MULTIVALENT COMPOSITION FOR NUCLEAR ACID ANALYSIS

Abstract

Multivalent binding compositions are described that include a particle-nucleotide conjugate with multiple copies of a nucleotide bound to the particle. These multivalent binding compositions enable the localization of detectable signals for active regions of biochemical interaction, such as protein-protein interaction sites, protein-nucleic acid interaction sites, nucleic acid hybridization sites, or enzymatic reaction sites. They can also be used to identify sites of base incorporation during nucleic acid chain elongation in polymerase reactions and to provide improved base discrimination for sequencing and array-based applications.

Description

CROSS-REFERENCE

[0001] This application is a continuation-in-part of U.S. Patent Application No. 16 / 579,794, filed September 23, 2019, and claims the benefit of U.S. Patent Application No. 62 / 897,172, filed September 6, 2019, and U.S. Patent Application No. 62 / 852,876, filed May 24, 2019, each of which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION

[0002] The present disclosure relates generally to multivalent binding compositions and their use in the analysis of nucleic acid molecules. More specifically, the inventive concept relates to a multivalent binding composition comprising multiple copies of a nucleotide bound to a particle or polymer core, which effectively increases the local concentration of the nucleotide and enhances binding signals. The multivalent binding composition can be used, for example, in the field of sequencing and biosensor microarrays. BACKGROUND

[0003] Nucleic acid sequencing can be used to obtain information in a wide variety of biomedical contexts, including diagnostics, prognosis, biotechnology, and forensic biology. Various sequencing methods have been developed, including Maxam-Gilbert sequencing and the chain termination method, or de novo sequencing methods including shotgun sequencing and Bridge PCR, or next-generation methods such as Polony sequencing, 454 pyrosequencing, Illumina sequencing, SOLiD sequencing, Ion semiconductor sequencing, HeliScope single-molecule sequencing, SMRT® sequencing, and others. Despite advances in DNA sequencing, many challenges remain on the path to cost-effective, high-throughput sequencing. The present disclosure provides new solutions and approaches to address many of the shortcomings of existing technologies. SUMMARY

[0004] Disclosed herein are methods for determining an 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; ii. two or more primer nucleic acid molecules complementary to one or more regions of the target nucleic acid sequence; and iii. two or more polymerase molecules; b. contacting the composition with a polymer-nucleotide conjugate under conditions sufficient to allow the 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 two or more copies of a nucleotide residue 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. In some embodiments, the target nucleic acid sequence is DNA. In some embodiments, detecting the multivalent binding complex is performed in the absence of unbound or in-solution 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 measuring fluorescence. In some embodiments, contacting comprises using one type of polymer-nucleotide conjugate. In some embodiments, contacting comprises using 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 residue. In some embodiments, the contacting comprises using three types of polymer-nucleotide conjugates, and wherein each of the three types of polymer-nucleotide conjugates comprises a different type of nucleotide residue. In some embodiments, the polymer-nucleotide conjugate comprises a blocked nucleotide residue. 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 occurs in the presence of strontium ions, magnesium ions, calcium ions, or any combination thereof.In some embodiments, the polymerase molecules are catalytically inactive. In some embodiments, the polymerase molecules have been rendered catalytically inactive by mutation or chemical modification. In some embodiments, the polymerase molecules have been rendered catalytically inactive by the absence of a necessary ion or cofactor. In some embodiments, the polymerase molecules are catalytically active. In some embodiments, the polymer-nucleotide conjugate does not comprise a blocked nucleotide residue. In some embodiments, the multivalent binding complex has a persistence time of greater than 2 seconds. In some embodiments, the method can be performed at a temperature within a range of 25°C to 62°C.In some embodiments, 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 a fluorescent image of the multivalent binding complex on the surface has a contrast-to-noise ratio in the detection step of greater than 20. In some embodiments, the composition of (a) is deposited on a surface using a buffer containing a polar aprotic solvent. In some embodiments, the contacting is performed under a condition that stabilizes the multivalent binding complex when the nucleotide residue is complementary to a nearest base of the target nucleic acid sequence and destabilizes the multivalent binding complex when the nucleotide residue is not complementary to the nearest 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 residues are attached to the branches. In some embodiments, the polymer has a star, comb, cross-linked, bottlebrush, or dendrimer configuration. In some embodiments, the polymer-nucleotide conjugate comprises one or more binding groups selected from the group consisting of an avidin, a 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, wherein the dissociation step enables removal of the polymer-nucleotide conjugate.In some embodiments, the method further comprises an extension step to incorporate a nucleotide complementary to a nearest base of the target nucleic acid sequence into the two or more primer nucleic acid molecules. In some embodiments, the extension step occurs concurrently with or after the dissociation step.

[0005] Disclosed herein are methods for determining an 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; ii. two or more primer nucleic acid molecules complementary to one or more regions of the target nucleic acid sequence; and iii. two or more polymerase molecules; b. contacting the composition with a polymer-nucleotide conjugate under conditions sufficient to allow the 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 of (a), wherein the polymer-nucleotide conjugate comprises two or more copies of a reversibly terminated nucleotide residue 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. In some embodiments, the target nucleic acid sequence is DNA. In some embodiments, the method further comprises contacting the composition of (a) with reversibly terminated nucleotides or polymer-nucleotide conjugates comprising two or more copies of a reversibly terminated nucleotide after detecting 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 measuring fluorescence. In some embodiments, contacting comprises using some type of polymer-nucleotide conjugate.In some embodiments, contacting comprises using 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 residue. In some embodiments, contacting comprises using three types of polymer-nucleotide conjugates, and wherein each of the three types of polymer-nucleotide conjugates comprises a different type of nucleotide residue. In some embodiments, the polymer-nucleotide conjugate comprises a blocked nucleotide residue. In some embodiments, the blocked nucleotide is a 3'-O-azidomethyl, 3'-O-methyl, or 3'-O-alkylhydroxylamine. In some embodiments, contacting occurs in the presence of an ion that stabilizes the multivalent binding complex. In some embodiments, the polymerase molecules are catalytically inactive.In some embodiments, the polymerase molecules have been rendered catalytically inactive by mutation or chemical modification. In some embodiments, the polymerase molecules are catalytically active. In some embodiments, the polymer-nucleotide conjugate does not comprise a blocked nucleotide residue. In some embodiments, the method can be performed at a temperature within a range of 25°C to 80°C. In some embodiments, 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 on, attached to, or hybridized to a surface, wherein a fluorescent image of the multivalent binding complex on the surface has a contrast-to-noise ratio in the detection step of greater than 20.

[0006] Also disclosed herein are systems comprising: a) one or more computer processors programmed individually or jointly to implement a method comprising: i) contacting a substrate comprising multiple copies of a target nucleic acid sequence bound 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 a multivalent binding complex to be formed between the polymer-nucleotide conjugate and 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 residue 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 a 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 for specified time intervals. In some embodiments, the system further comprises an imaging module configured to acquire images of the substrate surface. In some embodiments, (ii) and (iii) are repeated two or more times,whereby the identity of a series of two or more nucleotides in the target nucleic acid sequence is determined. In some embodiments, the series of steps further comprises a dissociation step that destabilizes the multivalent binding complex, wherein the dissociation step enables removal of the polymer-nucleotide conjugate. In some embodiments, the series of steps further comprises an extension step to add a nucleotide that is complementary to a nearest base of the target nucleic acid sequence.into the two or more primer nucleic acid molecules. In some embodiments, the extension step occurs simultaneously with or after the dissociation step. In some embodiments, the detectable label comprises a fluorophore, and the images comprise fluorescent images. In some embodiments, the fluorescent images of the multivalent binding complex on the substrate surface have a contrast-to-noise ratio greater than 20 when the fluorophore is Cyanine Dye 3 (Cy3) and the image is acquired using an inverted fluorescence microscope equipped with a 20X objective, NA = 0.75, a dichroic mirror optimized for 532 nm light, a bandpass filter optimized for the emission of Cyanine Dye 3, and a camera under non-saturating conditions while the surface is immersed in a buffer of 25 mM ACES, pH 7.4. 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, base calling accuracy is characterized by a Q-value of greater than 25 for at least 80% of the determined nucleotide identities. In some embodiments, base calling accuracy is characterized by a Q-value of greater than 30 for at least 80% of the determined nucleotide identities. In some embodiments, base calling accuracy is characterized by a Q-value of greater than 40 for at least 80% of the determined nucleotide identities.

[0007] Disclosed herein are compositions comprising: a) a polymer core; and b) two or more nucleotide, nucleotide analog, nucleoside, or nucleoside analog units linked to the polymer core; wherein the length of the linker depends on the nucleotide, nucleotide analog, nucleoside, or nucleoside analog unit linked 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 polymer core; and ii) two or more nucleotide, nucleotide analog, nucleoside, or nucleoside analog units linked to the polymer core, wherein the length of the linker depends on the nucleotide, nucleotide analog, nucleoside, or nucleoside analog unit linked to the polymer core; and wherein the mixture comprises polymer-nucleotide conjugates having at least two different types of linked nucleotide, nucleotide analog, nucleoside, or nucleoside analog units. In some embodiments, the polymer core comprises a polymer having a plurality of branches and the two or more nucleotide, nucleotide analog, nucleoside, or nucleoside analog units are attached to the branches.In some embodiments, the polymer has a star, comb, cross-linked, bottlebrush, or dendrimer configuration. In some embodiments, the polymer-nucleotide conjugate comprises one or more binding groups selected from the group consisting of an avidin, a biotin, an affinity tag, and combinations thereof. In some embodiments, the polymer core comprises a branched polyethylene glycol (PEG) molecule. 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.

[0008] In some embodiments, the present disclosure provides methods for determining the identity of a nucleotide in a target nucleic acid, comprising the steps, without regard to any particular order of operations, 1) providing a composition comprising: a target nucleic acid comprising two or more repeats of an identical 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 the 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 present disclosure provides the method, wherein the target nucleic acid is DNA, and / or wherein the target nucleic acid has been replicated, such as 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, in particular 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 occurs in the presence of strontium ions, barium ions, magnesium ions, and / or calcium ions. In some embodiments, the present disclosure provides the method wherein the polymerase molecule is catalytically inactive, for example, when the polymerase molecule has been rendered catalytically inactive by mutation, by chemical modification, or by the absence of a necessary 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 persistence time of more than 2 seconds and / or wherein the method is carried out at a temperature of 15°C or more, 20°C or more, 25°C or more, 35°C or more, 37°C or more, 42°C or more, 55°C or more,60°C or more or 72°C or within a range defined by any of the above statements. In some embodiments, the present disclosure provides the method, wherein the binding complex is deposited on, attached to, or hybridized to a surface that exhibits a contrast-to-noise ratio in the detection step of more than 20. In some embodiments, the present disclosure provides the method, wherein the composition is deposited under buffer conditions that include a polar aprotic solvent. In some embodiments, the present disclosure provides the method, wherein the contacting is carried out under a condition that stabilizes the binding complex when the nucleotide is complementary to a nearest base of the target nucleic acid and destabilizes the binding complex,if the nucleotide is not complementary to the nearest 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 linked to the branches, in particular wherein the first polymer has a star, comb, cross-linked, bottlebrush, 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 the group consisting of avidin, biotin, an affinity tag, and combinations thereof. In some embodiments, the present disclosure provides the method, further comprising a dissociation step,which destabilizes 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 to incorporate a nucleotide complementary to the nearest base of the target nucleic acid into the primer nucleic acid, and wherein the extension step optionally occurs simultaneously with or after the dissociation step.

[0009] In some embodiments, the present disclosure provides a composition comprising a branched polymer having two or more branches and two or more copies of a nucleotide, wherein the nucleotide is attached to a first plurality of the branches or arms, and optionally wherein one or more interaction moieties are attached to a second plurality of the branches or arms. In some embodiments, the composition may further comprise one or more labels 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 composition comprising or incorporating a nucleotide or nucleotide analog that is modified to prevent its incorporation into an extended nucleic acid chain during a polymerase reaction. In some embodiments, the composition may comprise or incorporate a nucleotide or nucleotide analog that is reversibly modified to prevent its incorporation into an extended nucleic acid chain during a polymerase reaction. In some embodiments, the present disclosure provides the composition wherein 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 extend from a central moiety. In some embodiments, the composition may comprise one or more interaction moieties, wherein the interaction moieties may comprise avidin or streptavidin; a biotin moiety; an affinity tag; an enzyme, an antibody, a minibody, a receptor, or other protein; 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 analogue is attached to the branch or arm by a linker; and in particular wherein the linker comprises PEG, and wherein the PEG linker moiety has an average molecular weight of about 1 kDa, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 50 kDa, about 100 kDa, about 150 kDa, or about 200 kDa, or greater than about 200 kDa. 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 between about 5 kDa and about 20 kDa.In some embodiments, the present disclosure provides the composition, wherein at least one nucleotide or nucleotide analog comprises a deoxyribonucleotide, a ribonucleotide, a deoxyribonucleoside, or a ribonucleoside; and / or wherein the nucleotide or nucleotide analog is conjugated to the linker through the 5' end of the nucleotide or nucleotide analog. 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-methyluridine, and / or cytidine; and wherein the length of the linker is between 1 nm and 1000 nm.In some embodiments, the present disclosure provides the composition, wherein at least one nucleotide or nucleotide analogue is a nucleotide that has been modified to inhibit extension during a polymerase reaction or a sequencing reaction, such as in a case where the at least one nucleotide or nucleotide analogue is a nucleotide lacking a 3'-hydroxyl group; is a nucleotide that has been modified to contain a blocking group at the 3'-position; and / or is 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 at least one nucleotide or nucleotide analog is a nucleotide that has not been modified at the 3' position.

