels (such as, for example, glucose or other reducing sugars, or thiols or other redox-active moieties), luminescent labels, chemiluminescent labels, spin labels, radioactive labels, steric labels, affinity tags, and the like.

A. Particle-nucleotide conjugates

In particle-nucleotide conjugates, multiple copies of the same nucleotide can be covalently or non-covalently bound to the particle. Examples of particles may include branched polymers; a dendritic polymer; cross-linked polymer particles, such as agarose, polyacrylamide, acrylate, methacrylate, cyanoacrylate, methyl methacrylate particles; glass particles; ceramic particles; metal particles; quantum dots; a liposome; emulsion particles, or any other particles known in the art (e.g., nanoparticles, microparticles, etc.). In a preferred embodiment, the particles are branched polymers.

In some cases, a particle-nucleotide conjugate (e.g., a polymer-nucleotide conjugate) can comprise 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 copies of a nucleotide, nucleotide analog, nucleoside, or nucleoside analog tethered to the particle.

The nucleotide may be attached to the particle by a linker, and the nucleotide may be attached to one end or one position of the polymer. The nucleotides may be conjugated to the particles through the 5' end of the nucleotides. In some particle-nucleotide conjugates, one nucleotide is attached to one end or one position of the polymer. In some particle-nucleotide conjugates, multiple nucleotides are attached to one end or position of the polymer. The conjugated nucleotides are sterically accessible to one or more proteins, one or more enzymes, and nucleotide binding or incorporation moieties. In some embodiments, the nucleotides may be provided separately from the nucleotide binding or incorporation moiety, e.g., a polymerase. In some embodiments, the linker does not comprise a light emitting or light absorbing group.

The particles may also have binding or incorporation moieties. In some embodiments, the particles can self-associate without the use of a separate interacting moiety. In some embodiments, the particles may self-associate due to buffer conditions or salt conditions, for example, in the case of calcium-mediated hydroxyapatite particle interactions, lipid or polymer-mediated micelle or liposome interactions, or salt-mediated aggregation of metal (e.g., iron or gold) nanoparticles.

The particle-nucleotide conjugate may have one or more labels. Examples of labels include, but are not limited to, fluorophores, spin labels, metals or metal ions, colorimetric labels, nanoparticles, PET labels, radioactive labels, or other such labels that can render the composition detectable by such methods known in the art for detecting macromolecular or molecular interactions. Labels can be attached to the nucleotides (e.g., by attachment to the 5' phosphate moiety of the nucleotide), to the particle itself (e.g., to the PEG subunit), to the end of the polymer, to a central portion, or to any other location within the polymer-nucleotide conjugate that would be deemed sufficient by one of skill in the art for the composition, e.g., particle, to be detectable by such methods known in the art or described elsewhere herein. In some embodiments, one or more labels are provided to correspond to or distinguish particular particle-nucleotide conjugates.

In some embodiments, the label is a fluorophore. Non-limiting examples of fluorescent moieties include, but are not limited to, fluorescein and fluorescein derivatives, such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthylfluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, maleimidofluorescein, SAMSA-fluorescein, thiosemicarbazide fluorescein, carbohydrazide methylthioacetamidofluorescein, rhodamine and rhodamine derivatives such as TRITC, TMR, lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, Lisamine rhodamine B sulfonyl chloride, Lisamine rhodamine B sulfonyl hydrazide, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarins and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivatives such as BODIPY FL C3-SE, BODIPY 530/550C 3, BODIPY 530/550C 3-SE, BODIPY 530/550C 3 hydrazide, BODIPY 493/503C 3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FL IA, BODIPY 530/551 IA, Br-BODIPY 493/503, cascade blue and derivatives such as cascade blue acetyl azide, cascade blue cadaverine, cascade blue ethylenediamine, cascade blue hydrazide, fluorescein and derivatives such as fluoroiodoacetamide, fluorescein CH, cyanines and derivatives such as indolium cyanine dye, benzindolinium cyanine dye, pyridinium cyanine dye, thiazolium cyanine dye, quinolinium cyanine dye, imidazolium cyanine dye, Cy3, Cy5, lanthanide chelates and derivatives such as BCOT, PDA TBP, TMT, HCT, BCOT, europium chelate and derivatives such as BCOT, BHP, Terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCycler red dyes, CAL Flour dyes, JOE and its derivatives, oregon green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, stilbene, DEG dyes, NR dyes, near infrared dyes, and other dyes known in the art, for example in Haugland, Molecular Probes Handbook, (Eugene, org.) 6th Edition; lakowicz, Principles of Fluorescence Spectroscopy,2nd Ed., Plenum Press New York (1999) or Hermanson, Bioconjugate Techniques,2nd Edition, or derivatives thereof or any combination thereof. Cyanine dyes can exist in sulfonated or non-sulfonated form and consist of two indolenine (indolenin), benzindolinium, pyridinium, thiazolium and/or quinolinium groups separated by a polymethine bridge between the two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3, (which may comprise 1- [6- (2, 5-dioxopyrrolidin-1-yloxy) -6-oxohexyl ] -2- (3- {1- [6- (2, 5-dioxopyrrolidin-1-yloxy) -6-oxohexyl ] -3, 3-dimethyl-1, 3-dihydro-2H-indol-2-ylidene } prop-1-en-1-yl) -3, 3-dimethyl-3H-indolium or 1- [6- (2, 5-dioxopyrrolidin-1-yloxy) -6-oxohexyl ] -2- (3- {1- [6- (2, 5-dioxopyrrolidin-1-yloxy) -6-oxohexyl ] -3, 3-dimethyl-5-sulfo-1, 3-dihydro-2H-indol-2-ylidene } prop-1-en-1-yl) -3, 3-dimethyl-3H-indolium-5-sulfonate, Cy5 (which may include 1- (6- ((2, 5-dioxopyrrolidin-1-yl) oxy) -6-oxohexyl) -2- ((1E,3E) -5- ((E) -1- (6- ((2, 5-dioxopyrrolidin-1-yl) oxy) -6-oxohexyl) -3, 3-dimethyl-5-indolin-2-ylidene) pentan-1, 3-dien-1-yl) -3, 3-dimethyl-3H-indol-1-ium or 1- (6- ((2, 5-dioxopyrrolidin-1-yl) oxy) -6-oxohexyl) -2- ((1E,3E) -5- ((E) -1- (6- ((2, 5-dioxopyrrolidin-1-yl) oxy) -6-oxohexyl) -3, 3-dimethyl-5-sulfoindolin-2-ylidene) pentan-1, 3-dien-1-yl) -3, 3-dimethyl-3H-indol-1-ium-5-sulfonate) and Cy7 (which may include 1- (5-carboxypentyl) -2- [ (1E,3E,5E,7Z) -7- (1-ethyl-1, 3-dihydro-2H-indol-2-ylidene) hept-1, 3, 5-trien-1-yl ] -3H-indolium or 1- (5-carboxypentyl) -2- [ (1E,3E,5E,7Z) -7- (1-ethyl-5-sulfo-1, 3-dihydro-2H-indol-2-ylidene) hept-1, 3, 5-trien-1-yl ] -3H-indolium-5-sulfonate), wherein "Cy" represents "cyanine", and the first number represents the number of carbon atoms between the two isoindolyl groups. Cy2 is an oxazole derivative rather than indolenine (indolenin), and benzo-derived cy3.5, cy5.5 and cy7.5 are exceptions to this rule.

In some embodiments, the detection label may be a FRET pair, such that multiple classifications may be made under a single excitation and imaging step. As used herein, FRET may include excitation exchange (Forster) transfer or electron exchange (Dexter) transfer.

B. Polymer-nucleotide conjugates

One example of a particle-nucleotide conjugate is a polymer-nucleotide conjugate. Some non-limiting examples of polymer-nucleotide conjugates are shown in fig. 5A-5C. For example, fig. 5A shows polymer-nucleotide conjugates having various configurations, e.g., a "starburst" configuration comprising a fluorescently labeled streptavidin core and four nucleotides bound to the core via biotinylated, linear PEG linkers having a molecular weight in the range of 1K daltons to 10K daltons; figure 5B shows a polymer-nucleotide conjugate having a dendrimer core, e.g., 12, 24, 48, or 96 arms, and a linear PEG linker radiating from the center with a molecular weight ranging from 1K daltons to 10K daltons; and figure 5C shows an example of a polymer-nucleotide conjugate comprising a network of, for example, streptavidin cores linked together by a branched PEG linker comprising a binding or incorporation moiety, such as biotin.

Examples of suitable linear or branched polymers include linear or branched polyethylene glycol (PEG), linear or branched polypropylene glycol, linear or branched polyvinyl alcohol, linear or branched polylactic acid, linear or branched polyglycolic acid, linear or branched polyglycine, linear or branched polyvinyl acetate, dextran, or other such polymers, or copolymers incorporating any two or more of the foregoing or incorporating other polymers known in the art. In one embodiment, the polymer is PEG. In another embodiment, the polymer may have PEG branches.

Suitable polymers may be characterized by incorporating repeating units of functional groups suitable for derivatization, such as amine, hydroxyl, carbonyl, or allyl groups. The polymer may also have one or more pre-derivatized substituents such that one or more particular subunits will incorporate a derivatization site or branching site, whether or not other subunits incorporate the same site, substituent or moiety. The pre-derivatized substituents may or may further comprise, for example, nucleotides, nucleosides, nucleotide analogs, labels such as fluorescent labels, radioactive labels, or spin labels, interacting moieties, additional polymeric moieties, or the like, or any combination of the foregoing.

In the polymer-nucleotide conjugate, the polymer may have a plurality of branches. The branched polymer may have a variety of configurations, including but not limited to a star ("starburst") form, an aggregated star ("helter skilter") form, a bottle brush, or a dendrimer. The branched polymer may radiate from a central point of attachment or central portion, or may incorporate a plurality of branch points, such as, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more branch points. In some embodiments, each subunit of the polymer may optionally constitute a separate branch point.

The length and size of the branches may vary depending on the type of polymer. In some branched polymers, the branches may have a length of 1 to 1,000nm, 1 to 100nm, 1 to 200nm, 1 to 300nm, 1 to 400nm, 1 to 500nm, 1 to 600nm, 1 to 700nm, 1 to 800nm, or 1 to 900nm or more, or a length that falls within or between any value disclosed herein.

In some polymer-nucleotide conjugates, the polymeric core can have a size corresponding to an apparent molecular weight of 1K Da, 2K Da, 3K Da, 4K Da, 5K Da, 10K Da, 15K Da, 20K Da, 30K Da, 50K Da, 80K Da, 100K Da, or any value within a range defined by any two of the above. The apparent molecular weight of a polymer can be calculated from the known molecular weights of a representative number of subunits, as determined by size exclusion chromatography, by mass spectrometry, or by any other method known in the art.

In some branched polymers, the branches may have a size corresponding to an apparent molecular weight of 1K Da, 2K Da, 3K Da, 4K Da, 5K Da, 10K Da, 15K Da, 20K Da, 30K Da, 50K Da, 80K Da, 100K Da, or any value within a range defined by any two of the foregoing. The apparent molecular weight of a polymer can be calculated from the known molecular weights of a representative number of subunits, as determined by size exclusion chromatography, by mass spectrometry, or by any other method known in the art. The polymer may have multiple branches. The number of branches in the polymer may be 2, 3, 4, 5, 6, 7, 8, 12, 16, 24, 32, 64, 128 or more, or a number falling within a range defined by any two of these.

For polymer-nucleotide conjugates comprising a branched polymer, such as a branched PEG comprising 4, 8, 16, 32, or 64 branches, the polymer-nucleotide conjugate can have nucleotides attached to the ends of the PEG branches such that each end has attached thereto 0, 1,2, 3, 4, 5, 6, or more nucleotides. In one non-limiting example, a branched PEG polymer of 3 to 128 PEG arms can be attached to the terminal one or more nucleotides of the polymer branches such that each terminal has attached to it 0, 1,2, 3, 4, 5, 6 or more nucleotides or nucleotide analogs. In some embodiments, the branched polymer or dendrimer has an even number of arms. In some embodiments, the branched polymer or dendrimer has an odd number of arms.

In some cases, the length of the linker (e.g., PEG linker) can be in the range of about 1nm to about 1,000 nm. In some cases, the linker can have a length of at least 1nm, at least 10nm, at least 25nm, at least 50nm, at least 75nm, at least 100nm, at least 200nm, at least 300nm, at least 400nm, at least 500nm, at least 600nm, at least 700nm, at least 800nm, at least 900nm, or at least 1,000 nm. In some cases, the length of the linker can be between any two values in this paragraph. For example, in some cases, the length of the linker can be in the range of about 75nm to about 400 nm. One skilled in the art will recognize that in some cases the length of the linker may have any value within the range of values in this paragraph, such as 834 nm.

In some cases, the length of the linker is different for different nucleotides (including deoxyribonucleotides and ribonucleotides), nucleotide analogs (including deoxyribonucleotide analogs and ribonucleotide analogs), nucleosides (including deoxyribonucleoside or ribonucleoside), or nucleoside analogs (including deoxyribonucleoside analogs or ribonucleoside analogs). In some cases, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs includes, for example, deoxyadenosine, and the length of the linker is 1nm to 1,000 nm. In some cases, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs includes, for example, deoxyguanosine, and the length of the linker is from 1nm to 1,000 nm. In some cases, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs includes, for example, thymidine, and the length of the linker is 1nm to 1,000 nm. In some cases, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs includes, for example, deoxyuridine, and the length of the linker is 1nm to 1,000 nm. In some cases, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs includes, for example, deoxycytidine, and the length of the linker is from 1nm to 1,000 nm. In some cases, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs includes, for example, adenosine, and the length of the linker is 1nm to 1,000 nm. In some cases, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs includes, for example, guanosine, and the length of the linker is from 1nm to 1,000 nm. In some cases, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs includes, for example, 5-methyl-uridine, and the length of the linker is 1nm to 1,000 nm. In some cases, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs comprises, for example, uridine, and the length of the linker is 1nm to 1,000 nm. In some cases, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs includes, for example, cytidine, and the length of the linker is 1nm to 1,000 nm.

In polymer-nucleotide conjugates, each branch or subset of branches of the polymer can have attached thereto a moiety comprising a nucleotide (e.g., an adenine, thymine, uracil, cytosine, or guanine residue, or a derivative or mimetic thereof) and which is capable of binding to or incorporating into a polymerase, reverse transcriptase, or other nucleotide binding or incorporation domain. Optionally, the moiety is capable of being incorporated into an extended nucleic acid strand during a polymerase reaction. In some cases, the moiety may be blocked so that it cannot be incorporated into an extended nucleic acid strand during a polymerase reaction. In some other cases, the moiety may be reversibly blocked such that it cannot be incorporated into an extended nucleic acid strand during a polymerase reaction until such blocking is removed, and then the moiety can be incorporated into the extended nucleic acid strand during the polymerase reaction.

Nucleotides may be conjugated to the polymer branch via the 5' end of the nucleotide. In some cases, nucleotides may be modified to inhibit or prevent incorporation of the nucleotide into an extended nucleic acid strand during a polymerase reaction. For example, a nucleotide may include a 3' deoxyribonucleotide, a 3' azido nucleotide, a 3' -methyl azido nucleotide, or another such nucleotide known in the art or that may be known in the art, and thus cannot be incorporated into a nucleic acid strand that becomes extended during a polymerase reaction. In some embodiments, nucleotides may include 3 '-O-azido, 3' -O-azidomethyl, 3 '-phosphorothioate, 3' -O-malonyl, 3 '-O-alkylhydroxylamine, or 3' -O-benzyl. In some embodiments, the nucleotide lacks a 3' hydroxyl group.

The polymer may also have a binding or incorporation moiety in each branch or subset of branches. Some examples of binding or incorporating moieties include, but are not limited to, biotin, avidin, streptavidin, and the like, polyhistidine domains, complementary paired nucleic acid domains, G-quadruplex-forming nucleic acid domains, calmodulin, maltose binding protein, cellulase, maltose, sucrose, glutathione-S-transferase, glutathione, O-6-methylguanine-DNA methyltransferase, benzylguanine and derivatives thereof, benzylcysteine and derivatives thereof, antibodies, epitopes, protein a, protein G. The binding or incorporation moiety may "interact" with any interacting molecule or fragment thereof known in the art that binds or facilitates an interaction between proteins, proteins and ligands, proteins and nucleic acids, or small molecule interaction domains or moieties.

In some embodiments, the compositions provided herein can comprise one or more elements of complementary interacting moieties. Non-limiting examples of complementary interacting moieties include, for example, biotin and avidin; SNAP-benzyl guanosine; antibodies or FABs and epitopes; IgG FC and protein A, protein G, protein A/G or protein L; maltose binding protein and maltose; lectins and homologous polysaccharides; an ion-chelating moiety, a complementary nucleic acid, a nucleic acid capable of forming a triplex or triplex helix interaction; nucleic acids capable of forming a G-quadruplex, and the like. One skilled in the art will readily recognize that there are many partial pairs and that they are commonly used due to the nature of their strong and specific interactions with each other; and thus any such complementary pair or group is deemed suitable for this purpose in constructing or contemplating the compositions of the present disclosure. In some embodiments, the compositions disclosed herein can include compositions in which one element of the complementary interacting moiety is attached to one molecule or multivalent ligand, and another element of the complementary interacting moiety is attached to a separate molecule or multivalent ligand. In some embodiments, a composition as disclosed herein can include a composition in which two or all elements of a complementary interacting moiety are attached to a single molecule or a multivalent ligand. In some embodiments, a composition as disclosed herein may include a composition in which two or all elements of a complementary interacting moiety are attached to separate arms or positions on a single molecule or multivalent ligand. In some embodiments, a composition as disclosed herein can include a composition in which two or all elements of a complementary interacting moiety are attached to the same arm or position on a single molecule or multivalent ligand. In some embodiments, a composition comprising one element of a complementary interacting moiety and a composition comprising another element of a complementary interacting moiety may be mixed simultaneously or sequentially. In some embodiments of the present invention, the substrate is,the interaction between molecules or particles as disclosed herein allows for the association or aggregation of multiple molecules or particles such that, for example, the detectable signal is increased. In some embodiments, the fluorescent, colorimetric, or radioactive signal is enhanced. In other embodiments, other interacting moieties disclosed herein or known in the art are contemplated. In some embodiments, a composition as provided herein can be provided such that one or more molecules comprising a first interacting moiety, such as, for example, one or more imidazole or pyridine moieties, and one or more additional molecules comprising a second interacting moiety, such as, for example, a histidine residue, are mixed, simultaneously or sequentially. In some embodiments, the composition comprises 1,2, 3, 4, 5, 6, or more imidazole or pyridine moieties. In some embodiments, the composition comprises 1,2, 3, 4, 5, 6, or more histidine residues. In such embodiments, the interaction between the provided molecules or particles may be facilitated by the presence of divalent cations such as nickel, manganese, magnesium, calcium, strontium, and the like. In some embodiments, for example, (His)₃The group can coordinate to another molecule or particle through nickel or manganese ion (His)₃The groups interact.

The multivalent binding or incorporation composition may comprise one or more buffers, salts, ions, or additives. In some embodiments, representative additives may include, but are not limited to, betaine, spermidine, detergents such as Triton X-100, Tween 20, SDS or NP-40, ethylene glycol, polyethylene glycol, dextran, polyvinyl alcohol, vinyl alcohol, methyl cellulose, heparin, heparan sulfate, glycerol, sucrose, 1, 2-propanediol, DMSO, N-trimethylglycine, ethanol, ethoxyethanol, propylene glycol, polypropylene glycol, block copolymers such as pluronic (r) series polymers, arginine, histidine, imidazole, or any combination thereof, or any substance known in the art as a DNA "relaxant" (a compound that has the effect of altering DNA persistence length, altering the number of linkages or crossovers within a polymer, or altering the conformational kinetics of a DNA molecule such that accessibility of an intrachain site to a DNA binding or incorporation moiety is increased).

The multivalent binding or incorporation composition may include a zwitterionic compound as an additive. Other representative additives may be known in Lorenz, t.c.j.vis.exp. (63), e3998, doi:10.3791/3998(2012), the disclosure of which is incorporated herein by reference with respect to additives that facilitate nucleic acid binding or kinetics, or processes involving manipulation, use or storage of nucleic acids. In some embodiments, representative cations may include, but are not limited to, sodium, magnesium, strontium, potassium, manganese, calcium, lithium, nickel, cobalt, or other such cations known in the art to facilitate nucleic acid interactions, e.g., such as self-association, secondary or tertiary structure formation, base pairing, surface association, peptide association, protein binding, and the like.

Binding between target nucleic acids and multivalent binding or incorporation compositions

When using multivalent binding or incorporation compositions instead of single unconjugated or untethered nucleotides to form complexes with one or more copies of the polymerase and target nucleic acid, the local concentration of nucleotides and the binding affinity of the complexes (in the case of complexes comprising two or more target nucleic acid molecules) is increased many-fold, which in turn enhances signal strength, particularly correct signals and mismatches. The present disclosure contemplates contacting a multivalent binding or incorporation composition with a polymerase and a primed target nucleic acid to determine formation of a ternary binding or incorporation complex.

Figure 6 illustrates the use of the disclosed polymer-nucleotide conjugates to achieve increased signal intensity during binding, persistence, and washing/removal steps. Binding between the polymerase, the primed target strand and the polymer-conjugated nucleotide becomes more advantageous when the nucleotide is complementary to the next base of the target nucleic acid due to the local concentration increase of nucleotides on the polymer-nucleotide conjugate and/or the formation of non-covalent bonds with two or more primed target nucleic acid molecules. The binding complex formed has a longer duration, which in turn helps to increase the signal and shorten the imaging step. The high signal intensity resulting from the use of the disclosed polymer-nucleotide conjugates remains stable throughout the binding and imaging steps. The strong binding between the polymerase, the primed target strand and the polymer-conjugated nucleotide or nucleotide analogue also means that the binding complex thus formed will remain stable during the washing step, as other reaction mixture components and unmatched nucleotide analogues are washed away. After the imaging step, the binding complex can be destabilized (e.g., by changing the buffer composition), and the primed target nucleic acid can then be extended by one base. After extension, the binding and imaging steps can be repeated using the disclosed polymer-nucleotide conjugates to determine the identity of the next base.

For example, a graphical depiction of the increase in signal intensity during binding, persistence, and washing/removal of a multivalent substrate as described herein is provided in fig. 6, which represents the experimentally observed change in signal intensity. Thus, the compositions and methods of the present disclosure provide a robust and controllable means of establishing and maintaining ternary enzyme complexes, as well as a greatly improved means by which the presence of such complexes can be identified and/or measured, and by which the persistence of such complexes can be controlled. This provides an important solution to problems such as determining the identity of N +1 bases in nucleic acid sequencing applications.

Without wishing to be bound by any particular theory, it has been observed that the multivalent binding compositions disclosed herein associate with polymerase nucleotide complexes to form ternary binding complexes with time-dependent rates, although significantly slower than the association rates known to be obtainable with nucleotides in free solution. Thus, the association Rate (K)_on) Significantly and surprisingly slower than the association rate of a single nucleotide or a nucleotide not attached to a multivalent ligand complex. However, importantly, the dissociation rate (K) of the multivalent ligand complex_off) Significantly slower than the dissociation rates observed for nucleotides in free solution. Thus, the multivalent ligand complexes of the present disclosure provide a surprising and beneficial improvement in the persistence of ternary polymerase-polynucleotide-nucleotide complexes (particularly with respect to such complexes formed from free nucleotides) that allows for significant improvements in imaging quality for nucleic acid sequencing applications such as currently available methods and reagents. Importantly, this disclosure providesSuch properties of the multivalent binding composition of (a) allow the formation of visible ternary complexes to be controllable such that subsequent visualization, modification or processing steps can be performed substantially irrespective of dissociation of the complex-that is, the complex can be otherwise formed, imaged, modified or used as desired, and will remain stable until the user performs a positive dissociation step, such as exposing the complex to a dissociation buffer.

In some cases, the duration of a multivalent binding complex formed using the disclosed particle-nucleotide or polymer-nucleotide conjugates can range from about 0.1 seconds to about 600 seconds under non-labile conditions. In some cases, the duration may be at least 0.1 seconds, at least 1 second, at least 2 seconds, at least 3 seconds, at least 4 seconds, at least 5 seconds, at least 6 seconds, at least 7 seconds, at least 8 seconds, at least 9 seconds, at least 10 seconds, at least 20 seconds, at least 30 seconds, at least 40 seconds, at least 50 seconds, at least 60 seconds, at least 120 seconds, at least 180 seconds, at least 240 seconds, at least 300 seconds, at least 360 seconds, at least 420 seconds, at least 480 seconds, at least 540 seconds, or at least 600 seconds. In some cases, the duration may be between any two values specified in this paragraph. For example, in some cases, the duration may be in a range of about 10 seconds to about 360 seconds. Those skilled in the art will recognize that in some cases, the duration may have any value within the range of values specified in this paragraph, such as 78 seconds.