[0010] In some embodiments, the present disclosure provides a method for determining the sequence of a nucleic acid molecule, comprising the steps, without regard to any particular order, of 1) providing a nucleic acid molecule comprising a template strand and a complementary strand that is at least partially complementary to the template strand; 2) contacting the nucleic acid molecule with the one or more nucleic acid binding compositions according to any of the embodiments disclosed herein; 3) detecting binding of the nucleic acid binding composition to the nucleic acid molecule; and 4) determining an identity of a terminal nucleotide to be incorporated into the complementary strand of the nucleic acid molecule.In some embodiments, the present disclosure provides a method for determining the sequence of a nucleic acid molecule, comprising the steps, without regard to any particular order, of 1) providing a nucleic acid molecule comprising a template strand and a complementary strand that is at least partially complementary to the template strand; 2) contacting the nucleic acid molecule with the one or more nucleic acid binding compositions according to any of the embodiments disclosed herein; 3) detecting partial or complete incorporation of the nucleic acid binding composition into the nucleic acid molecule; and 4) determining an identity of a 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 steps of contacting, detecting, and incorporating 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 bound to a solid support; and in particular, wherein the solid support comprises a glass or polymer substrate, at least one hydrophilic polymer coating layer, and a plurality of oligonucleotide molecules bound to at least one hydrophilic polymer coating layer.In some embodiments, the present disclosure provides the method, further comprising embodiments wherein at least one hydrophilic polymer coating layer 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 have a surface density of at least 500 molecules / mm. 2 , at least 1,000 molecules / mm 2 , at least 5,000 molecules / mm 2 , at least 10,000 molecules / mm 2 , at least 20,000 molecules / mm 2 , at least 50,000 molecules / mm 2 , at least 100,000 molecules / mm 2 or at least 500,000 molecules / mm 2is present. 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 the clonal amplification comprises using a 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), circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, single-strand binding protein-dependent amplification (SSB), or a combination thereof.In some embodiments, the present disclosure provides the method, wherein the one or more nucleic acid binding compositions are labeled with fluorophores, and the detecting step comprises using fluorescence imaging; and in particular, wherein the fluorescence imaging comprises two excitation wavelengths / four emission wavelengths fluorescence imaging.In some embodiments, the present disclosure provides the method wherein four different nucleic acid binding compositions, each comprising a different nucleotide or nucleotide analogue, are used to determine the identity of the terminal nucleotide, wherein the four different nucleic acid binding compositions are labelled with separate respective fluorophores, and wherein the detecting step comprises simultaneously exciting at a wavelength sufficient to excite all four fluorophores and imaging the fluorescence emission at wavelengths sufficient to detect each respective fluorophore.In some embodiments, the present disclosure provides the method wherein four different nucleic acid binding compositions, each comprising a different nucleotide or nucleotide analogue, are used to determine the identity of the terminal nucleotide, 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), respectively, and wherein the detection step comprises simultaneously exciting at any two of 532 nm, 568 nm, and 633 nm and imaging the fluorescence emission at about 570 nm, 592 nm, 670 nm, and 702 nm, respectively; and / or wherein the fluorescence imaging comprises fluorescence imaging with two excitation wavelengths / two emission wavelengths.In some embodiments, the present disclosure provides the method wherein four different nucleic acid binding compositions, each comprising a different nucleotide or nucleotide analogue, are used to determine the identity of the terminal nucleotide, wherein one, two, three or four different nucleic acid binding compositions are each labelled, each with a different fluorophore or set of fluorophores, and wherein the detecting step comprises simultaneously exciting at a wavelength sufficient to excite one, two, three or four fluorophores or sets of fluorophores and imaging the fluorescence emission at wavelengths sufficient to detect each respective fluorophore.In some embodiments, the present disclosure provides the method wherein three different nucleic acid binding or incorporation compositions, each comprising a different nucleotide or nucleotide analogue, are used to determine the identity of the terminal nucleotide, wherein one, two, or three different nucleic acid binding or incorporation compositions are each labeled with a different fluorophore or set of fluorophores, and wherein the detecting step comprises simultaneously exciting at a wavelength sufficient to excite one, two, or three fluorophores or sets of fluorophores and imaging the fluorescence emission at wavelengths sufficient to detect each respective fluorophore, and wherein the detection of the fourth nucleotide is determined or determinable with respect to the location of "dark" or unlabeled spots or target nucleotides.In some embodiments, the present disclosure provides the method wherein the multivalent binding or incorporation composition can comprise three types of polymer-nucleotide conjugates, and wherein each of the three types of polymer-nucleotide conjugates comprises a different type of nucleotide. In some embodiments, the present disclosure provides the method wherein the detection of the binding or incorporation complex is performed in the absence of unbound or in-solution polymer-nucleotide conjugates.

[0011] In some embodiments, the present disclosure provides the method wherein four different nucleic acid binding compositions or three different nucleic acid binding or incorporation compositions, each comprising a different nucleotide or nucleotide analogue, are used to determine the identity of the terminal nucleotide, 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 both the first and second fluorophore, and one is unlabeled or missing, and wherein the detecting step comprises simultaneous excitation at a first excitation wavelength and a second excitation wavelength and images are acquired at a first fluorescence emission wavelength and a second fluorescence emission wavelength.In some embodiments, the present disclosure provides the method, wherein the first fluorophore is Cy3, the second fluorophore is Cy5, the first excitation wavelength is 532 nm or 568 nm, the second excitation wavelength is 633 nm, the first fluorescence emission wavelength is about 570 nm, and the second fluorescence emission wavelength is about 670 nm. In some embodiments, the present disclosure provides the method, wherein the detection label may comprise one or more portions of a fluorescence resonance energy transfer (FRET) pair, such that multiple classifications can be performed under a single excitation and imaging step.In some embodiments, the present disclosure provides the method, wherein a sequencing reaction cycle comprising the steps of contacting, detecting, and incorporating / extending is performed in less than 30 minutes, in less than 20 minutes, or in less than 10 minutes. In some embodiments, the present disclosure provides the method, wherein an average Q-value for the base call accuracy over 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-value greater than 30 and / or greater than or equal to 40.In some embodiments, the present disclosure provides the method wherein at least 95% of the identified terminal nucleotides have a Q value greater than 30.

[0012] In some embodiments, the present disclosure provides a reagent comprising one or more nucleic acid binding compositions as disclosed herein and a buffer. For example, in some embodiments, the present disclosure provides a reagent, wherein the reagent 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, a reagent of the present disclosure 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 each comprises one or more of the group consisting of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), deoxyadenosine triphosphate (dATP),Deoxyadenosine diphosphate (dADP) and deoxyadenosine monophosphate (dAMP); one or more selected from the group consisting of 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 (dUDP) and deoxyuridine monophosphate (dUMP); one or more selected from the group consisting of cytidine triphosphate (CTP), cytidine diphosphate (CDP), cytidine monophosphate (CMP), deoxycytidine triphosphate (dCTP), deoxycytidine diphosphate (dCDP) and deoxycytidine monophosphate (dCMP); and one or more selected from the group consisting of guanosine triphosphate (GTP), guanosine diphosphate (GDP), guanosine monophosphate (GMP), deoxyguanosine triphosphate (dGTP),Deoxyguanosine diphosphate (dGDP) and deoxyguanosine monophosphate (dGMP). In some other examples or some further examples, the present disclosure provides a reagent 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 each correspond to one or more of the group consisting of 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.

[0013] Disclosed herein are kits comprising the nucleic acid binding or incorporation composition of any of the embodiments disclosed herein and / or a reagent of any of the embodiments disclosed herein and / or one or more buffers; and instructions for their use.

[0014] Disclosed herein are systems for performing the method according to any embodiment disclosed herein, comprising a nucleic acid binding or incorporation composition according to any embodiment disclosed herein and / or a reagent according to any embodiment disclosed herein. In some embodiments, a system is configured to iteratively perform the sequential contacting of tethered, primed nucleic acid molecules with the nucleic acid binding or incorporation compositions and / or the reagents; and to detect the binding or incorporation of the disclosed nucleic acid binding or incorporation compositions to the one or more primed nucleic acid molecules.

[0015] In some embodiments, the present disclosure provides a composition comprising a particle (e.g., a nanoparticle or a polymer core), wherein the particle comprises a plurality of enzyme or protein binding or incorporation substrates, wherein the enzyme or protein binding or incorporation substrates bind with one or more enzymes or proteins to form one or more binding or incorporation complexes (e.g., a multivalent binding or incorporation complex), and wherein the binding or incorporation can be monitored or identified by observing the location, presence, or persistence of the one or more binding or incorporation complexes. In some embodiments, the particle may comprise a polymer, branched polymer, dendrimer, liposome, micelle, nanoparticle, or quantum dot.In some embodiments, the substrate may comprise a nucleotide, a nucleoside, a nucleotide analog, or a nucleoside analog. In some embodiments, the enzyme or protein binding or incorporation substrate may comprise an agent capable of binding with a polymerase. In some embodiments, the enzyme or protein may comprise a polymerase. In some embodiments, observing the location, presence, or persistence of one or more binding or incorporation complexes 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 of a detectably different 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 bound nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs bound to the particle. In some embodiments, the present disclosure provides the composition, wherein the nucleoside or nucleoside analog is present at a surface density between 0.001 and 1,000,000 per µm. 2 , between 0.01 and 1,000,000 per µm 2 , between 0.1 and 1,000,000 per µm 2 , between 1 and 1,000,000 per µm 2 , between 10 and 1,000,000 per µm 2 , between 100 and 1,000,000 per µm 2 , between 1,000 and 1,000,000 per µm 2, between 1,000 and 100,000 per µm 2 , between 10,000 and 100,000 per µm 2 or between 50,000 and 100,000 per µm 2or is within a range defined by any two of the preceding values. In some embodiments, the present disclosure provides the composition, wherein the nucleoside or nucleoside analogue is present within a nucleotide or nucleotide analogue. In some embodiments, the present disclosure provides the composition, wherein the composition comprises or includes a nucleotide or nucleotide analogue that is modified to prevent its incorporation into an extended nucleic acid chain during a polymerase reaction. In some embodiments, the present disclosure provides the composition, wherein the composition comprises or includes a nucleotide or nucleotide analogue that is reversibly modified to prevent its incorporation into an extended nucleic acid chain during a polymerase reaction.In some embodiments, the present disclosure provides the composition wherein 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., nucleotide, nucleotide analog, nucleoside, or nucleoside analog) is attached to the particle by a linker.In some embodiments, the present disclosure provides the composition, wherein at least one nucleotide or nucleotide analog is a nucleotide that has been modified to inhibit extension during a polymerase reaction or a sequencing reaction, such as a nucleotide lacking a 3'-hydroxyl group; a nucleotide that has been modified to contain a blocking group at the 3'-position; 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; and / or a nucleotide that has not been modified at the 3'-position.

[0016] In some embodiments, the present disclosure provides a method for determining the sequence of a nucleic acid molecule, comprising the steps, without regard to the order, of 1) providing a nucleic acid molecule comprising a template strand and a complementary strand that is at least partially complementary to the template strand; 2) contacting the nucleic acid molecule with the one or more nucleic acid binding or incorporation compositions according to any of the embodiments disclosed herein; 3) detecting binding or incorporation of the nucleic acid binding or incorporation composition into the nucleic acid molecule, and 4) determining an identity of a terminal nucleotide to be incorporated into the complementary strand of the 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 the clonal amplification comprises using a 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, circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, single-strand binding protein-dependent amplification (SSB), or a combination thereof.In some embodiments, the present disclosure provides the method, wherein a sequencing reaction cycle comprising the steps of contacting, detecting, and incorporating is performed in less than 30 minutes, in less than 20 minutes, or in less than 10 minutes. In some embodiments, the present disclosure provides the method, wherein an average Q-value for the base call accuracy over 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 Q-value greater than 30; or greater than 40.In some embodiments, the present disclosure provides the method, wherein at least 95% of the identified terminal nucleotides have a Q value greater than 30.

[0017] In some embodiments, the present disclosure provides a reagent comprising one or more nucleic acid binding or incorporation compositions as disclosed herein and a buffer. In some embodiments, the present disclosure provides the reagent, wherein the reagent 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 analogue, and wherein the nucleotide or nucleotide analogue comprises a nucleotide, nucleotide analogue, nucleoside, or nucleoside analogue.In some embodiments, the present disclosure provides the method, wherein the reagent 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 can each correspond to: one or more of the group consisting of ATP, ADP, AMP, dATP, dADP, and dAMP; one or more of the group consisting of TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, and dUMP; one or more of the group consisting of CTP, CDP, CMP, dCTP, dCDP, and dCMP; and one or more of the group consisting of GTP, GDP, GMP, dGTP, dGDP and dGMP.In some embodiments, the present disclosure provides the method, wherein the reagent 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 each corresponds to one or more of the group consisting of 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.

[0018] In some embodiments, the present disclosure provides a kit comprising any of the compositions disclosed herein; and / or any of the reagents disclosed herein; one or more buffers; and instructions for their use.