In various embodiments, polymerases useful for the binding or incorporation interactions described herein can include any polymerase known or potentially known in the art. For example, it is well known that each organism encodes within its genome one or more DNA polymerases. Examples of suitable polymerases can include, but are not limited to: klenow DNA polymerase, Thermus aquaticus DNA polymerase I (Taq polymerase), Klenow Taq polymerase and bacteriophage T7 DNA polymerase; human alpha, delta and epsilon DNA polymerases; phage polymerases such as T4, RB69, and phi29 phage DNA polymerase, intense Pyrococcus DNA polymerase (Pfu polymerase); bacillus subtilis DNA polymerase III, and Escherichia coli DNA polymerase III alpha and epsilon; 9 degree N polymerase, reverse transcriptaseSuch as HIV type M or O reverse transcriptase, avian myeloblastosis virus reverse transcriptase, or Moloney Murine Leukemia Virus (MMLV) reverse transcriptase or telomerase. Further non-limiting examples of DNA polymerases can include those from various archaea, such as Aeropyrum (Aeropyrum), Archaeoglobus (Archaeoglobus), Desulfurococcus (Desulfocus), Pyrobaculum (Pyrobaculum), Pyrococcus (Pyrococcus), Pyrolobus (Pyrolobus), Pyrolusitum (Pyroditicum), Pyrococcus (Staphylothermus), Staphylothermus (Stetteria), Sulfolobus (Sulfolobus), Pyrococcus (Thermococcus), and Pyrococcus (Vulcanisaeta), and the like, or variants thereof, including polymerases known in the art, such as Vent^TM、Deep Vent^TM、Pfu、KOD、Pfx、Therminator^TMAnd Tgo polymerase. In some embodiments, the polymerase is a klenow polymerase.

When the nucleotides on the polymer-nucleotide conjugate are complementary to the target nucleic acid, the ternary complex has a longer duration than a non-complementary nucleotide. When the nucleotides on the polymer-nucleotide conjugate are complementary to the target nucleic acid, the ternary complex also has a longer duration than the unconjugated or tethered complementary nucleotides. For example, in some embodiments, the ternary complex may have a duration of less than 1 second, greater than 2 seconds, greater than 3 seconds, greater than 5 seconds, greater than 10 seconds, greater than 15 seconds, greater than 20 seconds, greater than 30 seconds, greater than 60 seconds, greater than 120 seconds, greater than 360 seconds, greater than 3600 seconds or more, or a time within a range defined by any two or more of these values.

The duration may be measured, for example, by observing the onset and/or duration of the binding complex, for example by observing a signal from a labeled component of the binding complex. For example, a labeled nucleotide or a labeling reagent comprising one or more nucleotides may be present in the binding complex, allowing detection of a signal from the label during the duration of the binding complex.

It has been observed that different ranges of durations can be obtained with different salts or ions, indicating, for example, in the case of magnesium ions (Mg)²⁺) In the presence ofThe complexes formed form faster than complexes formed with other ions. It has also been observed that in the case of, for example, strontium ions (Sr)²⁺) The complex formed in the presence of (a) readily forms and dissociates completely or has substantial integrity after ion withdrawal or after washing with a buffer lacking one or more components of the composition of the invention, such as, for example, a polymer and/or one or more nucleotides, and/or one or more interacting moieties, or a buffer containing, for example, a chelating agent that can cause or accelerate the removal of divalent cations from a polyvalent reagent containing the complex. Thus, in some embodiments, the compositions of the present disclosure comprise Mg²⁺. In some embodiments, the compositions of the present disclosure comprise Ca²⁺. In some embodiments, the compositions of the present disclosure comprise Sr²⁺. In some embodiments, the compositions of the present disclosure comprise cobalt ions (Co)²⁺). In some embodiments, the compositions of the present disclosure comprise MgCl₂. In some embodiments, the compositions of the present disclosure comprise CaCl₂. In some embodiments, the compositions of the present disclosure comprise SrCl₂. In some embodiments, the compositions of the present disclosure comprise CoCl₂. In some embodiments, the composition does not comprise, or substantially does not comprise, magnesium. In some embodiments, the composition is free or substantially free of calcium. In some embodiments, the methods of the present disclosure provide for contacting one or more nucleic acids with one or more compositions disclosed herein, wherein the composition lacks one or both of calcium or magnesium.

The dissociation of the ternary complex can be controlled by varying the buffer conditions. After the imaging step, the ternary complex is dissociated using a buffer with increased salt content so that the labeled polymer-nucleotide conjugate can be washed away, thereby providing a means by which the signal can be attenuated or terminated, for example, at the transition between one sequencing cycle and the next. In some embodiments, this dissociation can be affected by washing the complex with a buffer lacking the necessary metal or cofactor. In some embodiments, the wash buffer may comprise one or more compositions for maintaining pH control. In some embodiments, the wash buffer may comprise one or more monovalent cations, such as sodium. In some embodiments, the wash buffer lacks or substantially lacks divalent cations, e.g., does not contain or substantially contains strontium, calcium, magnesium, or manganese. In some embodiments, the wash buffer further comprises a chelating agent, such as, for example, EDTA, EGTA, nitrilotriacetic acid, polyhistidine, imidazole, and the like. In some embodiments, the wash buffer may maintain the pH of the environment at the same level as the binding complex. In some embodiments, the wash buffer can raise or lower the pH of the environment relative to the level seen by the binding complex. In some embodiments, the pH may be in the range of 2-4, 2-7, 5-8, 7-9, 7-10, or below 2, or above 10, or within a range defined by any two of the values provided herein.

The addition of a specific ion may affect the binding of the polymerase to the primed target nucleic acid, the formation of a ternary complex, the dissociation of a ternary complex, or the incorporation of one or more nucleotides into the extended nucleic acid, for example during a polymerase reaction. In some embodiments, the relevant anions can include chloride, acetate, gluconate, sulfate, phosphate, and the like. In some embodiments, the treatment may be carried out by adding one or more acids, bases or salts, for example NiCl₂、CoCl₂、MgCl₂、MnCl₂、SrCl₂、CaCl₂、CaSO₄、SrCO₃、BaCl₂And the like, into the compositions of the present disclosure. Representative salts, ions, solutions and conditions are known from Remington, The Science and Practice of Pharmacy,20th. edition, Gennaro, a.r., Ed. (2000), The entire contents of which are incorporated herein by reference, especially in connection with The 17 th article and related disclosure of salts, ions, salt solutions and ionic solutions.

The present disclosure contemplates contacting a multivalent binding or incorporation composition comprising at least one particle-nucleotide conjugate with one or more polymerases. The contacting can optionally be performed in the presence of one or more target nucleic acids. In some embodiments, the target nucleic acid is a single stranded nucleic acid. In some embodiments, the target nucleic acid is a primed single-stranded nucleic acid. In some embodiments, the target nucleic acid is a double-stranded nucleic acid. In some embodiments, the contacting comprises contacting the multivalent binding or incorporation composition with a polymerase. In some embodiments, the contacting comprises contacting the composition comprising one or more nucleotides with a plurality of polymerases. The polymerase may bind to a single nucleic acid molecule.

The binding between the target nucleic acid and the multivalent binding composition can be provided in the presence of a polymerase that has become catalytically inactive. In one embodiment, the polymerase may have been rendered catalytically inactive by mutation. In one embodiment, the polymerase may have been rendered catalytically inactive by chemical modification. In some embodiments, the polymerase may become catalytically inactive due to the lack of necessary substrates, ions, or cofactors. In some embodiments, the polymerase may become catalytically inactive due to the absence of magnesium ions.

Binding between the target nucleic acid and the multivalent binding composition occurs in the presence of a polymerase, wherein the binding solution, reaction solution, or buffer is devoid of magnesium or manganese. Alternatively, binding between the target nucleic acid and the multivalent binding composition occurs in the presence of a polymerase, wherein the binding solution, reaction solution, or buffer comprises calcium or strontium.

When a non-catalytically active polymerase is used to facilitate interaction of a nucleic acid with a multivalent binding composition, the interaction between the composition and the polymerase stabilizes the ternary complex, such that the complex can be detected by fluorescence or other methods disclosed herein or otherwise known in the art. Unbound polymer-nucleotide conjugate may optionally be washed away prior to detection of the ternary binding complex.

One or more nucleic acids are contacted with the polymer-nucleotide conjugates disclosed herein in a solution containing one of calcium or magnesium or both calcium and magnesium. Alternatively, one or more nucleic acids are contacted with the polymer-nucleotide conjugates disclosed herein in a solution lacking one or both of calcium or magnesium, and in a separate step, regardless of the order of the steps, one of calcium or magnesium, or both calcium and magnesium, are added to the solution. In some embodiments, one or more nucleic acids are contacted with the polymer-nucleotide conjugates disclosed herein in a solution lacking strontium, and including in a separate step, regardless of the order of the steps, adding strontium to the solution.

Use of multivalent binding or incorporation compositions in combination with low non-specific binding surfaces

Disclosed herein are solid supports comprising low non-specific binding surface compositions capable of improving nucleic acid hybridization and amplification performance. In general, the disclosed supports can comprise a substrate (or support structure), one or more covalently or non-covalently attached low binding chemical modification layers (e.g., silane layers, polymer films), and one or more covalently or non-covalently attached primer sequences that can be used to tether a single-stranded target nucleotide to the support surface. In some cases, the formulation of the surface, e.g., the chemical composition of one or more layers, coupling chemistry for crosslinking one or more layers to the support surface and/or to each other, and the total number of layers, can be varied to minimize or reduce non-specific binding of proteins, nucleic acid molecules, and other hybridization and amplification reaction components to the support surface relative to a comparable monolayer. In general, the formulation of the surface can be altered such that non-specific hybridization on the support surface is minimized or reduced relative to a comparable monolayer. The formulation of the surface can be altered such that non-specific amplification on the support surface is minimized or reduced relative to a comparable monolayer. The formulation of the surface may be altered so as to maximise the specific amplification rate and/or yield on the surface of the support. In some cases disclosed herein, a level of amplification suitable for detection is achieved in no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 30 or more amplification cycles.

Examples of materials that may be used to make the substrate or support structure include, but are not limited to, glass, fused silica, silicon, polymers (e.g., Polystyrene (PS), macroporous polystyrene (MPPS), Polymethylmethacrylate (PMMA), Polycarbonate (PC), polypropylene (PP), Polyethylene (PE), High Density Polyethylene (HDPE), Cyclic Olefin Polymer (COP), Cyclic Olefin Copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of glass and plastic substrates are contemplated.

The substrate or carrier structure may be present in any of a variety of geometries and dimensions known to those skilled in the art, and may comprise any of a variety of materials known to those skilled in the art. For example, in some cases, the substrate or carrier structure may be locally planar (e.g., including a microscope glass slide or a surface of a microscope glass slide). In general, the substrate or support structure can be cylindrical (e.g., including the inner surface of a capillary or capillary), spherical (e.g., including the outer surface of a non-porous bead), or irregular (e.g., including the outer surface of an irregular shape, a non-porous bead, or a particle). In some cases, the surface of the substrate or support structure for nucleic acid hybridization and amplification may be a solid, non-porous surface. In some cases, the surface of the substrate or support structure used for nucleic acid hybridization and amplification can be porous such that the coatings described herein penetrate the porous surface and nucleic acid hybridization and amplification reactions performed thereon can occur within the pores.

The substrate or support structure including one or more chemically modified layers (e.g., a layer of low non-specifically bound polymer) may be separate or integrated into another structure or component. For example, in some cases, a substrate or carrier structure may include one or more surfaces within an integrated or assembled microfluidic flow cell. The substrate or carrier structure may comprise one or more surfaces within the microplate format, such as the bottom surfaces of the wells in the microplate. As noted above, in some preferred embodiments, the substrate or carrier structure comprises an inner surface (e.g., a luminal surface) of the capillary tube. In an alternative preferred embodiment, the substrate or carrier structure comprises the inner surface (e.g. the lumen surface) of the capillaries etched into a planar chip.

As noted, the low non-specific binding vectors of the present disclosure exhibit reduced non-specific binding of proteins, nucleic acids, and other components of hybridization and/or amplification preparations for solid phase nucleic acid amplification. The degree of non-specific binding exhibited by a given support surface can be assessed qualitatively or quantitatively. For example, in some cases, a surface can be exposed to a fluorescent dye (e.g., a cyanine such as Cy3 or Cy5, etc., fluorescein, coumarin, rhodamine, etc., or other dyes disclosed herein), a fluorescently labeled nucleotide, a fluorescently labeled oligonucleotide, and/or a fluorescently labeled protein (e.g., polymerase) under a standard set of conditions, followed by a prescribed washing procedure and fluorescence imaging as a qualitative tool for comparing non-specific binding on a carrier containing different surface preparations. In some cases, the surface may be exposed to a fluorescent dye, fluorescently labeled nucleotide, fluorescently labeled oligonucleotide, and/or fluorescently labeled protein (e.g., polymerase) under a standard set of conditions, followed by a prescribed washing protocol and fluorescence imaging used as a quantitative tool for comparing non-specific binding on carriers containing different surface preparations, provided that it is ensured that fluorescence imaging is performed under conditions where the fluorescence signal is linearly related (or related in a predictable manner) to the number of fluorophores on the carrier surface (e.g., under conditions where signal saturation and/or self-quenching of fluorophores is not an issue). In some cases, other techniques known to those skilled in the art, such as radioisotope labeling and counting methods, can be used to quantitatively assess the degree of nonspecific binding exhibited by the different carrier surface formulations of the present disclosure.

Some surfaces disclosed herein exhibit a ratio of specific to non-specific binding of a fluorophore, such as Cy3, of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value within the ranges herein. Some surfaces disclosed herein exhibit a ratio of specific to non-specific fluorescence of a fluorophore, such as Cy3, of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value within the ranges herein.

As noted, in some cases, the degree of non-specific binding exhibited by the disclosed low-binding vectors can be assessed using a method for contacting the surface with a labeled protein (e.g., Bovine Serum Albumin (BSA), streptavidin, DNA polymerase, reverse transcriptase, helicase, single-stranded binding protein (SSB), or the like, or any combination thereof), a labeled nucleotide, a labeled oligonucleotide, or the like, under a standard set of incubation and rinsing conditions, followed by detecting the amount of label remaining on the surface, and comparing the resulting signal to an appropriate calibration standard. In some cases, the label may comprise a fluorescent label. In some cases, the label may comprise a radioisotope. In some cases, the label may comprise any other detectable label known to those of skill in the art. In some cases, the degree of non-specific binding exhibited by a given carrier surface preparation can thus be assessed in terms of the number of non-specifically bound protein molecules (or other molecules) per unit area. In some cases, the low-binding carriers of the present disclosure may exhibit less than 0.001 molecules/μm²Less than 0.01 molecules/. mu.m²0.1 molecules/. mu.m²0.25 molecules/. mu.m²0.5 molecules/. mu.m²1 molecule/. mu.m²10 molecules/. mu.m²100 molecules/. mu.m²Or 100 molecules/. mu.m²Non-specific protein binding (or non-specific binding of other specific molecules, such as Cy3 dye). One skilled in the art will recognize that a given support surface of the present disclosure may exhibit non-specific binding anywhere within this range, e.g., less than 86 molecules/μm². For example, some modified surfaces disclosed herein show less than 0.5 molecules/μ M after 15 minutes of contact with 1 μ M of Cy 3-labeled streptavidin (GE Amersham) in Phosphate Buffered Saline (PBS) buffer, followed by 3 washes with deionized water²Non-specific protein binding. Some of the modified surfaces disclosed herein exhibit less than 2 molecules/um²Non-specific binding of Cy3 dye molecules. In a separate non-specific binding assay, will1 μ M labeled Cy3 SA (ThermoFisher), 1 μ M Cy5 SA dye (ThermoFisher), 10 μ M aminoallyl-dUTP-ATTO-647N (Jena Biosciences), 10 μ M aminoallyl-dUTP-ATTO-Rhol 1(Jena Biosciences), 10 μ M aminoallyl-dUTP-ATTO-Rhol 1(Jena Biosciences), 10 μ M7-propargylamino-7-deaza-dGTP-Cy 5(Jena Biosciences and 10 μ M7-propargylamino-7-deaza-dGTP-Cy 3(Jena Biosciences) were incubated in 384 format on a low binding substrate for 15 minutes at 37 ℃ each well was rinsed with 50ul deionized RNase/free water 2-3 times and 25mm 673.26 mm pH 673 buffer (ACeS Biosciences) and rinsed with 26 times pH 26. 3. Cy3. manufactured using the indicated plate pH 673. Cy buffer, AF555 or Cy5 filter banks (according to the dye tests performed) 384 well plates were imaged on a GE Typhoon (Pittsburgh, PA) instrument specified by the manufacturer, and PMT gain was set at 800 and resolution 50-100 μm. For higher resolution imaging, images were collected on an Olympus IX83 microscope (Olympus corp., center valley, PA) with a Total Internal Reflection Fluorescence (TIRF) objective (20-fold, 0.75NA or 100-fold, 1.5NA, Olympus), a CCD camera (e.g., Olympus EM-CCD black and white camera, Olympus XM-10 black and white camera, or Olympus DP80 color and black and white camera), an illumination source (e.g., Olympus 100W Hg lamp, Olympus 75W Xe lamp, or Olympus U-HGLGPS fluorescence light source), a cmos Andor camera (zyma 4.2. dichroic mirror from Semrock (IDEX Health, PA)&Science, LLC, Rochester, LLC), such as 405, 488, 532, or 633nm dichroic mirrors/beam splitters, and a bandpass filter is selected as 532LP or 645LP, which is coincident with the appropriate excitation wavelength. Some of the modified surfaces disclosed herein exhibit less than 0.25 molecules/μm²Non-specific binding of the dye molecule of (a). In some cases, the surfaces disclosed herein exhibit a ratio of specific to non-specific binding of a fluorophore, e.g., Cy3, of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value within the ranges herein.

In some cases, the surfaces disclosed herein exhibit a ratio of specific to non-specific fluorescence signals of a fluorophore, e.g., Cy3, of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value within the ranges herein.

Low background surfaces consistent with the disclosure herein may exhibit a ratio of specific dye attachment (e.g., Cy3 attachment) to non-specific dye adsorption (e.g., Cy3 dye adsorption) or greater than 50 specific dye molecules per molecule adsorbed, of at least 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50: 1. Similarly, a low background surface having attached a fluorophore (e.g., Cy3) consistent with the disclosure herein may exhibit a ratio of a specific fluorescent signal (e.g., derived from a Cy3 labeled oligonucleotide attached to the surface) to the non-specific adsorption dye fluorescent signal of at least 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or greater than 50:1 when subjected to excitation energy.

In some cases, the degree of hydrophilicity (or "wettability" with aqueous solutions) of the disclosed support surfaces can be evaluated, for example, by measuring water contact angles (where a droplet of water is placed on a surface and the contact angle with the surface is measured using, for example, an optical tensiometer). In some cases, the static contact angle may be determined. In some cases, the advancing or receding contact angle may be determined. In some cases, the water contact angle of the hydrophilic, low-binding support surfaces disclosed herein can range from about 0 degrees to about 50 degrees. In some cases, the water contact angle of a hydrophilic, low-binding support surface disclosed herein can be no more than 50 degrees, 45 degrees, 40 degrees, 35 degrees, 30 degrees, 25 degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8 degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree. In many cases, the contact angle does not exceed any value within this range, such as not exceeding 40 degrees. One skilled in the art will recognize that a given hydrophilic, low binding support surface of the present disclosure may exhibit a water contact angle having any value within this range, such as about 27 degrees.

In some cases, the hydrophilic surfaces disclosed herein generally help to reduce wash time for bioassays due to reduced non-specific binding of biomolecules to low binding surfaces. In some cases, sufficient washing steps may be performed in less than 60 seconds, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 15 seconds, 10 seconds, or less than 10 seconds. For example, in some cases, sufficient washing steps may be performed in less than 30 seconds.

Some low-bonding surfaces of the present disclosure exhibit significant improvements in stability or durability to prolonged exposure to solvents and high temperatures, or repeated cycling of solvent exposure or temperature changes. For example, in some cases, the stability of the disclosed surfaces can be detected by fluorescently labeling functional groups on the surface or tethered biomolecules (e.g., oligonucleotide primers) on the surface and monitoring the fluorescent signal before, during, and after prolonged exposure to solvent and elevated temperatures, or repeated cycles of solvent exposure or temperature changes. In some cases, the degree of fluorescence change used to assess surface quality may be less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% over a period of time of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 100 hours (or any combination of these percentages measured over these periods of time) of exposure to solvent and/or elevated temperature. In some cases, the degree of fluorescence change used to assess surface quality can be less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% (or any combination of these percentages measured over the range of cycles) over 5 cycles, 10 cycles, 20 cycles, 30 cycles, 40 cycles, 50 cycles, 60 cycles, 70 cycles, 80 cycles, 90 cycles, 100 cycles, 200 cycles, 300 cycles, 400 cycles, 500 cycles, 600 cycles, 700 cycles, 800 cycles, 900 cycles, or 1,000 cycles of repeated exposure to solvent changes and/or temperature changes.

In some cases, the surfaces disclosed herein may exhibit a high ratio of specific signal to non-specific signal or other background. For example, when used for nucleic acid amplification, some surfaces can exhibit an amplification signal that is at least 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, or more than 100-fold greater than the signal of adjacent non-dense regions of the surface. Similarly, some surfaces exhibit amplification signals that are at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, or more than 100-fold greater than the signals of adjacent amplified nucleic acid population regions of the surface.

In some cases, the disclosed fluorescent images of low background surfaces exhibit a contrast to noise ratio (CNR) of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 210, 220, 230, 240, 250, or greater than 250 when used in nucleic acid hybridization or amplification applications to produce hybridized or clonally amplified nucleic acid molecule clusters (e.g., that have been directly or indirectly labeled with a fluorophore).

One or more types of primers may be attached or tethered to the support surface. One or more types of oligonucleotide molecules may be attached or tethered to the surface of the carrier. In some cases, one or more types of oligonucleotide adaptors or primers may comprise a spacer sequence, an adaptor sequence for hybridization to an adaptor-ligated template library nucleic acid sequence, a forward amplification primer, a reverse amplification primer, a sequencing primer, and/or a molecular barcode sequence, or any combination thereof. In some cases, 1 primer or adapter sequence may be tethered to at least one layer of the surface. In some cases, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different primers or adapter sequences can be tethered to at least one layer of the surface.

In some cases, the length of the tethered oligonucleotide adapter and/or primer sequence can range from about 10 nucleotides to about 100 nucleotides. In some cases, the tethered oligonucleotide adaptors and/or primer sequences can be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides in length. In some cases, the tethered oligonucleotide adaptors and/or primer sequences can be up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, or up to 10 nucleotides in length. Any lower and upper limit values described in this paragraph can be combined to form ranges encompassed by the present disclosure, e.g., in some cases, the length of the tethered oligonucleotide adapter and/or primer sequence can be in the range of about 20 nucleotides to about 80 nucleotides. One skilled in the art will recognize that the length of the tethered oligonucleotide adapter and/or primer sequence can have any value within this range, such as about 24 nucleotides.

In some cases, the resulting surface density of oligonucleotide adaptors or primers on the low-binding support surface of the present disclosure may be in the range of about 100 primer molecules/μm²To about 1,000,000 primer molecules/. mu.m²Within the range of (1). In some cases, the resulting surface density of primers on the surface of a low-binding support of the present disclosure can be about 1,000 primer molecules/μm²To about 1,000,000 primer molecules/. mu.m²Within the range of (1). In some cases, the surface density of the primers can be at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000 molecules/μm². In some cases, the surface density of the primer can be at most 1,000,000, at most 100,000, at most 10,000, or at most 1,000 molecules/μm². Any of the lower and upper values described in this paragraph can be combined to form ranges encompassed by the present disclosure, e.g., in some cases, the surface density of adapters or primers can be in the range of about 10,000 molecules/μm²To about 100,000 molecules/. mu.m²Within the range of (1). One skilled in the art will recognize that the surface density of adapter or primer molecules can have any value within this range, for example, in some cases about 3,800 molecules/μm²And in other cases about 455,000 molecules/μm². In some cases, as will be discussed further belowThe surface density of template library nucleic acid sequences (e.g., sample DNA molecules) that initially hybridize to the adapter or primer sequences on the surface of the support can be less than or equal to the density indicated by the surface density of tethered oligonucleotide primers. In some cases, as will be discussed further below, the surface density of clonally amplified template library nucleic acid sequences that hybridize to adapter or primer sequences on the surface of a vector may span the same or different range of densities as indicated by the surface density of tethered oligonucleotide adapters or primers.