[0019] In some embodiments, the present disclosure provides a system for performing any of the methods disclosed herein; wherein the methods may comprise: using one of the compositions as disclosed herein; and / or one of the reagents as disclosed herein; one or more buffers; and one or more nucleic acid molecules optionally bound or attached to a solid support, wherein the system is configured to iteratively perform: sequentially contacting the nucleic acid molecules with the composition and / or the reagent; and detecting binding or incorporation of the nucleic acid binding or incorporation compositions to the one or more nucleic acid molecules.

[0020] In some embodiments, the present disclosure provides a composition as disclosed herein for use in increasing the contrast to noise ratio (CNR) of a labeled nucleic acid complex bound to or associated with a surface.

[0021] In some embodiments, the present disclosure provides a composition as disclosed herein for use in establishing or maintaining control over the persistence time of a signal from a labeled nucleic acid complex bound to or associated with a surface.

[0022] In some embodiments, the present disclosure provides a composition as disclosed herein for use in establishing or maintaining control over the persistence time of a fluorescent, luminescent, electrical, electrochemical, colorimetric, radioactive, magnetic, or electromagnetic signal from a labeled nucleic acid complex bound to or associated with a surface.

[0023] In some embodiments, the present disclosure provides a composition as disclosed herein for use in increasing the specificity, accuracy, or read length of a nucleic acid sequencing and / or genotyping application.

[0024] In some embodiments, the present disclosure provides a composition as disclosed herein for use in increasing specificity, accuracy, or read length in a method of sequencing by binding or incorporation, sequencing by synthesis, single molecule sequencing, or ensemble sequencing.

[0025] In some embodiments, the present disclosure provides a reagent as disclosed herein for use in increasing the contrast-to-noise ratio (CNR) of a labeled nucleic acid complex bound to or associated with a surface.

[0026] In some embodiments, the present disclosure provides a reagent as disclosed herein for use in establishing or maintaining control over the persistence time of a signal from a labeled nucleic acid complex bound to or associated with a surface.

[0027] In some embodiments, the present disclosure provides a reagent as disclosed herein for use in establishing or maintaining control over the persistence time of a fluorescent, luminescent, electrical, electrochemical, colorimetric, radioactive, magnetic, or electromagnetic signal from a labeled nucleic acid complex bound to or associated with a surface.

[0028] In some embodiments, the present disclosure provides a reagent as disclosed herein for use in increasing the specificity, accuracy, or read length of a nucleic acid sequencing and / or genotyping application.

[0029] In some embodiments, the present disclosure provides a reagent as disclosed herein for use in increasing specificity, accuracy, or read length in a method for sequencing by binding or incorporation, sequencing by synthesis, single molecule sequencing, or ensemble sequencing. INCLUSION BY REFERENCE

[0030] All publications, patents, and patent applications mentioned in this specification are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated as being incorporated by reference in its entirety. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein shall control. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The patent or application file contains at least one color drawing. Copies of publications of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the required fee.

[0032] The novel features of the inventive concepts disclosed herein are particularly described in the appended claims. A better understanding of the features and advantages of the disclosed compositions, methods, and systems will be obtained by reference to the following detailed description, which sets forth illustrative embodiments in which the principles of the inventive concepts are utilized, and to the accompanying drawings, in which: Fig. 1A-1H illustrate the steps using a non-limiting example of a multivalent binding composition for sequencing a target nucleic acid: Fig. Figure 1A illustrates a non-limiting example 4 of attaching target nucleic acid to a surface; Fig. Figure 1B illustrates clonal targeting of nucleic acid to form clusters of amplified target nucleic acid molecules; Fig. Figure 1C illustrates a non-limiting example of priming the target nucleic acid to produce a primed target nucleic acid; Fig. Figure 1D illustrates a non-limiting example of contacting the primed target nucleic acid with the multivalent binding composition and polymerase to form a binding complex; Fig. Figure 1E illustrates a non-limiting example of the images of the binding complex acquired on the surface; Fig. Figure 1F illustrates a non-limiting example of extending the primer strand by one nucleotide; Fig. Figure 1G illustrates a non-limiting example of another cycle of contacting the primed target nucleic acid with the multivalent binding composition and polymerase to form a binding complex; and Fig. Figure 1H illustrates non-limiting examples of the images of the binding complex acquired on the surface in subsequent sequencing cycles. Fig. Figure 2 shows a flow diagram outlining the steps for sequencing a target nucleic acid and extending the primer strand by a single base addition. Fig. Figure 3 shows a flow diagram outlining the steps for sequencing a target nucleic acid and extending the primer strand by incorporating the nucleotide into the particle-nucleotide conjugate. Fig. Figures 4A-4B illustrate a non-limiting example of target nucleic acid detection using the polymer-nucleotide conjugates. Fig. Figure 4A shows the step of contacting the polymerase and the polymer-nucleotide conjugates with some nucleic acid molecules; Fig. Figure 4B shows the binding complex formed between the polymerase, the polymer-nucleotide conjugates, and the target nucleic acid molecules. Fig. 5A-5C show schematic representations of non-limiting examples of varying configurations of the polymer-nucleotide conjugates: Fig. Figure 5A shows polymer-nucleotide conjugates with various multi-arm configurations; Fig. Figure 5B shows a polymer-nucleotide conjugate in which the polymer branch originates from the center; and Fig. Figure 5C shows polymer-nucleotide conjugates with the binding moiety biotin. Fig. Figure 6 shows a generalized graphical representation of the increase in signal intensity observed during binding, persistence, and washing and removal of multivalent substrates. Fig. 7A-7J show fluorescence images of the steps in a sequencing reaction using multivalent PEG substrate compositions. Fig. 7A. Red and green fluorescence images after exposure of DNA-RCA templates (G and A first base) to 500 nM base-labeled nucleotides (A-Cy3 and G-Cy5) in exposure buffer containing 20 nM Klenow polymerase and 2.5 mM Sr +2 The images were acquired after washing with imaging buffer with the same composition as the exposure buffer, but without nucleotides or polymerase. The contrast was scaled to maximize visualization of the weakest signals, but no signals remained after washing with imaging buffer ( Fig. 7A, insert). Fig. 7B-7E: Fluorescence images showing the multivalent (base-labeled) PEG-nucleotide ligands PB1 ( Fig. 7B), PB2 ( Fig. 7C), PB3 ( Fig. 7D) and PB5 ( Fig. 7E) with an effective nucleotide concentration of 500 nM after mixing in exposure buffer and imaging in imaging buffer as described above. Fig. Figure 7F: Fluorescence image showing the multivalent (base-labeled) PEG-nucleotide ligand PB5 at 2.5 µM after mixing in exposure buffer and imaging in imaging buffer as above. Fig. 7G-7I: Fluorescence images showing further base discrimination by exposing the multivalent binding composition to inactive mutants of Klenow polymerase ( Fig. 7G. D882H; Fig. 7H. D882E; Fig. 7I. D882A) vs. the wild-type Klenow enzyme (control) ( Fig. 7 years). Fig. Figures 8A-8B show the effectiveness of the multivalent reporter compositions in determining the base sequence of a DNA sequence over 5 sequencing cycles: Fig. Figure 8A shows images and expected sequences for templates acquired after each sequencing cycle; and Fig. Figure 8B shows aligned sequencing results using the Fig. 8A captured images. Fig. 9A-9J show fluorescence images of multivalent polyethylene glycol-polymer-nucleotide conjugates (PEG) (base-labeled) with an effective nucleotide concentration of 500 nM and varying PEG branch length after contacting with a support surface containing DNA templates (comprising G or A as the first base; prepared using rolling circle amplification (RCA)) in an exposure buffer containing 20 nM Klenow polymerase and 2.5 mM Sr +2The images were acquired after washing with an imaging buffer that had the same composition as the exposure buffer, but without nucleotides and polymers. Panels show images obtained using multivalent PEG-nucleotide ligands with arm lengths as follows. Fig. 9A: 1 K PEG. Fig. 9B: 2K PEG. Fig. 9C: 3 K PEG. Fig. 9D: 5K PEG. Fig. 9E: 10K PEG. Fig. 9F: 20K PEG. Fig. Figure 9G shows images obtained using 10 K PEG and an inactive Klenow polymerase comprising the mutation D882H. Fig. Figure 7H shows images obtained using 10 K PEG and an inactive Klenow polymerase comprising the mutation D882E. Fig. Figure 7I shows images obtained using 10 K PEG and an inactive Klenow polymerase comprising the mutation D882A. Fig. Figure 7J shows images obtained using 10 K PEG and an active wild-type Klenow polymerase. Fig. Figure 10 shows a quantitative representation of the fluorescence intensities in the Fig. Images shown in Figures 9A-9F, separated by color value, with orange trace corresponding to the red label (Cy3 label; A bases) and blue trace corresponding to the green label (Cy5 label; G bases). Fig. Figure 11 shows normalized fluorescence of multivalent substrates bound to DNA clusters as for Fig. 7A-7J, where the substrate complexes are formed in the presence (condition B) and absence (condition A) of Triton-X100 (0.016%). Fig. Figures 12 A-12B show plots of normalized fluorescence intensity measured for multivalent polymer-nucleotide conjugates and free nucleotides. Fig. 12A: Two replicates of a multivalent polymer-nucleotide conjugate bound to DNA clusters (conditions A and B) vs. binding complexes formed using labeled free nucleotides (condition C) after 1 minute; Fig. 12B: Time course of fluorescence of multivalent substrate complexes over 60 min. DETAILED DESCRIPTIONI. Definitions

[0033] As used herein, "nucleic acid" (also referred to as "polynucleotide," "oligonucleotide," ribonucleic acid (RNA), or deoxyribonucleic acid (DNA)) is a linear polymer of two or more nucleotides joined by covalent internucleoside bonds, or variants or functional fragments thereof. In naturally occurring examples of nucleic acids, the internucleoside linkage is a phosphodiester bond. However, other examples optionally include other internucleoside linkages, such as phosphorothiolate bonds, 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 (PNAs), hybrids between PNAs and DNA or RNA, and may also include other types of nucleic acid modifications.

[0034] As used herein, a "nucleotide" refers to a nucleotide, nucleoside, or analogue thereof. Nucleotide refers to both naturally occurring and chemically modified nucleotides and may include, but is not limited to, a nucleoside, a ribonucleotide, a deoxyribonucleotide, a protein nucleic acid residue, or derivatives. Examples of nucleotide include: an adenine, a thymine, a uracil, a cytosine, a guanine, or a residue thereof; a deoxyadenine, a deoxythymine, a deoxyuracil, a deoxycytosine, a deoxyguanine, or a residue thereof; an adenine PNA, a thymine PNA, a uracil PNA, a cytosine PNA, a guanine PNA or a residue or equivalent thereof, an N- or C-glycoside of a purine or pyrimidine base (e.g., a deoxyribonucleoside containing 2-deoxy-D-ribose or a ribonucleoside containing D-ribose).

[0035] "Complementary," as used here, refers to the topological compatibility or matching of the interacting surfaces of a ligand molecule and its receptor. Thus, the receptor and its ligand can be described as complementary, and furthermore, the contact surface properties are complementary to each other.

[0036] "Branched polymer," as used herein, refers to a polymer having a plurality of functional groups that contribute to conjugating a biologically active molecule, such as a nucleotide. The functional group can be located either on the side chain of the polymer or directly attached to a central core or backbone of the polymer. The branched polymer can have a linear backbone with one or more functional groups detached from the backbone for conjugation. The branched polymer can also be a polymer having one or more side chains, wherein the side chain has a site suitable for conjugation.Examples of the functional group include, but are not limited to, hydroxyl, ester, amine, carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, hydrazide, thiol, alkanoic acid, acid halide, isocyanate, isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, and tresylate.

[0037] "Polymerase," as used herein, refers to an enzyme containing a nucleotide binding moiety that assists in the formation of a binding complex between a target nucleic acid and a complementary nucleotide. The polymerase may exhibit one or more activities, including, but not limited to, base analog detection activities, DNA polymerization activity, reverse transcriptase activity, DNA binding or incorporation, strand displacement activity, and nucleotide binding or incorporation and recognition. The polymerase may include catalytically inactive polymerase, catalytically active polymerase, reverse transcriptase, and other enzymes containing a nucleotide binding or incorporation moiety.

[0038] "Persistence time," as used herein, refers to the period of time during which a binding complex formed between the target nucleic acid, a polymerase, a conjugated, or unconjugated nucleotide remains stable without any binding component dissociating from the binding complex. Persistence time is an indicator of the stability of the binding complex and the strength of the binding interactions. Persistence time can be measured by observing the initiation and / or duration of a binding complex, such as by observing a signal from a labeled component of the binding complex. For example, a labeled nucleotide or a labeled reagent comprising one or more nucleotides can be present in a binding complex, allowing the signal from the label to be detected during the persistence time of the binding complex. A non-limiting example of the label is a fluorescent label. II. Methods for analyzing target nucleic acid

[0039] 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 the binding or incorporation of a nucleotide to an enzyme (e.g., polymerase) or enzyme complex can be influenced by increasing the effective concentration of the nucleotide. The increase can be achieved by increasing the concentration of the nucleotide in free solution or by increasing the amount of the nucleotide near the relevant binding or incorporation site.The increase can also be achieved by physically confining a number of nucleotides into a confined volume, resulting in a local increase in concentration, and such a structure can thus bind or be incorporated into the binding or incorporation site with a higher apparent avidity than would be observed with an unconjugated, unbound, or otherwise unrestricted individual nucleotide. One non-limiting means of effecting such confinement is to provide a multivalent binding or incorporation composition in which multiple nucleotides are bound to a particle, such as a polymer, a branched polymer, a dendrimer, a micelle, a liposome, a microparticle, a nanoparticle, a quantum dot, or other suitable particle known in the art.