The local surface density of the adapter or primer molecules as listed above does not exclude variations in density over the entire surface, such that the surface may comprise molecules having, for example, 500,000/um²While also comprising at least a second region having a substantially different local density.

Illustrative alternative embodiments

The disclosed method for determining a target nucleic acid sequence comprises: a) contacting a double-stranded or partially double-stranded target nucleic acid molecule comprising a template strand to be sequenced and a primer strand to be extended with one or more of the disclosed nucleic acid binding compositions; and b) detecting binding of the nucleic acid binding composition to the nucleic acid molecule, thereby determining the presence of one of the one or more nucleic acid binding compositions on the nucleic acid molecule and the identity of the next nucleotide (i.e., N +1 or terminal nucleotide) to be incorporated into the complementary strand.

The sequencing method may further comprise incorporating N +1 or terminal nucleotides into the primer strand, and then repeating the contacting, detecting, and incorporating steps for one or more additional iterations to determine the sequence of the template strand of the nucleic acid molecule. After the step of detecting the ternary binding complex, the primed strand of the primed target nucleic acid is extended by one base before another round of analysis is performed. The primed target nucleic acid can be extended using conjugated nucleotides attached to a polymer in the multivalent binding composition or using unconjugated or untethered free nucleotides provided after removal of the multivalent binding composition.

Detection of the ternary complex is achieved before, simultaneously with, or after nucleotide residue incorporation. In some embodiments, the primed target nucleic acid may comprise a target nucleic acid having multiple primed positions for attachment of a polymerase and/or a nucleic acid binding moiety. In some embodiments, multiple polymerases can be attached to a single target nucleic acid molecule, e.g., at multiple sites within a target nucleic acid molecule. In some embodiments, multiple polymerases can bind to a multivalent binding composition comprising multiple nucleotides disclosed herein. In some embodiments, the target nucleic acid molecule can be the product of strand displacement synthesis, rolling circle amplification, tandem or fusion of multiple copies of a query sequence, or other such methods known in the art or disclosed elsewhere herein to produce products comprising multiple copies of the same sequence of the nucleic acid molecule. Thus, in some embodiments, multiple polymerases can attach at multiple identical or substantially identical positions within a target nucleic acid comprising multiple identical or substantially identical copies of a query sequence. In some embodiments, the plurality of polymerases can then participate in interactions with one or more multivalent binding complexes; however, in preferred embodiments, the number of binding sites within the target nucleic acid is at least two, and the number of nucleotides or substrate moieties present on the particle-nucleotide conjugate, e.g., polymer-nucleotide conjugate, is also greater than or equal to 2.

It may be advantageous to provide multivalent binding compositions in combination with other elements, for example to provide optimized signals, for example to provide identification of nucleotides at specific positions in a nucleic acid sequence. In some embodiments, the compositions disclosed herein are provided in combination with a surface that provides low background binding or low levels of protein binding, particularly a hydrophilic or polymer coated surface. Representative surfaces may be found, for example, in U.S. patent application No. 16/363,842, which is incorporated by reference herein in its entirety.

In some cases, the nucleic acid molecule is tethered to the surface of the solid support, for example, by hybridization of a template strand to an adaptor nucleic acid sequence or primer nucleic acid sequence tethered to the solid support. In some cases, the solid support comprises a glass, fused silica, silicon, or polymer substrate. In some cases, the solid support comprises a low non-specific binding coating comprising one or more hydrophilic polymer layers (e.g., PEG layers), wherein at least one hydrophilic polymer layer comprises branched polymer molecules (e.g., branched PEG molecules comprising 4, 8, 16, or 32 branches).

The solid support comprises about 1,000 primer molecules/. mu.m²To about 1,000,000 primer molecules/. mu.m²Is tethered to the oligonucleotide adaptors or primers of the at least one hydrophilic polymer layer. In some cases, the surface density of the oligonucleotide primers can be at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000 molecules/μm². In some cases, the surface density of the oligonucleotide primer can be at most 1,000,000, at most 100,000, at most 10,000, or at most 1,000 molecules/μm². Any of the lower and upper values described in this paragraph can be combined to form ranges encompassed by the present disclosure, e.g., in some cases, the surface density of the primers can range from about 10,000 molecules/μm²To about 100,000 molecules/. mu.m²Within the range of (1). One skilled in the art will recognize that the surface density of primer molecules can have any value within this range, for example, about 455,000 molecules/μm²。

One of ordinary skill will recognize that in a series of iterative sequencing reactions, sometimes one or more sites fail to incorporate nucleotides during a given cycle, resulting in one or more sites being out of sync with the mostly elongated nucleic acid strand. These incorporation failures will generate discrete errors in the output sequence under conditions in which the sequencing signal is derived from reactions occurring on a single copy of the target nucleic acid. It is an object of the present disclosure to describe methods for reducing such errors in sequencing reactions. For example, the frequency of "skip" cycles in which no base is incorporated can be reduced by providing an increased probability of re-association when the ternary polymerase complex dissociates prematurely, using a multivalent substrate capable of incorporation into the extended strand. Thus, in some embodiments, the present disclosure contemplates the use of a multivalent substrate as disclosed herein, wherein the nucleoside moiety is contained within a nucleotide having a free or reversibly modified 5' phosphate, diphosphate, or triphosphate moiety, and wherein the nucleotide is linked to a particle or polymer disclosed herein through a labile or cleavable linkage. In some embodiments, the present disclosure contemplates a reduction in the inherent error rate due to skipped incorporation resulting from the use of multivalent substrates as disclosed herein.

The present disclosure also contemplates sequencing reactions in which sequencing signals from or associated with a given sequence originate or originate from a definable region containing multiple copies of a target sequence. An advantage of a sequencing method incorporating multiple copies of the target sequence is that the signal can be amplified since there are multiple simultaneous sequencing reactions within a defined region, each providing its own signal. The presence of multiple signals within a defined region also reduces the effect of any single skip cycle, since a large number of correctly base detected signals may overwhelm a small number of skipped or incorrectly base detected signals. The present disclosure further contemplates including free, unlabeled nucleotides during the extension reaction or during separate portions of the extension cycle to provide for incorporation at sites that may have been skipped in previous cycles. For example, unlabeled blocked nucleotides can be added during or after the incorporation cycle so that they can be incorporated at skipped sites. The unlabeled blocked nucleotides can be of the same type or types as the nucleotides attached to the multivalent binding substrate or substrates that are or were present during a particular cycle, or can include a mixture of 1,2, 3, 4, or more types of unlabeled blocked nucleotides.

When each sequencing cycle is perfectly performed, every reaction within a defined region will provide the same signal. However, as described elsewhere herein, in a series of iterative sequencing reactions, sometimes one or more sites fail to incorporate nucleotides during a given cycle, resulting in one or more sites being out of synchronization with the mostly elongated nucleic acid strand. This problem, known as "phase splitting", results in degradation of the sequencing signal because the signal is contaminated with spurious signals from sites skipping one or more cycles. This in turn creates the possibility of base discrimination errors. The gradual accumulation of skipped cycles over multiple cycles also reduces the effective read length due to gradual degradation of the sequencing signal in each cycle. It is another object of the present disclosure to provide methods for reducing phase separation errors and/or increasing read length in sequencing reactions.

Sequencing methods can include contacting a target nucleic acid or nucleic acids comprising multiple linked or unlinked copies of a target sequence with a multivalent binding composition described herein. Contacting the target nucleic acid or nucleic acids comprising multiple linked or unlinked copies of the target sequence with one or more particle-nucleotide conjugates can provide a significantly increased local concentration of the correct nucleotides that are interrogated in a given sequencing cycle, thus suppressing signals from incorrectly incorporated or phase-separated nucleic acid strands (i.e., those having one or more skipped cycles of nucleic acid strands) from such nucleic acid strands.

Methods of obtaining nucleic acid sequence information can include contacting a target nucleic acid or a plurality of target nucleic acids with one or more particle-nucleotide conjugates, wherein the target nucleic acid or plurality of target nucleic acids comprise multiple linked or unlinked copies of a target sequence. This approach results in a reduction in the rate of sequencing errors, as indicated by a reduction in base misidentification, the absence of a base report, or the failure to report the correct base. In some embodiments, the reduction in sequencing error rate can comprise a 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200% or more reduction in error rate compared to the error rate observed using a monovalent ligand comprising free nucleotides, labeled free nucleotides, protein or peptide bound nucleotides, or labeled protein or peptide bound nucleotides.

Methods of obtaining nucleic acid sequence information can include contacting a target nucleic acid or nucleic acid targets with one or more particle-nucleotide conjugates, wherein the template nucleic acid or nucleic acid targets comprise multiple linked or unlinked copies of a target sequence. The method results in an increase in average read length of 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300% or more compared to the average read length observed using a monovalent ligand comprising free nucleotides, labeled free nucleotides, protein or peptide bound nucleotides, or labeled protein or peptide bound nucleotides.

Disclosed herein are methods of obtaining nucleic acid sequence information, the methods comprising contacting a target nucleic acid or a plurality of target nucleic acids with one or more particle-nucleotide conjugates, wherein the target nucleic acid or plurality of target nucleic acids comprise multiple linked or unlinked copies of a target sequence. The method results in an increase in average read length of 10 Nucleotides (NT), 20NT, 25NT, 30NT, 50NT, 75NT, 100NT, 125NT, 150NT, 200NT, 250NT, 300NT, 350NT, 400NT, 500NT or more compared to the average read length observed using a monovalent ligand comprising free nucleotides, labeled free nucleotides, protein or peptide bound nucleotides, or labeled protein or peptide bound nucleotides.

In some cases, the disclosed compositions and methods can result in an average read length for sequencing applications in the range of 100 nucleotides to 1,000 nucleotides. In some cases, the average read length can be at least 100 nucleotides, at least 200 nucleotides, at least 225 nucleotides, at least 250 nucleotides, at least 275 nucleotides, at least 300 nucleotides, at least 325 nucleotides, at least 350 nucleotides, at least 375 nucleotides, at least 400 nucleotides, at least 425 nucleotides, at least 450 nucleotides, at least 475 nucleotides, at least 500 nucleotides, at least 525 nucleotides, at least 550 nucleotides, at least 575 nucleotides, at least 600 nucleotides, at least 625 nucleotides, at least 650 nucleotides, at least 675 nucleotides, at least 700 nucleotides, at least 725 nucleotides, at least 750 nucleotides, at least 775 nucleotides, at least 800 nucleotides, at least 825 nucleotides, at least 850 nucleotides, at least 875 nucleotides, at least 900 nucleotides, at least 925 nucleotides, At least 950 nucleotides, at least 975 nucleotides, or at least 1,000 nucleotides. In some cases, the average read length can be a range bounded by any two values within the range, e.g., an average read length ranging from 375 nucleotides to 825 nucleotides. One skilled in the art will recognize that in some cases, the average read length can have any value within the range specified in this paragraph, e.g., 523 nucleotides.

Sequencing using multivalent binding compositions effectively shortens sequencing time. The cycle of the sequencing reaction, including the contacting, detecting, and incorporating steps, is performed over a total time range of about 5 minutes to about 60 minutes. In some cases, the sequencing reaction cycle is performed for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes. In some cases, the sequencing reaction cycle is performed in at most 60 minutes, at most 50 minutes, at most 40 minutes, at most 30 minutes, at most 20 minutes, at most 10 minutes, or at most 5 minutes. Any of the lower and upper values described in this paragraph can be combined to form ranges encompassed by the present disclosure, e.g., a sequencing reaction cycle can in some cases be performed over a total time range of about 10 minutes to about 30 minutes. One skilled in the art will recognize that the sequencing cycle time can have any value within this range, for example about 16 minutes.

In some cases, the disclosed compositions and methods for nucleic acid sequencing will provide an average base detection accuracy that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% correct during a sequencing run. In some cases, every 1,000 bases, 10,0000 bases, 25,000 bases, 50,000 bases, 75,000 bases, or 100,000 bases identified, the disclosed compositions and methods for nucleic acid sequencing will provide an average base detection accuracy that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% correct.

Sequencing using multivalent binding compositions can provide more accurate base reads. The disclosed compositions and methods for nucleic acid sequencing will provide an average Q-score for base detection accuracy in a sequencing run in the range of about 20 to about 50. In some cases, the average Q score is at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50. One skilled in the art will recognize that the average Q score may have any value within this range, such as about 32.