[0040] The multivalent binding or incorporation composition disclosed herein may include at least one particle-nucleotide conjugate, and the particle-nucleotide conjugate has multiple copies of the same nucleotide bound to the particle. When the nucleotide is complementary to the target nucleic acid, the particle-nucleotide conjugate forms a binding or incorporation complex with the polymerase and the target nucleic acid, and the binding or incorporation complex exhibits increased stability and a longer persistence time than the binding or incorporation complex formed using a single unconjugated or unbound nucleotide.Each of the nucleotide residues 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 molecules (or other enzyme molecules), and the multivalent binding composition (e.g., the polymer-nucleotide conjugate). Each of the nucleotide residues 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 molecules (or other enzyme molecules), and the multivalent binding composition (e.g., the polymer-nucleotide conjugate).From this bound complex, the nucleotide can interrogate the complementary base before incorporating a modified, reversibly blocked nucleotide that extends the replication strand by 1 base. Furthermore, one can imagine interrogating the N-nucleotide with a bound complex, proceeding with a reversibly terminated nucleotide, and then probing the N+1 base before and after unblocking. In this way, error checking could be performed and the overall accuracy of base calling could be improved by reading the interrogated data twice. The important distinguishing factor from conventional methods is that binding is used to interrogate the matching base, whereas the forward or incorporation step is used only for further movement along the extension strand.

[0041] The multivalent binding or incorporation composition can be used to localize detectable signals in active regions of biochemical interactions, such as sites of protein-nucleic acid interactions, nucleic acid hybridization reactions, or enzymatic reactions such as polymerase reactions. For example, the multivalent binding or incorporation composition described herein can be used to identify sites of base incorporation in the extension of nucleic acid chains during polymerase reactions and to provide base discrimination for sequencing and array-based applications. The increased binding or incorporation between the target nucleic acid and the nucleotide in the multivalent binding or incorporation composition, when the nucleotide is complementary to the target nucleic acid, provides an enhanced signal that greatly improves the accuracy of base calling and reduces imaging time.

[0042] Furthermore, the use of multivalent binding composition allows sequencing signals from a given sequence to originate from clustered regions containing multiple copies of the target sequence. Sequencing methods using multiple copies of a target sequence have the advantage that the signals can be amplified by the presence of multiple simultaneous sequencing reactions within the defined region, each providing its own signal. The presence of multiple signals within a defined area also reduces the impact of a single skipped cycle, as the signal from a large number of correct base calls can overwhelm the signal from a smaller number of skipped or incorrect base calls, thus providing methods for reducing phasing errors and / or improving read length in sequencing reactions.

[0043] The multivalent binding compositions disclosed herein and their use have one or more of the following effects: (i) a stronger signal for better base calling accuracy compared to conventional nucleic acid amplification methods and sequencing methods; ii) allowing greater discrimination of the sequence-specific signal from background signals; (iii) reduced requirements on the amount of starting material required; (iv) increased sequencing rate and shortened sequencing time; (v) reducing phase errors; and (vi) improving read length in sequencing reactions.

[0044] In some embodiments, the target nucleic acid may refer to a target nucleic acid sample having one or more nucleic acid molecules. In some embodiments, the target nucleic acid may include a plurality of nucleic acid molecules. In some embodiments, the target nucleic acid may include two or more nucleic acid molecules. In some embodiments, the target nucleic acid may include two or more nucleic acid molecules with the same sequences. A. Sequencing of target nucleic acid

[0045] Fig. 1A-1H illustrate an exemplary method in which the multivalent binding composition is used to sequence a target nucleic acid. As in Fig. As shown in Figure 1A, the target nucleic acid 102 can be bound to a solid support surface 101. The target nucleic acid can be bound to the surface directly or indirectly. Although in Fig. 1A, the target nucleic acid 102 can be hybridized to an adapter that is bound to the surface via a covalent or non-covalent bond. When one or more adapters are used to bind the target nucleic acid to the surface, the target surface can comprise a fragment that is complementary to the adapter and thus hybridizes to the adapter. In some cases, one adapter sequence can be bound to the surface. In some cases, a plurality of adapter sequences can be bound to the surface. In some cases, the target nucleic acid 102 can also be bound directly to the surface of the solid support without the use of an adapter. The solid support can be a surface with weak non-specific binding.

[0046] In Fig. 1B, after the initial step of attaching the target nucleic acid to the surface of a solid support (e.g., by hybridization to adapters), the target nucleic acid is then clonally amplified to form clusters of amplified nucleic acids. When the target nucleic acid is attached to the surface via an adapter, the surface density of clonally amplified nucleic acid sequences hybridized to the adapter on the support surface can span the same range as the surface density of attached 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, circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, single-strand binding protein-dependent amplification (SSB), or a combination thereof.

[0047] Fig. Figure 1C illustrates a non-limiting step of annealing a primer 103 to the target nucleic acid 102 to form a primed target nucleic acid 104. Fig. Figure 1B shows only one primer used in the annealing step, however, depending on the type of target nucleic acid, more than one primer may be used. In some cases, the adapter used to attach the target nucleic acid to the surface has the same sequence as the primer used to produce the primed target nucleic acid. The primer may include forward amplification primers, reverse amplification primers, sequencing primers, and / or molecular barcoding sequences, or any combination thereof. In some cases, one primer sequence may be used in the hybridization step. In some cases, a variety of different primer sequences may be used in the hybridization step.

[0048] As 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. 1D comprises four particle-nucleotide conjugates 105a, 105b, 105c, and 105d. Each particle-nucleotide conjugate has multiple copies of a nucleotide bound to the particle, and the four particle-nucleotide conjugates each cover four types of nucleotides. The particle-nucleotide conjugate, which has a nucleotide complementary to the nearest base on the primed target nucleic acid, forms a binding or incorporation complex with the polymerase and the target nucleic acid. In some cases, the multivalent binding or incorporation composition may include one, two, or three particle-nucleotide conjugates. In some embodiments, each different type of particle-nucleotide conjugate may be labeled with a separate label. In some embodiments, three of four types of nucleotide conjugates may be labeled, with a fourth being either unlabeled or conjugated to a non-detectable 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 their conjugated nucleotide, with 3, 2, 1, or no particle-nucleotide conjugates, each of which may be left unlabeled or conjugated with an undetectable label. In some embodiments, detection of a polymerase complex containing a particle-nucleotide conjugate may be performed using four-color detection, such that conjugates corresponding to all four nucleotides are present in a sample, with each conjugate having a separate label corresponding to the nucleotide conjugated to it.In some embodiments, the four particle-nucleotide conjugates can be exposed to or contacted with the target nucleic acid simultaneously; in some other embodiments, the four particle-nucleotide conjugates can be exposed to or contacted with the target nucleic acid sequentially, either individually or in groups of two or three. In some embodiments, detection of a polymerase complex containing a particle-nucleotide conjugate can be performed using three-color detection, such that conjugates corresponding to three of the four nucleotides are present in a sample, with three conjugates having a separate label corresponding to the nucleotide conjugated thereto, and one conjugate having no label or being 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 a sample, with three conjugates having a separate label corresponding to the nucleotide conjugated thereto and one conjugate missing. In some embodiments, the identity of nucleotides corresponding to an unlabeled or missing nucleotide conjugate can be determined with respect to the position and / or identity of "dark" spots or positions of known target nucleic acids that do not exhibit a fluorescent signal. In some embodiments, the present disclosure provides the method, wherein the detection of the binding or incorporation complex is performed in the absence of unbound or in-solution polymer-nucleotide conjugates.

[0049] In some embodiments where three of the four particle-nucleotide conjugates are labeled, or where 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 unlabeled complexes, such as by identifying "dark" spots or unlabeled regions in a sequencing reaction. In some embodiments, detection of a polymerase complex containing a particle-nucleotide conjugate can be performed using two-color detection, such that conjugates corresponding to two of the four nucleotides are present in a sample, with two conjugates having a separate label corresponding to the nucleotide conjugated thereto, and two conjugates having no label or being conjugated to an undetectable label.In some embodiments, only two of the four particle-nucleotide conjugates are labeled. In some embodiments where two of the four particle-nucleotide conjugates are labeled, the identity of the nucleotide corresponding to the unlabeled conjugate(s) can be determined by the absence of a signal or by monitoring the presence of unlabeled complexes, such as by identifying "dark" spots or unlabeled regions in a sequencing reaction. In some embodiments where two of the four particle-nucleotide conjugates are labeled, the four particle-nucleotide conjugates can be sequentially exposed to or contacted with the target nucleic acid, either individually or in groups of two or three.In some embodiments, two of the four particle-nucleotide conjugates may share a common label, and the four particle-nucleotide conjugates may be sequentially exposed to or contacted with the target nucleic acid, either individually or in groups of two or three, with each contacting step demonstrating the discrimination between two or more different bases, such that after two, three, four or more such contacting steps, the identities of all unknown bases have been determined.

[0050] Fig. Figure 1E illustrates the images captured on the surface after the binding or incorporation complex was formed between the polymerase, the target nucleic acid, and the particle-nucleotide conjugate containing a nucleotide complementary to the nearest 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. Each binding or incorporation complex has a different nucleotide that can be distinguished based on the label (e.g., fluorescence emission dye) on the particle-nucleotide conjugate. Because the use of the particle-nucleotide conjugate allows binding or incorporation signals from a given sequence to arise within cluster regions containing multiple copies of the target sequence, the sequencing signals are greatly enhanced. Although Fig. 1E includes four particle-nucleotide conjugates, each having a different type of nucleotide, some methods may use one, two, or three particle-nucleotide conjugates, each having a different type of nucleotide and label. In some embodiments, each different type of particle-nucleotide conjugate may be labeled either with the same label or each with a label corresponding to the identity of its conjugated nucleotide. In some embodiments, three of the four types of nucleotide conjugates may be labeled, with a fourth being either unlabeled or conjugated to a non-detectable label. In some embodiments, 1, 2, 3, or 4 particle-nucleotide conjugates may be labeled with a separate label, with 3, 2, 1, or no particle-nucleotide conjugates being either unlabeled or conjugated to a non-detectable label, respectively.In some embodiments, a detection step may comprise simultaneous and / or serial excitation of up to 4 different excitation wavelengths, for example, wherein fluorescence imaging is performed by detecting single and / or multiple fluorescence emission bands that uniquely classify each of the possible base pairings (A, G, C or T).In some embodiments, four different nucleic acid binding or incorporation compositions, each comprising a different nucleotide or nucleotide analogue, can be used to determine the identity of the terminal nucleotide, 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 both the first and second fluorophore, 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 a first fluorescence emission wavelength and a second fluorescence emission wavelength.

[0051] When the multivalent binding or incorporation composition is used to replace individual unconjugated or unbound nucleotides to form a binding or incorporation complex with the polymerase and the primed target nucleic acid, the local concentration of the nucleotide is increased severalfold, which in turn increases the signal intensity. The formed binding or incorporation complex also exhibits a longer persistence time, which in turn contributes to shortening the imaging step. The high signal intensity results from the high binding or incorporation avidity of the polymer-nucleotide conjugate (which may also include multiple fluorophores or other labels), which thus forms a complex that remains stable throughout the binding or incorporation and imaging step.The strong binding or incorporation between the polymerase, the primed target strand, and the polymer-nucleotide or nucleotide analogue conjugate also means that the resulting multivalent binding or incorporation complex remains stable during the washing steps, and the signal intensity remains high when other reaction mixture components and mismatched nucleotide analogues are washed away. After the imaging step, the binding or incorporation complex can be destabilized (e.g., by changing the buffer composition), and the primed target nucleic acid can then be extended by one base.

[0052] The sequencing method may further comprise incorporating the N+1 or terminal nucleotide into the primed strand as in Fig. 1F shown. In Fig. 1F, the primer strand of the primed target nucleic acid 108 can be extended by one base to form an extended nucleic acid 109. The extension step can occur after or simultaneously with the destabilization of the multivalent binding or incorporation complex. The primed target nucleic acid 108 can be extended using a complementary nucleotide bound to the particle in the particle-nucleotide conjugate or using an unconjugated or unbound free nucleotide provided after the multivalent binding or incorporation composition has been removed.

[0053] After the extension step, the contacting step can be carried out as in Fig. 1G, can be performed again to form binding or incorporation complexes and mimic the next sequencing cycle. The steps of contacting, detecting, and extending can be repeated for one or more cycles, thereby determining the sequence of the target nucleic acid molecule. For example, Fig. 1H the surface images obtained after performing multiple sequencing cycles, and the images can then be processed to determine the sequences of the target nucleic acid molecules.

[0054] Extension of the primed target nucleic acid may be prevented or inhibited due to a blocked nucleotide on the strand or the use of polymerase that is catalytically inactive. If the nucleotide in the polymer-nucleotide conjugate has a blocking group that prevents extension of the nucleic acid, incorporation of a nucleotide may be achieved by removing a blocking group from the nucleotide (such as by cleaving the nucleotide from its polymer, branched polymer, dendrimer, particle, or the like). If extension of the primed target nucleic acid is inhibited due to the use of polymerase that is catalytically inactive, incorporation of a nucleotide may be achieved by providing a cofactor or activator, such as a metal ion.

[0055] Also disclosed herein are systems configured to perform any of the disclosed nucleic acid sequencing or nucleic acid analysis methods. The system may comprise a fluid flow controller and / or a fluid delivery system configured to sequentially and iteratively contact the primed target nucleic acid molecules bound to a solid support with the disclosed polymerase and the multivalent binding or incorporation compositions and / or reagents. The contacting may be performed within one or more flow cells. In some cases, these flow cells may be fixed components of the system. In some cases, the flow cells may be removable and / or disposable components of the system.

[0056] The sequencing system may 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 bound to a solid support or the interior of a flow cell. The disclosed compositions, reagents, and methods may be used for 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.

[0057] The sequencing system may also include computer control systems programmed to implement methods of the disclosure. The computer system is programmed or otherwise configured to implement methods of the disclosure, including nucleic acid sequencing methods, interpretation of nucleic acid sequencing data, analysis of cellular nucleic acids, such as RNA (e.g., mRNA), and characterization of cells from sequencing data. The computer system may be a user's electronic device or a computer system remotely located with respect to the electronic device. The electronic device may be a mobile electronic device.

[0058] Fig. Figure 2 is a flow diagram outlining the steps in sequencing a target nucleic acid. 201 describes a step of attaching target library sequences to a solid support surface by hybridizing the target nucleic acid molecules to complementary adapters on the substrate surface. The target nucleic acid molecules may 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 the step of clonal amplification to generate clusters of target nucleic acid molecules on the surface. 203 describes the hybridization of sequencing primers to complementary primer binding or incorporation sequences on the target nucleic acid to form the primed target nucleic acid.204 describes combining the polymerase, the multivalent binding or incorporation composition containing labeled (e.g., fluorescently labeled) particle-nucleotide conjugates, and the primed target nucleic acid. 204 may also include a step of washing or removing the unbound reagents, including the polymerase and particle-nucleotide conjugate.

[0059] Again referring to Fig. 2, if the nucleotide on the particle-nucleotide conjugate is complementary to the nearest base of the primed target nucleic acid (205), the particle-nucleotide conjugate, the polymerase, and the primed target nucleic acid form a ternary binding or incorporation complex that can be detected by detection methods (e.g., fluorescence imaging) compatible with labeling on the particle-nucleotide conjugate. 205 may also include measuring the persistence time of the ternary binding or incorporation complex. In 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 placing the binding or incorporation complex in a state (e.g., addition of strontium ions) that changes the conformation of the polymerase and destabilizes the 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 sequences of the target nucleic acid. Fig. Figure 3 is another flow diagram outlining the steps in sequencing a target nucleic acid, which includes cleaving a nucleotide from the particle-nucleotide conjugate and incorporating the cleaved nucleotide. 301 describes a step of attaching target library sequences to a solid support surface by hybridizing the target nucleic acid molecules to complementary adapters on the substrate surface. The target nucleic acid molecules may 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 the step of clonal amplification to generate clusters of target nucleic acid molecules on the surface.303 describes the hybridization of sequencing primers to complementary primer binding or incorporation sequences on the target nucleic acid to form the primed target nucleic acid. 304 describes the combination of the polymerase, the multivalent binding or incorporation composition containing labeled (e.g., fluorescently labeled) particle-nucleotide conjugates, and the primed target nucleic acid. In the particle-nucleotide conjugates, the nucleotides are bound to the particle via chemical bonds or later separable interactions. 404 may also include a step of washing or removing the unbound reagents, including the polymerase and particle-nucleotide conjugate.

[0060] Again referring to Fig. 3, if the nucleotide on the particle-nucleotide conjugate is complementary to the nearest base of the primed target nucleic acid (305), the particle-nucleotide conjugate, the polymerase, and the primed target nucleic acid form a ternary binding or incorporation complex that can be detected by detection methods (e.g., fluorescence imaging) compatible with the labeling on the particle-nucleotide conjugate. 305 may also include measuring the persistence time of the ternary binding or incorporation complex. In 306, the polymerase is brought into a state that makes it catalytically active to incorporate a nucleotide. The state may include exposing the polymerase to Mg or Mn ions in the reaction solution. The nucleotide bound to the polymerase and the primed target nucleic acid is then cleaved from the particle and then incorporated into the primed strand of the primed target nucleic acid.The binding or incorporation complex is destabilized. 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 may be repeated in several cycles to determine the sequences of the target nucleic acid. B. Detection of target nucleic acid molecules

[0061] Fig. 4A-4B illustrate an exemplary method in which the multivalent binding or incorporation composition is used to detect a target nucleic acid. As in Fig. As shown in FIG. 4A, the polymer-nucleotide conjugate 401 is contacted with 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 core, some branches bound to nucleotide or oligonucleotide 402, and some branches bound to a label 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. B, and the strong binding or incorporation signal can help detect target nucleic acids with sequences that are 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 molecules, and the polymer-nucleotide conjugates is bound to a solid support.

[0062] The multivalent binding or incorporation composition described herein can be used in a method for 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 delivery system configured to sequentially and iteratively contact the nucleic acid molecules with the disclosed polymerase and the multivalent binding or incorporation compositions and / or reagents. The contacting can be performed within one or more flow cells. In some cases, these flow cells can be fixed components of the system. In some cases, the flow cells can be removable and / or disposable components of the system.The system may also include a cartridge comprising a sample collection unit and an assay assembly, wherein the sample collection unit is configured to collect a sample, and wherein the assay assembly comprises at least one reaction site containing a multivalent binding or incorporation composition adapted to interact with the analyte, thereby enabling the predetermined portion of the sample to react with assay reagents contained in the assay assembly to yield a signal indicative of the presence of the analyte in the sample, and detecting the signal generated by the analyte. III. Multivalent binding or incorporation composition

[0063] 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 a polymerase and multiple copies of a primed target nucleic acid can result in the formation of a ternary complex that can be detected, in turn allowing for more accurate determination of the bases of the target nucleic acid.

[0064] When the multivalent binding or incorporation composition is used as a replacement for a single unconjugated or unbound nucleotide to form a complex with the polymerase and one or more copies of the target nucleic acid, the local concentration of the nucleotide, as well as the binding avidity of the complex (in the case of a complex formed from two or more target nucleic acid molecules), is increased severalfold, which in turn increases the signal intensity, particularly the correct signal compared to the mismatch. The multivalent binding or incorporation composition described herein may include at least one particle-nucleotide conjugate (each particle-nucleotide conjugate comprising multiple copies of a single nucleotide residue) for interaction with the 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.

[0065] The multivalent binding or incorporation composition may comprise 1, 2, 3, 4, or more types of particle-nucleotide conjugates, each particle-nucleotide conjugate comprising a different type of nucleotide. A first type of particle-nucleotide conjugate may comprise a nucleotide selected from the group consisting of ATP, ADP, AMP, dATP, dADP, and dAMP. A second type of particle-nucleotide conjugate may comprise a nucleotide selected from the group consisting of TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, and dUMP. A third type of particle-nucleotide conjugate may comprise a nucleotide selected from the group consisting of CTP, CDP, CMP, dCTP, dCDP, and dCMP. A fourth type of particle-nucleotide conjugate may comprise a nucleotide selected from the group consisting of GTP, GDP, GMP, dGTP, dGDP, and dGMP.In some embodiments, each particle-nucleotide conjugate comprises a single type of nucleotide, each corresponding to one or more nucleotides selected from the group consisting of 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. Each multivalent binding or incorporation composition may further comprise one or more labels corresponding to the particular nucleotide conjugated to each respective conjugate. Non-limiting examples of labels include fluorescent labels, colorimetric labels, electrochemical labels (such as glucose or other reducing sugars or thiols or other redox-active moieties), luminescent labels, chemiluminescent labels, spin labels, radioactive labels, steric labels, affinity tags, or the like. A. Particle-nucleotide conjugate

[0066] In a particle-nucleotide conjugate, multiple copies of the same nucleotide can be covalently or non-covalently bound to the particle. Examples of the particle can include: a branched polymer; a dendrimer; a cross-linked polymer particle, such as an agarose, polyacrylamide, acrylate, methacrylate, cyanoacrylate, or methyl methacrylate particle; a glass particle; a ceramic particle; a metal particle; a quantum dot; a liposome; an emulsion particle; or any other particle (e.g., nanoparticles, microparticles, or the like) known in the art. In a preferred embodiment, the particle is a branched polymer.

[0067] In some cases, the particle-nucleotide conjugate (e.g., a polymer-nucleotide conjugate) may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 copies of a nucleotide, a nucleotide analog, a nucleoside, or a nucleoside analog attached to the particle.

[0068] The nucleotide may be connected to the particle via a linker, and the nucleotide may be attached to an end or site of a polymer. The nucleotide may be conjugated to the particle via the 5' end of the nucleotide. In some particle-nucleotide conjugates, one nucleotide is attached to an end or site of a polymer. In some particle-nucleotide conjugates, multiple nucleotides are attached to an end or site of a polymer. The conjugated nucleotide is sterically accessible to one or more proteins, one or more enzymes, and nucleotide binding moieties or nucleotide incorporation units. In some embodiments, a nucleotide may be provided separately from a nucleotide binding moiety or nucleotide incorporation unit, such as a polymerase. In some embodiments, the linker does not comprise a photoemissive or photoabsorbing group.

[0069] The particle may also comprise a binding or incorporation moiety. In some embodiments, particles may self-associate without the use of a separate interaction component. In some embodiments, particles may self-associate due to buffer or salt conditions, e.g., as in the case of calcium-mediated interactions of hydroxyapatite particles, lipid- or polymer-mediated interactions of micelles or liposomes, or salt-mediated aggregation of metallic (such as iron or gold) nanoparticles.

[0070] The particle-nucleotide conjugate may comprise 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 capable of making the composition detectable by methods known in the art of detecting macromolecules or molecular interactions. The label may be attached to the nucleotide (e.g., by binding to the 5'-phosphate moiety of a nucleotide), to the particle itself (e.g.,to the PEG subunits), to an end of the polymer, to a central moiety, or to any other location within the polymer-nucleotide conjugate that would be recognized by one of ordinary skill in the art as sufficient to make the composition, such as a particle, detectable by methods known in the art or described elsewhere herein. In some embodiments, one or more labels are provided to correspond to or distinguish a particular particle-nucleotide conjugate.

[0071] In some embodiments, the label is a fluorophore. Non-limiting examples of fluorescent moieties include, but are not limited to, the following: fluorescein and fluorescein derivatives such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-amino-fluorescein, rhodamine and rhodamine derivatives such as TRITC, TMR, lissamine rhodamine, Texas Red, Rhodamine B, Rhodamine 6G, Rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, Lissamine-rhodamine-B-sulfonylhydrazine, Texas red sulfonyl chloride, Texas red hydrazide, coumarin 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 / 550 C3, BODIPY 530 / 550 C3-SE, BODIPY 530 / 550 C3 hydrazide,BODIPY 493 / 503 C3 Hydrazide, BODIPY FL C3 Hydrazide, BODIPY FL IA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503, Cascade Blue and derivatives such as Cascade Blue Acetylazide, Cascade Blue Cadaverine, Cascade Blue Ethylenediamine, Cascade Blue Hydrazide, Lucifer Yellow and derivatives such as Lucifer Yellow Iodoacetamide, Lucifer Yellow CH, Cyanine and derivatives such as indolium-based cyanine dyes, benzoindolium-based cyanine dyes, pyridium-based cyanine dyes, thiozolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy3, Cy5, lanthanide chelates and derivatives such as BCPDA, TBP, TMT, BHHCT, BCOT, europium chelates, Terbium chelates, Alexa fluoro dyes, DyLight dyes, Atto dyes, LightCycler red dyes, CAL flour dyes, JOE and derivatives thereof, 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, such as those described in Haugland, Molecular Probes Handbook (Eugene, Oregon), 6th ed.; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed., Plenum Press, New York (1999); or Hermanson, Bioconjugate Techniques, 2nd ed., or derivatives thereof, or any combination thereof. Cyanine dyes can be in sulfonated or nonsulfonated form and consist of two indolenine, benzoindolium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethine bridge between two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3, (which is 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 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)penta-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)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium 5-sulfonate), and Cy7 (which may comprise 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-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)hepta-1,3,5-trien-1-yl]-3H-indolium 5-sulfonate), where “Cy” stands for ,Cyanine' and the first digit indicates the number of carbon atoms between two indolenine groups. Cy2, which is not indolenine but an oxazole derivative, and the benzo-derivatized Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule.

[0072] In some embodiments, the detection label may be a FRET pair, allowing multiple classifications to be performed in a single excitation and imaging step. As used herein, FRET may involve excitation exchange (Forster) or electron exchange (Dexter) transfers. B. Polymer-nucleotide conjugate

[0073] An example of a particle-nucleotide conjugate is a polymer-nucleotide conjugate. Some non-limiting examples of polymer-nucleotide conjugates are described in Fig. 5A-5C. For example, Fig. 5A Polymer-nucleotide conjugates with various configurations, e.g., a “starburst” configuration, comprising a fluorescently labeled streptavidin core and four nucleotides attached to the core via biotinylated, linear PEG linkers with a molecular weight ranging from 1 K Dalton to 10 K Dalton; Fig. Figure 5B shows a polymer-nucleotide conjugate with a dendrimer core of, for example, 12, 24, 48, or 96 arms and linear PEG linkers with a molecular weight in the range of 1 K Dalton to 10 K Dalton emanating from the center; and Fig. Figure 5C shows an example of polymer-nucleotide conjugates comprising a network of, e.g., streptavidin cores connected by branched PEG linkers comprising a binding or incorporation moiety, such as a biotin.