In some cases, the disclosed compositions and methods for nucleic acid sequencing will provide a Q score of greater than 30 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the terminal (or N +1) nucleotides identified. In some cases, the disclosed compositions and methods for nucleic acid sequencing will provide a Q score of greater than 35 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the terminal (or N +1) nucleotides identified. In some cases, the disclosed compositions and methods for nucleic acid sequencing will provide a Q score of greater than 40 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the terminal (or N +1) nucleotides identified. In some cases, the disclosed compositions and methods for nucleic acid sequencing will provide a Q score of greater than 45 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the terminal (or N +1) nucleotides identified. In some cases, the disclosed compositions and methods for nucleic acid sequencing will provide a Q score greater than 50 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the terminal (or N +1) nucleotides identified.

The disclosed low non-specific binding vectors and related nucleic acid hybridization and amplification methods can be used to analyze nucleic acid molecules derived from any of a number of different cell, tissue, or sample types known to those of skill in the art. For example, nucleic acids can be extracted from cells derived from eukaryotes (e.g., animals, plants, fungi, protists), archaea, or eubacteria, or tissue samples containing one or more types of cells. In some cases, nucleic acids can be extracted from prokaryotic or eukaryotic cells, such as adherent or non-adherent eukaryotic cells. Nucleic acids are extracted from a variety of, e.g., primary or immortalized rodent, porcine, feline, canine, bovine, equine, primate, or human cell lines. Nucleic acids can be extracted from a variety of different cell, organ, or tissue types (e.g., leukocytes, erythrocytes, platelets, epithelial cells, endothelial cells, neurons, glial cells, astrocytes, fibroblasts, skeletal muscle cells, smooth muscle cells, gametes, or cells from the heart, lung, brain, liver, kidney, spleen, pancreas, thymus, bladder, stomach, colon, or small intestine). Nucleic acids can be extracted from normal or healthy cells. Alternatively or in combination, the acid is extracted from diseased cells (e.g., cancer cells) or pathogenic cells that infect the host. Certain nucleic acids can be extracted from different subsets of cell types, such as immune cells (e.g., T cells, cytotoxic (killer) T cells, helper T cells, α β T cells, γ δ T cells, T cell progenitors, B cells, B cell progenitors, lymphoid stem cells, myeloid progenitors, lymphocytes, granulocytes, natural killer cells, plasma cells, memory cells, neutrophils, eosinophils, basophils, mast cells, monocytes, dendritic cells and/or macrophages, or any combination thereof), undifferentiated human stem cells, human stem cells that have been induced to differentiate, rare cells (e.g., Circulating Tumor Cells (CTCs), circulating epithelial cells, circulating endothelial cells, circulating endometrial cells, myeloid cells, progenitor cells, foam cells, and/or cells, Mesenchymal cells or trophoblasts). Nucleic acids may also include nucleic acids derived from viral samples and from subviral pathogens such as viroids and infectious RNA. Nucleic acids can be derived from clinical or other samples, such as sputum, saliva, ocular fluid, synovial fluid, blood, stool, urine, tissue exudate, sweat, pus, drainage fluids, and the like. The nucleic acid may also be derived from a plant or fungal sample, such as a leaf, cambium, root, meristem, pollen, ovum, seed, spore, inflorescence, mycelium, and the like. Nucleic acids can also be derived from environmental or industrial samples, such as water, air, dust, food, and the like. Other cells, tissues, and samples are contemplated and are consistent with the disclosure herein.

Nucleic acids can be extracted from cells or other biological samples using any of a variety of techniques known to those of skill in the art. For example, a typical DNA extraction procedure involves (i) collecting a cell sample or tissue sample from which DNA is to be extracted, (ii) disrupting the cell membrane (i.e., cell lysis) to release DNA and other cytoplasmic components, (iii) treating the lysed sample with a concentrated salt solution to precipitate proteins, lipids, and RNA, followed by centrifugation to separate the precipitated proteins, lipids, and RNA, and (iv) purifying the DNA from the supernatant to remove detergents, proteins, salts, or other reagents used in the cell membrane lysis step.

A variety of suitable commercially available nucleic acid extraction and purification kits are consistent with the disclosure herein. Examples include, but are not limited to, the QIAamp kit from Qiagen (Germantown, Md) (for isolating genomic DNA from human samples) and the DNAasy kit (for isolating genomic DNA from animal or plant samples), or from Promega (Madison, Wis.)

And ReliaPrep^TMA series of kits.

VII. System

A system module: also disclosed herein is a system configured to perform any of the disclosed nucleic acid sequencing or nucleic acid analysis methods, as described herein. In some cases, the disclosed systems can comprise one or more multivalent binding compositions described herein, one or more buffers, and/or one or more nucleic acid molecules tethered to a solid support.

In some cases, the system can further include a fluid flow controller and/or a fluid dispensing system configured to sequentially and repeatedly contact a template nucleic acid molecule that hybridizes to a nucleic acid molecule (e.g., an adaptor or primer) tethered to a solid support having the disclosed multivalent binding compositions and/or reagents. In some cases, the contacting may be performed in one or more flow cells. In some cases, the flow cell may be a stationary component of the system. In some cases, the flow cell may be a removable and/or disposable component of the system.

In some cases, the system can further include an imaging module, wherein the imaging module includes, for example, one or more light sources, one or more optical components (e.g., lenses, mirrors, prisms, optical filters, colored glass filters, narrow band interferometers, broadband interferometers, dichroic mirrors, diffraction gratings, apertures, optical fibers or optical waveguides, etc.), and one or more image sensors (e.g., Charge Coupled Device (CCD) sensors or cameras, Complementary Metal Oxide Semiconductor (CMOS) image sensors or cameras, or negative channel metal oxide semiconductor (NMOS) image sensors or cameras) for imaging and detecting binding of the disclosed multivalent binding compositions to target (or template) nucleic acid molecules tethered to the interior of a solid support or flow cell.

Processor and computer system: one or more processors can be employed to implement a system for nucleic acid sequencing or other nucleic acid detection and analysis methods disclosed herein. The one or more processors may include a hardware processor, such as a Central Processing Unit (CPU), a Graphics Processing Unit (GPU), a general purpose processing unit, or a computing platform. The one or more processors may be comprised of any of a variety of suitable integrated circuits (e.g., an Application Specific Integrated Circuit (ASIC) or Field Programmable Gate Array (FPGA) designed specifically for implementing a deep learning network architecture to accelerate computation time, etc., and/or to facilitate deployment), microprocessors, emerging next generation microprocessor designs (e.g., memristor-based processors), logic devices, and the like. Although the present disclosure is described with reference to a processor, other types of integrated circuits and logic devices may also be suitable. The processor may have any suitable data manipulation capability. For example, the processor may perform 512-bit, 256-bit, 128-bit, 64-bit, 32-bit, or 16-bit data operations. The one or more processors may be single-core or multi-core processors, or multiple processors configured for parallel processing.

One or more processors or computers for implementing the disclosed methods may be part of a larger computer system and/or may be operatively coupled to a computer network ("network") with the aid of a communication interface to facilitate data transmission and sharing. The network may be a local area network, an intranet and/or extranet in communication with the internet, or the internet. In some cases, the network is a telecommunications and/or data network. The network may include one or more computer servers, which in some cases may enable distributed computing, such as cloud computing. Networks a peer-to-peer network may be implemented with the aid of a computer system in some cases, which may enable devices coupled to the computer system to act as clients or servers.

The computer system also includes a memory or memory location (e.g., random access memory, read only memory, peripheral devices, and/or the like) for communicating with one or more other systems and peripheral devices, such as a cache, other memory, a data store, and/or an electronic display adapter,

OptaneTM technology), an electronic storage unit (e.g., hard disk), a communication interface (e.g., network adapter). The memory, storage unit, interface, and peripherals may communicate with one or more processors (e.g., CPUs) over a communication bus, such as found on a motherboard. The storage unit may be a data storage unit (or data repository) for storing data.

One or more processors, such as a CPU, execute a series of machine-readable instructions embodied in a program (or software). The instructions are stored in a storage location. The instructions are directed to a CPU, which is then programmed or otherwise configured to implement the methods of the present disclosure. Examples of operations performed by the CPU include fetch, decode, execute, and write back. The CPU may be part of a circuit such as an integrated circuit. One or more other components of the system may be included in the circuit. In some cases, the circuit is an Application Specific Integrated Circuit (ASIC).

The storage unit stores files such as drivers, libraries, and saved programs. The storage unit stores user data such as user-specified preferences and user-specified programs. In some cases, the computer system may include one or more other data storage units external to the computer system, for example, on a remote server in communication with the computer system via an intranet or the internet.

Some aspects of the methods and systems provided herein may be implemented by machine (e.g., processor) executable code stored in an electronic storage location of a computer system, such as, for example, in a memory or electronic storage unit. The machine executable or machine readable code may be provided in the form of software. In use, the code is executed by one or more processors. In some cases, code is retrieved from a storage unit and stored in memory for ready access by one or more processors. In some cases, electronic storage units may be eliminated, and machine-executable instructions stored in memory. The code may be precompiled and configured for use with a machine having one or more processors adapted to execute the code or may be compiled at runtime. The code may be provided in a programming language of choice to enable the code to be executed in a pre-compiled or compiled form.

Various aspects of the technology may be considered as an "article of manufacture" or "article of manufacture," e.g., "a computer program or software product," typically in the form of machine (or processor) executable code and/or associated data stored in one type of machine-readable medium, where the executable code comprises a plurality of instructions for controlling a computer or computer system to perform one or more of the methodologies disclosed herein. The machine executable code may be stored in an optical storage unit including an optically readable medium such as a compact disc, CD-ROM, DVD, or blu-ray disc. The machine executable code may be stored in an electronic storage unit, such as a memory (e.g., read only memory, random access memory, flash memory) or on a hard disk. "storage" type media include any or all tangible memory of a computer, processor, etc., or associated modules thereof, such as various semiconductor memory chips, optical drives, tape drives, disk drives, etc., that may provide non-transitory storage of software encoding the methods and algorithms disclosed herein at any time.

All or part of the software code may sometimes be communicated over the internet or other various telecommunications networks. For example, such communication enables loading of software from one computer or processor to another computer or processor, such as from a management server or host to the computer platform of an application server. Thus, other types of media for communicating software-encoded instructions include optical, electrical, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical fixed-line networks, and through various air links. The physical elements carrying such waves, e.g., wired or wireless links, optical links, etc., are also considered to be media conveying software-encoded instructions for performing the methods disclosed herein. As used herein, unless limited to a non-transitory tangible "storage" medium, terms such as a computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

Computer systems typically include or may be in communication with an electronic display for providing images, for example, captured by a machine vision system. The display is also typically capable of providing a User Interface (UI). Examples of UIs include, but are not limited to, Graphical User Interfaces (GUIs), Web-based user interfaces, and the like.

System control software: in some cases, the disclosed systems may include a computer (or processor) and a computer readable medium including code for providing a user interface and manual, semi-automatic, or fully automatic control of all system functions, such as control of fluid flow controllers and/or fluid dispensing systems (or subsystems), temperature control systems (or subsystems), imaging systems (or subsystems), and the like. In some cases, the system computer or processor may be an integrated component of the instrument system (e.g., a microprocessor or motherboard embedded in the instrument). In some cases, the system computer or processor may be a stand-alone module, such as a personal computer or laptop computer. Examples of fluid flow control functions that may be provided by instrument control software include, but are not limited to, volumetric fluid flow rates, timing and duration of sample and reagent addition, washing steps, and the like. Examples of temperature control functions that may be provided by instrument control software include, but are not limited to, specifying temperature set point(s) and control for the timing, duration, and ramp rate of temperature changes. Examples of imaging system control functions that may be provided by instrument control software include, but are not limited to, auto-focus capability, control of illumination or excitation light exposure time and intensity, control of image acquisition rate, exposure time, data storage options, and the like.

Image processing software: in some examples of the disclosed system, the system may also include a computer-readable medium including code for providing image processing and analysis capabilities. Examples of image processing and analysis capabilities that software may provide include, but are not limited to, manual, semi-automatic or fully automatic image exposure adjustment (e.g., white balance, contrast adjustment, signal averaging and other noise reduction capabilities, etc.), manual, semi-automatic or fully automatic edge detection and target identification (e.g., for identifying clusters of template nucleic acid molecules amplified on a substrate surface), manual, semi-automatic or fully automatic signal intensity measurement and/or thresholding in one or more detection channels (e.g., one or more fluorescence emission channels), manual, semi-automatic or fully automatic statistical analysis (e.g., for comparing signal intensity to reference values for base detection purposes).