[0074] 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, a dextran, or other such polymers or copolymers containing any two or more of the foregoing or containing other polymers as are known in the art. In one embodiment, the polymer is a PEG. In another embodiment, the polymer may have PEG branches.

[0075] Suitable polymers may be characterized by a repeating unit that includes a functional group suitable for derivatization, such as an amine, a hydroxyl, a carbonyl, or an allyl group. The polymer may also have one or more pre-derivatized substituents such that one or more particular subunits include a derivatization site or a branching point, regardless of whether or not other subunits include the same site, substituent, or moiety. A pre-derivatized substituent may, for example, comprise a nucleotide, a nucleoside, a nucleotide analogue, a label such as a fluorescent label, a radioactive label, or a spin label, an interaction moiety, an additional polymer unit, or the like, or any combination of the foregoing.

[0076] In the polymer-nucleotide conjugate, the polymer may have a variety of branches. The branched polymer may have various configurations, including, but not limited to, star-shaped shapes ("starburst"), aggregated star-shaped shapes ("helterskelter"), bottlebrush, or dendrimer. The branched polymer may originate from a central attachment point or unit or include multiple branch points, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more branch points. In some embodiments, each subunit of a polymer may optionally form a separate branch point.

[0077] The length and size of the branch may vary depending on the type of polymer. In some branched polymers, the branch may have a length between 1 and 1,000 nm, between 1 and 100 nm, between 1 and 200 nm, between 1 and 300 nm, between 1 and 400 nm, between 1 and 500 nm, between 1 and 600 nm, between 1 and 700 nm, between 1 and 800 nm, or between 1 and 900 nm, or more, or may have a length that falls within or between any of the values ​​disclosed herein.

[0078] In some polymer-nucleotide conjugates, the polymer core may have a size corresponding to an apparent molecular weight of 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 30 kDa, 50 kDa, 80 kDa, 100 kDa, or any value within a range defined by any two of the foregoing. The apparent molecular weight of a polymer may be calculated from the known molecular weight of a representative number of subunits, as determined by size-exclusion chromatography, as determined by mass spectrometry, or as determined by any other method known in the art.

[0079] In some branched polymers, the branching may have a size corresponding to an apparent molecular weight of 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 30 kDa, 50 kDa, 80 kDa, 100 kDa, or any value within a range defined by any two of the foregoing. The apparent molecular weight of a polymer may be calculated from the known molecular weight of a representative number of subunits, as determined by size exclusion chromatography, as determined by mass spectrometry, or as determined by any other method consistent with the art. The polymer may be multiply branched. The number of branches in the polymer can be 2, 3, 4, 5, 6, 7, 8, 12, 16, 24, 32, 64, 128 or more, or a number that falls within a range defined by any two of these values.

[0080] For polymer-nucleotide conjugates comprising a branched polymer of, for example, 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 0, 1, 2, 3, 4, 5, 6, or more nucleotides are attached to each end. In one non-limiting example, a branched PEG polymer having between 3 and 128 PEG arms can have one or more nucleotides attached to it such that 0, 1, 2, 3, 4, 5, 6, or more nucleotides or nucleotide analogs are attached to each end. In some embodiments, a branched polymer or dendrimer has an even number of arms. In some embodiments, a branched polymer or dendrimer has an odd number of arms.

[0081] In some cases, the length of the linker (e.g., a PEG linker) may range from about 1 nm to about 1,000 nm. In some cases, the length of the linker may be at least 1 nm, at least 10 nm, at least 25 nm, at least 50 nm, at least 75 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 400 nm, at least 500 nm, at least 600 nm, at least 700 nm, at least 800 nm, at least 900 nm, or at least 1,000 nm. In some cases, the length of the linker may be between any two of the values ​​in this paragraph. For example, in some cases, the length of the linker may range from about 75 nm to about 400 nm. Those skilled in the art will recognize that in some cases, the length of the linker may be any value within the range of values ​​in this paragraph, e.g., 834 nm.

[0082] In some cases, the length of the linker is different for different nucleotides (including deoxyribonucleotides and ribonucleotides), nucleotide analogues (including deoxyribonucleotide analogues and ribonucleotide analogues), nucleosides (including deoxyribonucleosides or ribonucleosides) or nucleoside analogues (including deoxyribonucleoside analogues or ribonucleoside analogues). In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, deoxyadenosine, and the length of the linker is between 1 nm and 1,000 nm. In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, deoxyguanosine, and the length of the linker is between 1 nm and 1,000 nm. In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, thymidine, and the length of the linker is between 1 nm and 1,000 nm.In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, deoxyuridine, and the length of the linker is between 1 nm and 1,000 nm. In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, deoxycytidine, and the length of the linker is between 1 nm and 1,000 nm. In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, adenosine, and the length of the linker is between 1 nm and 1,000 nm. In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, guanosine, and the length of the linker is between 1 and 1,000 nm. In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, 5-methyluridine, and the length of the linker is between 1 nm and 1,000 nm.In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, uridine, and the length of the linker is between 1 nm and 1,000 nm. In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, cytidine, and the length of the linker is between 1 nm and 1,000 nm.

[0083] In the polymer-nucleotide conjugate, a moiety comprising a nucleotide (e.g., an adenine, a thymine, a uracil, a cytosine, or a guanine residue, or a derivative or mimetic thereof) can be bound to each branch or a subset of branches of the polymer, and the moiety is capable of binding to or being incorporated by a polymerase, reverse transcriptase, or other nucleotide binding or incorporation domain. Optionally, the moiety may be capable of being incorporated into an elongating nucleic acid chain during a polymerase reaction. In some cases, the moiety may be blocked such that it cannot be incorporated into an elongating nucleic acid chain during a polymerase reaction.In some other cases, the moiety may be reversibly blocked so that it is unable to be incorporated into an elongating nucleic acid chain during a polymerase reaction until this block is removed, after which the moiety is then able to be incorporated into an elongating nucleic acid chain during a polymerase reaction.

[0084] The nucleotide may be conjugated to the polymer branch via the 5' end of the nucleotide. In some cases, the nucleotide may be modified to inhibit or prevent incorporation of the nucleotide into an extending nucleic acid chain during a polymerase reaction. For example, the nucleotide may include a 3'-deoxyribonucleotide, a 3'-azidonucleotide, a 3'-methylazidonucleotide, or any other such nucleotide as is or may be known in the art to be incapable of being incorporated into an extending nucleic acid chain during a polymerase reaction. In some embodiments, the nucleotide may include a 3'-O-azido group, a 3'-O-azidomethyl group, a 3'-phosphorothioate group, a 3'-O-malonyl group, a 3'-O-alkylhydroxylamine group, or a 3'-O-benzyl group. In some embodiments, the nucleotide lacks a 3'-hydroxyl group.

[0085] The polymer may further comprise a binding or incorporation moiety in each branch or a subset of branches. Some examples of the binding or incorporation moiety include, but are not limited to, biotin, avidin, streptavidin, or the like, polyhistidine domains, complementary paired nucleic acid domains, G-quartet-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, an antibody, an epitope, a protein A, a protein G.The binding or incorporation moiety may be any interactive molecule or fragment thereof known to bind to or facilitate interactions between proteins, between proteins and ligands, between proteins and nucleic acids, between nucleic acids, or between small molecule interaction domains or moieties.

[0086] In some embodiments, a composition as provided herein may comprise one or more elements of a complementary interaction moiety. Non-limiting examples of complementary interaction moieties include, for example, the following: biotin and avidin; SNAP-benzylguanosine; antibody or FAB and epitope; IgG FC and Protein A, Protein G, Protein A / G, or Protein L; maltose binding protein and maltose; lectin and related polysaccharide; ion chelating moieties, complementary nucleic acids, nucleic acids capable of forming triple or triple helix interactions; nucleic acids capable of forming G-quartets, and the like.One of ordinary skill in the art will readily recognize that many pairs of moieties exist and are commonly used for their ability to interact strongly and specifically with one another; therefore, any such complementary pair or set is considered suitable for constructing or designing the compositions of the present disclosure. In some embodiments, a composition as disclosed herein may include compositions in which one member of a complementary interaction moiety is bound to one molecule or multivalent ligand, and the other member of the complementary interaction moiety is bound to a separate molecule or multivalent ligand.In some embodiments, a composition as disclosed herein may comprise compositions in which both or all elements of a complementary interaction moiety are bound to a single molecule or multivalent ligand. In some embodiments, a composition as disclosed herein may comprise compositions in which both or all elements of a complementary interaction moiety are bound to separate arms or sites on a single molecule or multivalent ligand. In some embodiments, a composition as disclosed herein may comprise compositions in which both or all elements of a complementary interaction moiety are bound to the same arm or sites on a single molecule or multivalent ligand.In some embodiments, compositions comprising one member of a complementary interaction moiety and compositions comprising another member of a complementary interaction moiety can be mixed simultaneously or sequentially. In some embodiments, interactions between molecules or particles, as disclosed herein, enable the association or aggregation of multiple molecules or particles such that, for example, detectable signals are enhanced. In some embodiments, fluorescent, colorimetric, or radioactive signals are enhanced. In other embodiments, other interaction moieties, as disclosed herein or as 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 interaction moiety, such as one or more imidazole or pyridine moieties, and one or more additional molecules comprising a second interaction moiety, such as histidine residues, 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 can be facilitated by the presence of a divalent cation, such as nickel, manganese, magnesium, calcium, strontium, or the like.For example, in some embodiments, a (His)3 group may interact with a (His)3 group on another molecule or particle via coordination of a nickel or manganese ion.

[0087] 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, methylcellulose, heparin, heparan sulfate, glycerin, sucrose, 1,2-propanediol, DMSO, N,N,N-trimethylglycine, ethanol, ethoxyethanol, propylene glycol, polypropylene glycol, block copolymers such as the Pluronic® series polymers, arginine, histidine, imidazole, or any combination thereof, or any substance known in the art as a DNA “relaxer” (a compound having the effect of altering the persistence length of DNA, altering the number of inter-polymer linkages or junctions, or altering the conformational dynamics of a DNA molecule, thereby increasing the accessibility of sites within the strand to DNA binding or insertion units).

[0088] The multivalent binding or incorporation composition may include zwitterionic compounds as additives. Further representative additives can be found in Lorenz, TCJ Vis. Exp. (63), e3998, doi: 10.3791 / 3998 (2012), which is hereby incorporated by reference for its disclosure of additives for facilitating nucleic acid binding or dynamics or for facilitating 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 such as self-association, secondary or tertiary structure formation, base pairing, surface association, peptide association, protein binding, or the like. IV. Binding between target nucleic acid and the multivalent binding or incorporation composition

[0089] When the multivalent binding or incorporation composition is used as a replacement for a single unconjugated or unbound nucleotide to form a complex with the polymerase and one or more copies of the target nucleic acid, the local concentration of the nucleotide, as well as the binding avidity of the complex (in the case of a complex formed by two or more target nucleic acid molecules), is increased many-fold, which in turn increases the signal intensity, particularly the correct signal compared to the mismatch. The present disclosure contemplates contacting the multivalent binding or incorporation composition with a polymerase and a primed target nucleic acid to determine the formation of a ternary binding or incorporation complex.

[0090] Fig. Figure 6 illustrates the use of the disclosed polymer-nucleotide conjugates to achieve increased signal intensity during binding, persistence, and washing / removal steps. Due to the increased local concentration of the nucleotide on the polymer-nucleotide conjugate and / or the formation of noncovalent bonds with two or more primed target nucleic acid molecules, binding between the polymerase, the primed target strand, and the polymer-conjugated nucleotide is favored when the nucleotide is complementary to the nearest base of the target nucleic acid. The formed binding complex has a longer persistence time, 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 bond between the polymerase, the primed target strand, and the polymer-conjugated nucleotide or nucleotide analogue also ensures that the resulting binding complex remains stable during the washing steps, while other reaction mixture components and inappropriate 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.

[0091] As an example, a graphical representation of the increase in signal intensity during binding, persistence and washing / removal of a multivalent substrate as described herein is shown in Fig. 6, which is representative of the changes in signal intensity observed experimentally. Therefore, the compositions and methods of the present disclosure provide a robust and controllable means for producing and maintaining a ternary enzyme complex, as well as a greatly improved means by which the presence of the complex can be identified and / or measured, and a means by which the persistence of the complex can be controlled. This provides important solutions to problems such as determining the identity of the N+1 base in nucleic acid sequencing applications.

[0092] Without wishing to be bound by any particular theory, it has been observed that multivalent binding compositions disclosed herein associate with polymerase-nucleotide complexes to form ternary binding complexes at a rate that is time-dependent, although substantially slower than the association rate known to be achievable by nucleotides in free solution. Thus, the on-rate (K on ) is significantly and surprisingly slower than the on-rate for single nucleotides or nucleotides not bound to multivalent ligand complexes. Importantly, however, the off-rate (K off) of the multivalent ligand complex is significantly slower than that observed for nucleotides in free solution. Therefore, the multivalent ligand complexes of the present disclosure offer a surprising and advantageous improvement in the persistence of ternary polymerase-polynucleotide-nucleotide complexes (particularly over those complexes formed with free nucleotides), enabling, for example, significant improvements in imaging quality for nucleic acid sequencing applications over currently available methods and reagents.Importantly, this property of the multivalent binding compositions disclosed herein makes the formation of visible ternary complexes controllable, so that subsequent visualization, modification, or processing steps can be performed substantially without regard to the dissociation of the complex; the complex can be formed, imaged, modified, or otherwise used as needed, and remains stable until a user performs a confirmatory dissociation step, such as exposing the complexes to a dissociation buffer.

[0093] In some cases, the persistence times for the multivalent binding complexes formed using the disclosed particle-nucleotide or polymer-nucleotide conjugates may range from about 0.1 seconds to about 600 seconds under non-destabilizing conditions. In some cases, the persistence time 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 persistence time may be between any two of the values ​​specified in this paragraph. For example, in some cases, the persistence time may range from about 10 seconds to about 360 seconds. Those skilled in the art will recognize that in some cases, the persistence time may be any value within the range specified in this paragraph, for example, 78 seconds.

[0094] In various embodiments, polymerases suitable for the binding or incorporation interaction described herein may include any polymerase as is or may be known in the art. For example, it is known that every organism encodes one or more DNA polymerases within its genome. Examples of suitable polymerases may include, but are not limited to: Klenow DNA polymerase, Thermus aquaticus DNA polymerase I (Taq polymerase), KlenTaq polymerase, and bacteriophage T7 DNA polymerase; human alpha, delta, and epsilon DNA polymerases; bacteriophage polymerases such as T4, RB69, and phi29 bacteriophage DNA polymerases; Pyrococcus furiosus DNA polymerase (Pfu polymerase); Bacillus subtilis DNA polymerase III, and E.coli DNA polymerase III alpha and epsilon; 9 degree N polymerase; reverse transcriptases, such as HIV type M or O reverse transcriptases, avian myeloblastosis virus reverse transcriptase, or Moloney Murine Leukemia Virus (MMLV) reverse transcriptase; or telomerase. Other non-limiting examples of DNA polymerases may include those from various Archaea genera, such as Aeropyrum, Archaeglobus, Desulfurococcus, Pyrobaculum, Pyrococcus, Pyrolobus, Pyrodictium, Staphylothermus, Stetteria, Sulfolobus, Thermococcus, and Vulcanisaeta, and the like, or variants thereof, including such polymerases as are known in the art, such as Vent™, Deep Vent™, Pfu, KOD, Pfx, Therminator™, and Tgo polymerases. In some embodiments, the polymerase is a Klenow polymerase.

[0095] The ternary complex exhibits a longer persistence time when the nucleotide on the polymer-nucleotide conjugate is complementary to the target nucleic acid than when it is a non-complementary nucleotide. The ternary complex also exhibits a longer persistence time when the nucleotide on the polymer-nucleotide conjugate is complementary to the target nucleic acid than when it is a complementary nucleotide that is not conjugated or bound. For example, in some embodiments, the ternary complexes may have a persistence time of less than 1 s, more than 1 s, more than 2 s, more than 3 s, more than 5 s, more than 10 s, more than 15 s, more than 20 s, more than 30 s, more than 60 s, more than 120 s, more than 360 s, more than 3600 s, or more, or a time that is within a range defined by any two or more of these values.

[0096] The persistence time can be measured, for example, by observing the onset and / or duration of a binding complex, such as by observing a signal from a labeled component of the binding complex. For example, a labeled nucleotide or a labeled reagent comprising one or more nucleotides can be present in a binding complex, allowing the signal from the label to be detected during the persistence time of the binding complex.

[0097] It has been observed that different ranges of persistence times can be achieved with different salts or ions. For example, it has been shown that in the presence of magnesium ions (Mg 2+ ) form complexes faster than complexes formed with other ions. It has also been observed that complexes formed in the presence of, for example, strontium ions (Sr 2+) are formed, readily form, and dissociate completely or with substantial completeness after the ion is removed or after washing with buffer lacking one or more components of the present compositions, such as a polymer and / or one or more nucleotides and / or one or more interaction moieties, or a buffer containing, for example, a chelating agent capable of effecting or accelerating the removal of a divalent cation from the complex containing a multivalent reagent. Thus, in some embodiments, a composition of the present disclosure comprises Mg 2+ . In some embodiments, a composition of the present disclosure comprises Ca 2+ . In some embodiments, a composition of the present disclosure comprises Sr 2+ . In some embodiments, a composition of the present disclosure comprises cobalt ions (Co 2+). In some embodiments, a composition of the present disclosure comprises MgCl2. In some embodiments, a composition of the present disclosure comprises CaCl2. In some embodiments, a composition of the present disclosure comprises SrCl2. In some embodiments, a composition of the present disclosure comprises CoCl2. In some embodiments, the composition comprises no or substantially no magnesium. In some embodiments, the composition comprises no or substantially no calcium. In some embodiments, the methods of the present disclosure provide for contacting one or more nucleic acids with one or more of the compositions disclosed herein, wherein the composition lacks either calcium or magnesium or lacks both calcium and magnesium.

[0098] The dissociation of ternary complexes can be controlled by altering the buffer conditions. Following the imaging step, a buffer with increased salt content is used to cause dissociation of the ternary complexes so that labeled polymer-nucleotide conjugates can be washed out, providing a means by which signals can be attenuated or terminated, such as during the transition between one sequencing cycle and the next. This dissociation can, in some embodiments, be influenced by washing the complexes with a buffer lacking a necessary metal or cofactor. In some embodiments, a wash buffer can comprise one or more compositions for the purpose of maintaining pH control. In some embodiments, a wash buffer can comprise one or more monovalent cations, such as sodium.In some embodiments, a wash buffer substantially lacks or entirely lacks a divalent cation, for example, having no or substantially no strontium, calcium, magnesium, or manganese. In some embodiments, a wash buffer further comprises a chelating agent, such as EDTA, EGTA, nitrilotriacetic acid, polyhistidine, imidazole, or the like. In some embodiments, a wash buffer can maintain the ambient pH at the same level as for the bound complex. In some embodiments, a wash buffer can increase or decrease the ambient pH relative to the level seen for the bound complex. In some embodiments, the pH can be within a range of 2-4, 2-7, 5-8, 7-9, 7-10, or lower than 2 or higher than 10, or a range defined by any two of the values ​​provided herein.

[0099] The addition of a particular ion may affect the binding of the polymerase to a 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 an extending nucleic acid, such as during a polymerase reaction. In some embodiments, relevant anions may include chloride, acetate, gluconate, sulfate, phosphate, or the like. In some embodiments, an ion may be incorporated into the compositions of the present disclosure through the addition of one or more acids, bases, or salts, such as NiCl2, CoCl2, MgCl2, MnCl2, SrCl2, CaCl2, CaSO4, SrCO3, BaCl2, or the like. Representative salts, ions, solutions, and conditions can be found in Remington: The Science and Practice of Pharmacy, 20th ed., Gennaro, AR: Ed.(2000), which is hereby incorporated by reference in its entirety and particularly with respect to Chapter 17 and related disclosure of salts, ions, salt solutions and ionic solutions.

[0100] The present disclosure contemplates contacting the multivalent binding or incorporation composition comprising at least one particle-nucleotide conjugate with one or more polymerases. The contacting may optionally occur in the presence of one or more target nucleic acids. In some embodiments, the target nucleic acids are single-stranded nucleic acids. In some embodiments, the target nucleic acids are primed single-stranded nucleic acids. In some embodiments, the target nucleic acids are double-stranded nucleic acids. 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 multiple polymerases. The polymerase may be bound to a single nucleic acid molecule.

[0101] The binding between target nucleic acid and multivalent binding composition can be provided in the presence of a polymerase that has been rendered catalytically inactive. In one embodiment, the polymerase can be rendered catalytically inactive by mutation. In one embodiment, the polymerase can be rendered catalytically inactive by chemical modification. In some embodiments, the polymerase can be rendered catalytically inactive by the absence of a necessary substrate, ion, or cofactor. In some embodiments, the polymerase enzyme can be rendered catalytically inactive by the absence of magnesium ions.

[0102] The 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 lacks magnesium or manganese. Alternatively, the 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.

[0103] When the catalytically inactive polymerases are used to promote interaction of a nucleic acid with a multivalent binding composition, the interaction between the composition and the polymerase stabilizes a ternary complex to make the complex detectable by fluorescence or other methods as disclosed herein or otherwise known in the art. Unbound polymer-nucleotide conjugates can optionally be washed away prior to detection of the ternary binding complex.

[0104] Contacting one or more nucleic acids with the polymer-nucleotide conjugates disclosed herein in a solution containing either calcium or magnesium or both calcium and magnesium. Alternatively, contacting one or more nucleic acids with the polymer-nucleotide conjugates disclosed herein is performed in a solution lacking either calcium or magnesium or both calcium and magnesium, and in a separate step, regardless of the order of the steps, adding calcium or magnesium or both calcium and magnesium to the solution. In some embodiments, contacting one or more nucleic acids with the polymer-nucleotide conjugates disclosed herein is performed in a solution lacking strontium and comprises adding strontium to the solution in a separate step, regardless of the order of the steps. V. Use of multivalent binding or incorporation compositions in combination with a surface with weak nonspecific binding

[0105] Disclosed herein are solid supports comprising surface compositions with weak nonspecific binding that enable improved performance in nucleic acid hybridization and amplification. In general, the disclosed supports may comprise a substrate (or support structure), one or more layers of covalently or non-covalently bound weakly binding chemical modification layers, e.g., silane layers, polymer films, and one or more covalently or non-covalently bound primer sequences that can be used to attach single-stranded target nucleic acid(s) to the support surface. In some cases, the formulation of the surface, e.g.,the chemical composition of one or more layers, the coupling chemistry used to crosslink the one or more layers to the support surface and / or to each other, and the total number of layers can be varied so that nonspecific binding of proteins, nucleic acid molecules, and other components of hybridization and amplification reactions to the support surface is minimized or reduced compared to a comparable monolayer. Often, the formulation of the surface can be varied so that nonspecific hybridization on the support surface is minimized or reduced compared to a comparable monolayer. The formulation of the surface can be varied so that nonspecific amplification on the support surface is minimized or reduced compared to a comparable monolayer.The surface formulation can be varied to maximize specific amplification rates and / or yields on the support surface. Amplification levels suitable for detection are achieved in some cases disclosed herein in no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30 amplification cycles.

[0106] Examples of materials from which the substrate or support structure can be made include, but are not limited to, glass, fused silica, silicon, a polymer (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are contemplated.

[0107] The substrate or support structure may be provided in a variety of geometries and dimensions known to those of skill in the art and may comprise any of a variety of materials known to those of skill in the art. For example, in some cases, the substrate or support structure may be locally planar (e.g., comprising a slide or the surface of a slide). Overall, the substrate or support structure may be cylindrical (e.g., comprising a capillary or the inner surface of a capillary), spherical (e.g., comprising the outer surface of a non-porous bead), or irregular (e.g., comprising the outer surface of an irregularly shaped, non-porous bead or particle). In some cases, the surface of the substrate or support structure used 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 may be porous, so that the coatings described herein can penetrate the porous surface and nucleic acid hybridization and amplification reactions performed thereon can take place within the pores.

[0108] The substrate or support structure comprising one or more chemically modified layers, e.g., layers of a polymer with weak nonspecific binding, may be independent or integrated into another structure or assembly. For example, in some cases, the substrate or support structure may comprise one or more surfaces within an integrated or assembled microfluidic flow cell. The substrate or support structure may comprise one or more surfaces within a microplate format, e.g., the bottom surface of the wells in a microplate. As mentioned above, in some preferred embodiments, the substrate or support structure comprises the interior surface (such as the lumen surface) of a capillary.In alternative preferred embodiments, the substrate or support structure comprises the inner surface (such as the lumen surface) of a capillary etched into a planar chip.

[0109] As mentioned, the weak nonspecific binding supports of the present disclosure exhibit reduced nonspecific binding of proteins, nucleic acids, and other components of the hybridization and / or amplification formulation used for solid-phase nucleic acid amplification. The degree of nonspecific binding exhibited by a given support surface can be assessed either qualitatively or quantitatively. For example, in some cases, exposure of the surface to fluorescent dyes (e.g., cyanines such as Cy3 or Cy5, etc., fluoresceins, coumarins, rhodamines, etc., or other dyes disclosed herein), fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, and / or fluorescently labeled proteins (e.g.,Polymerases) under a standardized set of conditions, followed by a specified rinsing protocol, and fluorescence imaging can be used as a qualitative tool for comparing nonspecific binding on supports containing different surface formulations. In some cases, exposure of the surface to fluorescent dyes, fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, and / or fluorescently labeled proteins (e.g.,Polymerases) under a standardized set of conditions, followed by a specified rinsing protocol and fluorescence imaging can be used as a quantitative tool to compare non-specific binding on supports comprising different surface formulations - provided that care has been taken to ensure that fluorescence imaging is performed under conditions where the fluorescence signal is linearly (or predictably) related to the number of fluorophores on the support surface (e.g., under conditions where signal saturation and / or self-attenuation of the fluorophore is not a factor) and that appropriate calibration standards are used.In some cases, other techniques known to those skilled in the art, for example, radioisotope labeling and counting methods, may be used to quantitatively assess the degree to which the various support surface formulations of the present disclosure exhibit nonspecific binding.

[0110] Some surfaces disclosed herein have a ratio of specific to nonspecific 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 more than 100, or any intermediate value spanned by the range described herein. Some surfaces disclosed herein have 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 more than 100, or any intermediate value spanned by the range described herein.