In some cases, the system software may provide integrated real-time image analysis and instrument control so that sample loading, reagent addition, washing, and/or imaging/base detection steps may be extended, modified, or repeated as needed until, for example, optimal base detection results are achieved. Real-time or post-processing image analysis capabilities may be implemented using any of a variety of image processing and analysis algorithms known to those skilled in the art. Examples include, but are not limited to, Canny edge detection methods, Canny-Deriche edge detection methods, first-order gradient edge detection methods (e.g., Sobel operators), second-order differential edge detection methods, phase consistency (phase coherence) edge detection methods, other image segmentation algorithms (e.g., intensity thresholding, intensity clustering methods, intensity histogram-based methods, etc.), feature and pattern recognition algorithms (e.g., generalized hough transform for detecting arbitrary shapes, circular hough transform, etc.), and mathematical analysis algorithms (e.g., fourier transform, fast fourier transform, wavelet analysis, autocorrelation, etc.), or combinations thereof.

In some cases, the system control and image processing/analysis software may be written as separate software modules. In some cases, the system control and image processing/analysis software may be incorporated into an integrated software package.

VIII example

1. Preparation of multivalent binding compositions

As shown in fig. 5A, one type of multi-arm substrate is prepared by reacting propargylamine dntps with biotin-PEG-NHS. Driving the aqueous reaction to completion and purification; producing pure biotin-PEG-dNTP species. In different reactions, several different PEG lengths were used, corresponding to average molecular weights varying from 1K Da to 20K Da. The biotin-PEG-dNTP species was mixed with dye-labeled Streptavidin (SA), either freshly prepared or commercially available, using a dye to SA ratio of 3-5: 1. Mixing of the biotin-PEG-dNTPs with the dye-labeled streptavidin is done in the presence of excess biotin-PEG-dNTPs to ensure saturation of the biotin binding sites on each streptavidin tetramer. The complete complex was purified from excess biotin-PEG-dNTPs by size exclusion chromatography. Each nucleotide type was conjugated and purified separately and then mixed together to create a four base mixture for sequencing.

Another type of multi-arm substrate is shown in FIG. 5A by contacting a multi-arm PEG NHS with excess dye-NH₂Reacted with propargylamine dNTP, prepared in a single pot. Various multi-arm PEG NHS variants were used, ranging from 4-16 arms, with molecular weights ranging from 5K Da to 40K Da. After the reaction, excess small molecule dye and dntps were removed by size exclusion chromatography. Each nucleotide type was conjugated and purified separately and then mixed together to create a four base mixture for sequencing.

A class II substrate as shown in figure 5B was prepared using a one-pot reaction to conjugate the dye and dntps simultaneously. alkyne-PEG-NHS was reacted with excess propargylamine dNTP. The product (alkyne-PEG-dNTP) was then purified to homogeneity by chromatography. A variety of PEG lengths were used, with average molecular weights varying between 1K Da and 20K Da. Dendrimer cores containing variable, discrete numbers (12, 24, 48, 96) of azide conjugation sites were used. Conjugation of alkyne-dye and alkyne-PEG-dNTP to the dendrimer core occurs in a one-pot reaction containing excess dye and dNTP species by a copper-mediated click chemistry reaction. After the reaction, excess small molecule dye and dntps were removed by size exclusion chromatography. Each nucleotide type was conjugated and purified separately and then mixed together to create a four base mixture for sequencing. We note that this approach allows for easy replacement of replacement cores, such as dextran, other polymers, proteins, etc.

Class III polymer-nucleotide conjugates as shown in figure 5C were constructed by reacting 4-arm or 8-arm PEG NHS with a saturated mixture of biotin and propargylamine dNTP. The reaction was then purified by size exclusion chromatography. The result of this reaction is a multi-armed PEG containing discrete distributions of biotin and nucleotides. This heterogeneous population is then reacted with dye-labeled streptavidin and purified by size exclusion chromatography. Each nucleotide type was conjugated and purified separately and then mixed together to create a four base mixture for sequencing. We note that the distribution of biotin and nucleotides can be determined by biotin-NH₂The input ratio of the propargylamine dNTP is adjusted.

2. Detection of ternary complexes

Assay Using multivalent binding compositions with PEG Polymer-nucleotide conjugatesTo detect possible formation of a ternary binding complex, and fluorescence images of the various steps are shown in fig. 7A-7J. In FIG. 7A, the DNA fragment was amplified in a medium containing 20nM Klenow polymerase and 2.5mM Sr⁺²In exposure buffer (c), red and green fluorescence images of DNA Rolling Circle Application (RCA) template (G and a first base) after exposure to 500nM base-labeled nucleotides (a-Cy3 and G-Cy 5). Multivalent PEG-base compositions were prepared using different ratios of 4-arm PEG-amine (4ArmPEG-NH), biotin-PEG-amine (biotin-PEG-NH), and nucleotides (Nuc) as follows: samples PB1 and PB5, 4 ArmPEG-NH: biotin-PEG-NH: nuc is 0.25:1: 0.5; sample PB2, 4 ArmPEG-NH: biotin-PEG-NH: nuc is 0.125:0.5: 0.25; sample PB3, 4 ArmPEG-NH: biotin-PEG-NH: nuc ═ 0.25:1:0.5 images were collected after washing with imaging buffer, the composition of which was the same as the exposure buffer, but which contained no nucleotides or polymerase.

The contrast was scaled to maximize visualization of the darkest signal, but no signal persisted after washing with imaging buffer (fig. 7A, inset). In FIGS. 7B-7E, fluorescence images of multivalent PEG-nucleotide (base-labeled) ligands at 500nM after mixing in exposure buffer and imaging in imaging buffer as described above are shown (FIG. 7B: PB 1; FIG. 7C: PB 2; FIG. 7D: PB 3; FIG. 7E: PB 5). FIG. 7F: fluorescence images of multivalent PEG-nucleotide (base-labeled) ligand PB5 at 2.5uM after mixing in exposure buffer and imaging in imaging buffer as described above are shown. In FIGS. 7G-7I, fluorescence images showing further base identifications by exposing multivalent ligands to inactive mutants of Klenow polymerase (FIG. 7G: D882H; FIG. 7H: D882E; FIG. 7I: D882A), and wild-type Klenow (control) enzyme are shown in FIG. 7J.

Using multivalent ligand formulations, base discrimination can be achieved by providing polymerase-ligand interactions with increased affinity. Furthermore, it was demonstrated that an increase in multivalent ligand concentration can produce higher signals, and that various Klenow mutations that knock out catalytic activity can be used for affinity-based sequencing.

3. Sequencing of target nucleic acid molecules using ternary complexes

To prove based onSequencing of the valency ligand reporter gene, 4 known templates were amplified on a low binding substrate using the RCA method. Continuous cycle exposure to Klenow polymerase containing 20nM and 2.5mM Sr⁺²And washed with imaging buffer and imaged. After imaging, the substrate is washed with wash buffer (EDTA and high salt) and blocked nucleotides are added to continue to the next base. This cycle was repeated for 5 cycles. Spots were detected using standard imaging processing and spot detection, and sequences were identified using a two-color scheme of green and red (G-Cy3 and A-Cy5) to identify the template that was cycled. As shown in fig. 8A and 8B, multivalent ligands were able to provide base discrimination in all 5 sequencing cycles.

4. Control of nucleotide dissociation in ternary complexes

Ternary complexes were prepared and imaged as described in example 2. The composites were imaged for different lengths of time to demonstrate the persistence of the ternary composites, for example, up to 60 seconds. After a period of time, the complex is washed with the same buffer as used to form the complex, except in the absence of any divalent cation, e.g., 10mM Tris pH 8.0,0.5mM EDTA,50mM NaCl, 0.016% Triton X100 (without SrOAc), or, alternatively, in the absence of any divalent cation, e.g., 10mM Tris pH 8.0,0.5mM EDTA,50mM NaCl, 0.016% Triton X100 (without SrOAc), 100nm to 100mM EDTA. Fluorescence from the complex is observed over time so that dissociation of the ternary complex can be observed and quantified. A representative time course of such dissolution is shown in figure 6.

5. Extension of the complementary sequence of a target nucleic acid

After preparation, imaging and dissociation of the ternary complex as in example 4, the deblock solution is flowed into the chamber containing the bound DNA molecules sufficient to remove the blocking moiety, e.g., O-azidomethyl, O-alkylhydroxylamine, or O-amino, from the 3' end of the extended DNA strand. After this or simultaneously, the extension solution is flowed into the chamber containing the bound DNA molecules. The extension solution comprises a buffer, a divalent cation sufficient to support polymerase activity, an active polymerase, and appropriate amounts of all four nucleotides, wherein the nucleotides are blocked such that they cannot support further extension after addition of a single nucleotide to an extended DNA strand, for example by incorporation of a 3' -O-azidomethyl group, a 3' -O-alkylhydroxylamine group, or a 3' -O-amino group. Thus, the extended strand is extended by one and only one base, and the binding of the non-catalytically active polymerase and the multivalent binding substrate can be used to recognize the next base in the cycle.

Alternatively, nucleotides attached to multivalent substrates may be attached by labile bonds, such that the buffer may flow into a chamber containing bound DNA molecules containing sufficient divalent cations or other cofactors to render the polymerase catalytically inactive. Before, after, or simultaneously with this, conditions may be provided sufficient to cleave the base from the multivalent substrate so that it may be incorporated into the extended strand. This cleavage and incorporation leads to dissociation of the label and the polymer backbone of the multivalent substrate, while extending the extended DNA strand by exactly one base. Washing is performed to remove the used polymer backbone and a new multivalent substrate is flowed into the chamber containing the bound DNA molecule, allowing the new base to be recognized as described in example 1.

6. Use of polymer-nucleotide conjugates with PEG branches of different lengths

The polymer-nucleotide conjugates with different PEG arm lengths described in example 3 were subjected to a single sequencing cycle and imaged as described in example 1. As shown in fig. 9A-9J, increasing the length of the PEG branches resulted in an increase in signal up to a length corresponding to an apparent average PEG MW of 5K Da (fig. 9A-9D). Using a longer PEG arm than this resulted in a decrease in the fluorescence signal of Cy3-A and Cy5-G (FIGS. 9E-9G). The quantitative measurement of signal intensity is shown in graphical form in fig. 10.

7. Enhancement of multivalent substrate binding by addition of detergents

Preparing multivalent substrates and assembling into a binding complex in the presence and absence of detergent: one group used 10mM Tris pH 8.0,0.5mM EDTA,50mM NaCl, 5mM SroAc, 0% TritonX100 (condition A), and one group used 10mM Tris pH 8.0,0.5mM EDTA,50mM NaCl, 5mM SroAc, 0.016% Triton X100. FIG. 11 shows the normalized fluorescence of these multivalent substrates bound to DNA clusters, where the substrate complex formed in the presence of Triton-X100 (0.016%) (Condition B) shows significantly enhanced fluorescence intensity.

8. Evaluation of multivalent substrate binding time course

Multivalent substrates were prepared and assembled into binding complexes as in example 2. Complexes were also formed using free labeled nucleotides under the same buffer conditions. The duration of the complex was characterized by imaging the complex over the course of 60 minutes. Representative results are shown in fig. 12A-12B. The multivalent binding complex is stable over a time scale of greater than 60 minutes (fig. 12B) while the labeled free nucleotides dissociate in less than one minute (fig. 12A).

Conclusion VIII

The present disclosure provides greatly improved methods and compositions for DNA sequencing and biosensor applications. It is to be understood that the above description is intended to be illustrative, and not restrictive. Many embodiments will be apparent to those of skill in the art upon reading the above description. For example, the concepts of the present invention are described primarily with reference to the use of polymer-nucleotide conjugates, but those skilled in the art will readily recognize that other types of particle-nucleotide conjugates may also be used. For example, in some embodiments, it may be desirable to use a composition that includes quantum dots; a liposome; or particle-nucleotide conjugates of emulsion particles. Alternatively, conjugation may be achieved through non-covalent bonds such as hydrogen bonding or other interactions. The scope of the disclosed inventive concepts should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. A method of determining the identity of a nucleotide in a target nucleic acid sequence, comprising:

a. providing a composition comprising:

i. two or more copies of the target nucleic acid sequence;

two or more primer nucleic acid molecules complementary to one or more regions of the target nucleic acid sequence; and

two or more polymerase molecules;

b. contacting a polymer-nucleotide conjugate with the polymer-nucleotide conjugate under conditions sufficient to allow formation of a multivalent binding complex between the polymer-nucleotide conjugate and the two or more copies of the target nucleic acid sequence in the composition of (a), wherein the polymer-nucleotide conjugate comprises copies of two or more nucleotide moieties and optionally one or more detectable labels; and

c. detecting the multivalent binding complex, thereby determining the identity of the nucleotide in the target nucleic acid sequence.

2. The method of claim 1, wherein the target nucleic acid sequence is DNA.

3. The method of claim 1 or claim 2, wherein the detection of the multivalent binding complex is performed in the absence of unbound or solution-borne polymer-nucleotide conjugates.

4. The method of any one of claims 1 to 3, wherein the target nucleic acid sequence has been replicated or amplified or has been produced by replication or amplification.

5. The method of any one of claims 1 to 4, wherein the one or more detectable labels are fluorescent labels.

6. The method of any one of claims 1 to 5, wherein detecting the multivalent complex comprises fluorescence measurement.

7. The method of any one of claims 1 to 6, wherein the contacting comprises the use of one type of polymer-nucleotide conjugate.

8. The method of any one of claims 1 to 7, wherein the contacting comprises the use of two or more types of polymer-nucleotide conjugates.

9. The method of claim 8, wherein each of the two or more types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety.

10. The method of claim 9, wherein the contacting comprises the use of three types of polymer-nucleotide conjugates, and wherein each of the three types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety.

11. The method of any one of claims 1-10, wherein the polymer-nucleotide conjugate comprises a blocked nucleotide moiety.

12. The method of claim 11, wherein the blocked nucleotide is a 3' -O-azidomethyl nucleotide, a 3' -O-methyl nucleotide, or a 3' -O-alkylhydroxylamine nucleotide.

13. The method of any one of claims 1 to 12, wherein the contacting occurs in the presence of an ion that stabilizes the multivalent binding complex.

14. The method of any one of claims 1 to 13, wherein the contacting is performed in the presence of strontium ions, magnesium ions, calcium ions, or any combination thereof.

15. The method of any one of claims 1 to 14, wherein the polymerase molecule is non-catalytically active.

16. The method of any one of claims 1 to 15, wherein the polymerase molecule has been rendered non-catalytically active by mutation or chemistry.

17. The method of any one of claims 1 to 16, wherein the polymerase molecule has been rendered non-catalytically active by the absence of an essential ion or cofactor.

18. The method of any one of claims 1 to 17, wherein the polymerase molecule is catalytically active.

19. The method of any one of claims 1-18, wherein the polymer-nucleotide conjugate does not comprise a blocked nucleotide moiety.

20. The method of any one of claims 1-19, wherein the multivalent binding complex has a duration of greater than 2 seconds.

21. The method of any one of claims 1 to 20, wherein the method may be carried out at a temperature in the range of from 25 ℃ to 62 ℃.

22. The method of any one of claims 1 to 21, wherein the polymer-nucleotide conjugate further comprises one or more fluorescent labels and the two or more copies of the target nucleic acid sequence are deposited, attached or hybridized to a surface, wherein the contrast to noise ratio of a fluorescence image of the multivalent binding complex on the surface in the detecting step is greater than 20.

23. The method of any one of claims 1 to 22, wherein the composition of (a) is deposited onto a surface using a buffer incorporating a polar aprotic solvent.

24. The method of any one of claims 1 to 23, wherein the contacting is performed under the following conditions: stabilizing the multivalent binding complex when the nucleotide moiety is complementary to the next base of the target nucleic acid sequence, and destabilizing the multivalent binding complex when the nucleotide moiety is not complementary to the next base of the target nucleic acid sequence.

25. The method of any one of claims 1-24, wherein the polymer-nucleotide conjugate comprises a polymer having a plurality of branches and the two or more nucleotide moieties are attached to the branches.

26. The method of claim 25, wherein the polymer has a star, comb, cross-linked, bottle brush, or dendrimer configuration.

27. The method of any one of claims 1-26, wherein the polymer-nucleotide conjugate comprises one or more binding groups selected from avidin, biotin, an affinity tag, and combinations thereof.

28. The method of any one of claims 1 to 27, further comprising a dissociation step that destabilizes the multivalent binding complex formed between the composition of (a) and the polymer-nucleotide conjugate, the dissociation step being capable of removing the polymer-nucleotide conjugate.

29. The method of claim 28, further comprising an extension step of incorporating a nucleotide complementary to the next base of the target nucleic acid sequence into the two or more primer nucleic acid molecules.

30. The method of claim 29, wherein the extending step occurs simultaneously with or after the dissociating step.

31. A method of determining the identity of a nucleotide in a target nucleic acid sequence, comprising:

a. providing a composition comprising:

i. two or more copies of the target nucleic acid sequence;

two or more polymerase molecules;

b. contacting a polymer-nucleotide conjugate with the composition of (a) under conditions sufficient to allow formation of a multivalent complex between the polymer-nucleotide conjugate and the two or more copies of the target nucleic acid sequence in the composition, wherein the polymer-nucleotide conjugate comprises two or more copies of a reversibly terminated nucleotide moiety and optionally one or more cleavable detectable labels; and

c. detecting the multivalent complex, thereby determining the identity of the nucleotide in the target nucleic acid sequence.

32. The method of claim 31, wherein the target nucleic acid sequence is DNA.

33. The method of claim 31 or claim 32, further comprising contacting the composition of (a) with a reversibly terminated nucleotide or a polymer-nucleotide conjugate comprising two or more copies of a reversibly terminated nucleotide after detection of the multivalent binding complex.

34. The method of any one of claims 31-33, wherein the target nucleic acid sequence has been replicated or amplified or has been produced by replication or amplification.

35. The method of any one of claims 31-34, wherein the one or more detectable labels are fluorescent labels.

36. The method of any one of claims 31-35, wherein detecting the multivalent complex comprises fluorescence measurement.

37. The method of any one of claims 31-36, wherein the contacting comprises the use of one type of polymer-nucleotide conjugate.

38. The method of any one of claims 31-37, wherein the contacting comprises the use of two or more types of polymer-nucleotide conjugates.

39. The method of claim 38, wherein each of the two or more types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety.

40. The method of claim 39, wherein the contacting comprises the use of three types of polymer-nucleotide conjugates, and wherein each of the three types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety.

41. The method of any one of claims 31-40, wherein the polymer-nucleotide conjugate comprises a blocked nucleotide moiety.

42. The method of claim 41, wherein the blocked nucleotide is 3' -O-azidomethyl, 3' -O-methyl, or 3' -O-alkylhydroxylamine.

43. The method of any one of claims 31-42, wherein the contacting occurs in the presence of an ion that stabilizes the multivalent binding complex.

44. The method of any one of claims 31-43, wherein the polymerase molecule is non-catalytically active.

45. The method of any one of claims 31-44, wherein the polymerase molecule has been rendered non-catalytically active by mutation or chemical modification.

46. The method of any one of claims 31-45, wherein the polymerase molecule is catalytically active.

47. The method of any one of claims 31-46, wherein the polymer-nucleotide conjugate does not comprise a blocked nucleotide moiety.

48. The method of any one of claims 31 to 47, wherein the method can be carried out at a temperature in the range of 25 ℃ to 80 ℃.

49. The method of any one of claims 31-48, wherein the polymer-nucleotide conjugate further comprises one or more fluorescent labels and the two or more copies of the target nucleic acid sequence are deposited, attached, or hybridized to a surface, wherein the contrast to noise ratio of a fluorescence image of the multivalent binding complex on the surface in the detecting step is greater than 20.

50. A system, comprising:

a) one or more computer processors individually or collectively programmed to implement a method comprising:

i) contacting a substrate comprising multiple copies of a target nucleic acid sequence tethered to a surface of the substrate with a reagent comprising a polymerase and one or more primer nucleic acid sequences complementary to one or more regions of the target nucleic acid sequence to form a primed target nucleic acid sequence;

ii) contacting the substrate surface with a reagent comprising a polymer-nucleotide conjugate under conditions sufficient to allow formation of a multivalent binding complex between the polymer-nucleotide conjugate and the two or more copies of the primed target nucleic acid sequence, wherein the polymer-nucleotide conjugate comprises two or more copies of a known nucleotide moiety and a detectable label; and

iii) acquiring and processing an image of the substrate surface to detect the multivalent binding complex, thereby determining the identity of the nucleotide in the target nucleic acid sequence.

51. The system of claim 50, further comprising a fluidics module configured to deliver a series of reagents to the substrate surface in a specified order and at specified time intervals.

52. The system of claim 50 or claim 51, further comprising an imaging module configured to acquire an image of the substrate surface.

53. The system of any one of claims 50 to 52, wherein (ii) and (iii) are repeated two or more times, thereby determining the identity of a series of two or more nucleotides in the target nucleic acid sequence.

54. The system of any one of claims 50-53, wherein the series of steps further comprises a dissociation step that destabilizes the multivalent binding complex, the dissociation step being capable of removing the polymer-nucleotide conjugate.

55. The system of claim 54, wherein the series of steps further comprises an extension step of incorporating a nucleotide complementary to the next base of the target nucleic acid sequence into the two or more primer nucleic acid molecules.

56. The system of claim 55, wherein the extending step occurs simultaneously with or after the dissociating step.

57. The system of any one of claims 50 to 56, wherein the detectable label comprises a fluorophore and the image comprises a fluorescent image.

58. The system of claim 57, wherein when the fluorophore is cyanine dye 3(Cy3) and the image is acquired using an inverted fluorescence microscope equipped with a 20X objective, a dichroic mirror with NA 0.75, optimized for 532nm light, a band pass filter optimized for cyanine dye 3 emission, and a camera under non-signal saturation conditions while the surface is immersed in 25mMACES, pH 7.4 buffer, the contrast-to-noise ratio of the fluorescence image of the multivalent binding complex on the substrate surface is greater than 20.

59. The system of any one of claims 50 to 58, wherein the series of steps is completed in less than 60 minutes.

60. The system of any one of claims 50 to 59, wherein the series of steps is completed in less than 30 minutes.

61. The system of any one of claims 50 to 60, wherein the series of steps is completed in less than 10 minutes.

62. The system of any one of claims 50 to 61, wherein the accuracy of base detection is characterized by a Q score greater than 25 for at least 80% of the nucleotide identities determined.

63. The system of any one of claims 50 to 62, wherein the accuracy of the base detection is characterized by a Q score greater than 30 for at least 80% of the nucleotide identities determined.

64. The system of any one of claims 50 to 63, wherein the accuracy of the base detection is characterized by a Q score greater than 40 for at least 80% of the nucleotide identities determined.

65. A composition, comprising:

a) a polymeric core; and

b) two or more nucleotides, nucleotide analogs, nucleoside or nucleoside analog moieties attached to the polymeric core;

wherein the length of the linker is dependent on the nucleotide, nucleotide analogue, nucleoside or nucleoside analogue moiety attached to the polymer core.

66. A composition, comprising:

a) a mixture of polymer-nucleotide conjugates, wherein each polymer-nucleotide conjugate comprises:

i) a polymeric core; and

ii) two or more nucleotides, nucleotide analogs, nucleosides, or nucleoside analog moieties attached to the polymer core, wherein the length of the linker depends on the nucleotide, nucleotide analog, nucleoside, or nucleoside analog moieties attached to the polymer core.

Wherein the mixture comprises polymer-nucleotide conjugates having at least two different types of attached nucleotide, nucleotide analog, nucleoside, or nucleoside analog moieties.

67. The composition of claim 65 or claim 66, wherein the polymeric core comprises a polymer having a plurality of branches and the two or more nucleotides, nucleotide analogs, nucleosides, or nucleoside analog moieties are attached to the branches.

68. The composition of claim 67, wherein the polymer has a star, comb, cross-linked, bottle brush, or dendrimer configuration.

69. The composition of any one of claims 65-68, wherein the polymer-nucleotide conjugate comprises one or more binding groups selected from avidin, biotin, an affinity tag, and combinations thereof.

70. The composition of any one of claims 65-69, wherein the polymeric core comprises branched polyethylene glycol (PEG) molecules.

71. The composition of any one of claims 65-70, wherein the polymer-nucleotide conjugate comprises a blocked nucleotide moiety.

72. The composition of claim 71, wherein the blocked nucleotide is a 3' -O-azidomethyl nucleotide, a 3' -O-methyl nucleotide, or a 3' -O-alkylhydroxylamine nucleotide.

73. The composition of any one of claims 65-72, wherein the polymer-nucleotide conjugate further comprises one or more fluorescent labels.