[0111] As mentioned, in some cases, the degree of nonspecific binding exhibited by the disclosed low-binding supports can be assessed using a standardized protocol for contacting the surface with a labeled protein (e.g., bovine serum albumin (BSA), streptavidin, a DNA polymerase, a reverse transcriptase, a helicase, a single-stranded binding protein (SSB), etc., or any combination thereof), a labeled nucleotide, a labeled oligonucleotide, etc., under a standardized set of incubation and rinsing conditions, followed by detecting the amount of label remaining on the surface and comparing the resulting signal with 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 nonspecific binding exhibited by a given support surface formulation may thus be assessed in terms of the number of nonspecifically bound protein molecules (or other molecules) per unit area. In some cases, the weakly binding supports of the present disclosure may exhibit nonspecific protein binding (or nonspecific binding of other specified molecules (e.g., cyanine dyes such as Cy3 or Cy5, etc., fluoresceins, coumarins, rhodamines, etc., or other dyes disclosed herein) of less than 0.001 molecule per µm. 2 , less than 0.01 molecules per µm 2 , less than 0.1 molecules per µm 2 , less than 0.25 molecules per µm 2 , less than 0.5 molecules per µm 2 , less than 1 molecule per µm 2 , less than 10 molecules per µm 2, less than 100 molecules per µm 2 or less than 1000 molecules per µm 2 , Those skilled in the art will recognize that a given support surface of the present disclosure may exhibit nonspecific binding falling anywhere within this range, for example, less than 86 molecules per µm 2 For example, some modified surfaces disclosed herein exhibit nonspecific protein binding of less than 0.5 molecules / µm 2 after contact with a 1 µM solution of Cy3-labeled streptavidin (GE Amersham) in PBS buffer (phosphate-buffered saline) for 15 minutes, followed by three rinses with deionized water. Some modified surfaces disclosed herein exhibit nonspecific binding of Cy3 dye molecules of less than 0.25 molecules per µm 2In independent nonspecific binding assays, 1 µM labeled Cy3 SA (ThermoFisher), 1 µM Cy5 SA dye (ThermoFisher), 10 µM aminoallyl-dUTP - ATTO-647N (Jena Biosciences), 10 µM aminoallyl-dUTP - ATTO-Rho11 (Jena Biosciences), 10 µM aminoallyl-dUTP - ATTO-Rho11 (Jena Biosciences), 10 µM 7-propargylamino-7-deaza-dGTP - Cy5 (Jena Biosciences), and 10 µM 7-propargylamino-7-deaza-dGTP - Cy3 (Jena Biosciences) were incubated on the weakly binding substrates at 37 °C for 15 minutes in a 384-well plate format. Each well was rinsed 2-3 times with 50 μl of deionized RNase / DNase-free water and 2-3 times with 25 mM ACES buffer at pH 7.4. The 384-well plates were imaged on a GE Typhoon instrument using the Cy3, AF555, or Cy5 filter sets (depending on the dye assay performed) according to the manufacturer's instructions at a PMT gain setting of 800 and a resolution of 50-100 μm.For higher resolution imaging, images were acquired on an Olympus IX83 microscope (Olympus Corp., Center Valley, PA) with a total internal reflectance fluorescence (TIRF) objective (100X, 1.5 NA, Olympus), a CCD camera (e.g., an Olympus EM-CCD monochrome camera, Olympus XM-10 monochrome camera, or an Olympus DP80 color and monochrome camera), an illumination source (e.g., an Olympus 100 W Hg lamp, an Olympus 75 W Xe lamp, or an Olympus U-HGLGPS fluorescent light source), and excitation wavelengths of 532 nm or 635 nm. Dichroic mirrors were purchased from Semrock (IDEX Health & Science, LLC, Rochester, New York), e.g., from Olympus. B. 405, 488, 532 or 633 nm dichroic reflectors / beamsplitters, and bandpass filters were selected as 532 LP or 645 LP according to the corresponding excitation wavelength.Some modified surfaces disclosed herein exhibit nonspecific binding of dye molecules of less than 0.25 molecules per µm. 2 .

[0112] In some cases, the surfaces disclosed herein have a ratio of specific to nonspecific 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 spanned by the range described herein. In some cases, the surfaces disclosed herein have a ratio of specific to non-specific fluorescence signals for 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 spanned by the range described herein.

[0113] The low-background surfaces consistent with the disclosure herein can have specific ratios of dye binding (e.g., Cy3 binding) to nonspecific dye adsorption (e.g., Cy3 dye adsorption) of at least 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 more than 50 specific dye molecules nonspecifically adsorbed per molecule. Similarly, low-background surfaces consistent with the disclosure herein to which fluorophores, e.g., are attached can have specific ratios of dye binding (e.g., Cy3 binding) to nonspecific dye adsorption (e.g., Cy3 dye adsorption) of at least 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 more than 50 specific dye molecules nonspecifically adsorbed per molecule. B. Cy3, when exposed to excitation energy, have ratios of a specific fluorescence signal (e.g. resulting from Cy3-labelled oligonucleotides bound to the surface) to non-specific adsorbed dye fluorescence signals of at least 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 more than 50:1.

[0114] In some cases, the degree of hydrophilicity (or "wettability" with aqueous solutions) of the disclosed support surfaces can be assessed, for example, by measuring water contact angles, where a small droplet of water is placed on the surface and its contact angle with the surface is measured using, for example, an optical tensiometer. In some cases, a static contact angle can be determined. In some cases, an advancing or receding contact angle can be determined. In some cases, the water contact angle for the hydrophilic, weakly binding support surface disclosed herein can range from about 0 degrees to about 30 degrees. In some cases, the water contact angle for the hydrophilic, weakly binding support surface disclosed herein can be no more than 50 degrees, 40 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 is no more than 40 degrees. Those skilled in the art will recognize that a given hydrophilic, weakly binding support surface of the present disclosure may have a water contact angle value anywhere within this range.

[0115] In some cases, the hydrophilic surfaces disclosed herein facilitate reduced wash times for bioassays, often due to reduced nonspecific binding of biomolecules to the weakly binding surfaces. In some cases, adequate wash steps can be performed in less than 60, 50, 40, 30, 20, 15, 10, or less than 10 seconds. For example, in some cases, adequate wash steps can be performed in less than 30 seconds.

[0116] Some weakly binding surfaces of the present disclosure demonstrate significant improvement in stability or resistance to prolonged exposure to solvents and elevated temperatures, or repeated cycles of solvent exposure or temperature changes. For example, in some cases, the stability of the disclosed surfaces can be tested by fluorescently labeling a functional group on the surface or a bound biomolecule (e.g., an oligonucleotide primer) on the surface and monitoring the fluorescence signal before, during, and after prolonged exposure to solvents and elevated temperatures, or repeated cycles of solvent exposure or temperature changes.In some cases, the degree of change in fluorescence used to assess surface quality may be less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% over a period 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 of exposure to solvents and / or elevated temperatures (or any combination of these percentages as measured over these time periods).In some cases, the degree of change in fluorescence used to assess surface quality may be less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% 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 1000 cycles of repeated exposure to solvent changes and / or temperature changes (or any combination of these percentages as measured over this range of cycles).

[0117] In some cases, the surfaces disclosed herein may exhibit a high ratio of specific signal to nonspecific signal or other background. For example, when used for nucleic acid amplification, some surfaces may exhibit an amplification signal that is at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or more than 100 times greater than a signal from an adjacent unpopulated region of the surface. Likewise, some surfaces exhibit an amplification signal that is at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or more than 100 times greater than a signal from an adjacent amplified nucleic acid population region of the surface.

[0118] In some cases, low background fluorescence images of the disclosed surfaces, when used in nucleic acid hybridization or amplification applications to detect clusters of hybridized or clonally amplified nucleic acid molecules (e.g., directly or indirectly labeled with a fluorophore), exhibit contrast-to-noise ratios (CNRs) 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.

[0119] One or more primer types may be attached or tethered to the support surface. In some cases, the one or more adapter or primer types may include spacer sequences, adapter sequences for hybridizing to adapter-ligated target library nucleic acid sequences, forward amplification primers, reverse amplification primers, sequencing primers, and / or molecular barcoding sequences, or any combination thereof. In some cases, one primer or adapter sequence may be attached 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 primer or adapter sequences may be attached to at least one layer of the surface.

[0120] In some cases, the tethered adapter and / or primer sequences may range in length from about 10 nucleotides to about 100 nucleotides. In some cases, the tethered adapter and / or primer sequences may 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 long. In some cases, the tethered adapter and / or primer sequences may be at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, or at most 10 nucleotides long. Any of the lower and upper values ​​described in this paragraph may be combined to form a range encompassed by the present disclosure, for example, in some cases, the length of the tethered adapter and / or primer sequences may range from about 20 nucleotides to about 80 nucleotides.Those skilled in the art will recognize that the length of the tethered adapter and / or primer sequences may be any value within this range, e.g., about 24 nucleotides.

[0121] In some cases, the resulting surface density of primers on the weakly binding support surfaces of the present disclosure may be in the range of about 100 primer molecules per µm 2 up to about 100,000 primer molecules per µm 2 In some cases, the resulting surface density of primers on the weakly binding support surfaces of the present disclosure may be in the range of about 1,000 primer molecules per µm 2 up to about 1,000,000 primer molecules per µm 2 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 per µm 2In some cases, the surface density of primers can be at most 1,000,000, at most 100,000, at most 10,000 or at most 1,000 molecules per µm 2 Each of the lower and upper values ​​described in this paragraph can be combined to form a range encompassed by the present disclosure, for example, in some cases the surface density of primers can be in the range of about 10,000 molecules per µm 2 up to about 100,000 molecules per µm 2 Those skilled in the art will recognize that the surface density of primer molecules can be any value within this range, e.g., approximately 455,000 molecules per µm 2In some cases, the surface density of target library nucleic acid sequences initially hybridized to adapter or primer sequences on the support surface may be less than or equal to that specified for the surface density of tethered primers. In some cases, the surface density of clonally amplified target library nucleic acid sequences hybridized to adapter or primer sequences on the support surface may extend over the same range specified for the surface density of tethered primers.

[0122] Local densities, as listed above, do not exclude a variation of density across a surface, so that a surface may have an area with an oligo density of, for example, 500,000 / µm 2 while also comprising at least a second area with a substantially different local density. VI. Illustrative Alternative Embodiments

[0123] The disclosed methods for determining the sequence of a target nucleic acid comprise: a) contacting a double-stranded or partially double-stranded target nucleic acid molecule comprising the 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 the binding of a 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., the N+1 or terminal nucleotide) to be incorporated into the complementary strand.

[0124] The sequencing method may further comprise incorporating the N+1 or terminal nucleotide into the primer strand and then 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. Following the step of detecting the ternary binding complex, the primed strand of the primed target nucleic acid is extended by one base before performing another round of analysis. The primed target nucleic acid may be extended using the conjugated nucleotide bound to the polymer in the multivalent binding composition or using an unconjugated or unbound free nucleotide provided after the multivalent binding composition is removed.

[0125] Extension of the primed target nucleic acid may be prevented or inhibited due to a blocked nucleotide on the strand or the use of polymerase that is catalytically inactive. If the nucleotide in the polymer-nucleotide conjugate has a blocking group that prevents extension of the nucleic acid, incorporation of a nucleotide may be achieved by removing a blocking group from the nucleotide (such as by cleaving the nucleotide from its polymer, branched polymer, dendrimer, particle, or the like). If extension of the primed target nucleic acid is inhibited due to the use of polymerase that is catalytically inactive, incorporation of a nucleotide may be achieved by providing a cofactor or activator, such as a metal ion.

[0126] Detection of the ternary complex is achieved before, simultaneously with, or after incorporation of the nucleotide residue. In some embodiments, a primed target nucleic acid may comprise a target nucleic acid with multiple primed sites for attachment of polymerases and / or nucleic acid binding moieties. In some embodiments, multiple polymerases may be bound to a single target nucleic acid molecule, such as at multiple sites within a target nucleic acid molecule. In some embodiments, multiple polymerases may be bound to a multivalent binding composition disclosed herein comprising multiple nucleotides.In some embodiments, a target nucleic acid molecule may be a product of strand displacement synthesis, rolling circle amplification, concatenation or fusion of multiple copies of a query sequence, or other such methods known in the art or disclosed elsewhere herein to produce nucleic acid molecules comprising multiple copies of an identical sequence. Therefore, in some embodiments, multiple polymerases may be attached to multiple identical or substantially identical sites within a target nucleic acid comprising multiple identical or substantially identical copies of a query sequence.In some embodiments, the multiple polymerases may then be involved in interactions with one or more multivalent binding complexes; however, in preferred embodiments, the number of binding sites within a target nucleic acid is at least two, and the number of nucleotides or substrate moieties present on a particle-nucleotide conjugate, such as a polymer-nucleotide conjugate, is also greater than or equal to two.

[0127] It may be advantageous to provide the multivalent binding compositions in combination with other elements to provide optimized signals, for example, to provide identification of a nucleotide at a specific position 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 can be found, for example, in U.S. Patent Application No. 16 / 363,842, the contents of which are hereby incorporated by reference in their entirety.

[0128] In some cases, the nucleic acid molecule is bound to the surface of a solid support, e.g., by hybridization of the template strand to an adapter nucleic acid sequence or primer nucleic acid sequence bound to the solid support. In some cases, the solid support comprises a glass, quartz, silicon, or polymer substrate. In some cases, the solid support comprises a weak nonspecific binding coating comprising one or more hydrophilic polymer layers (e.g., PEG layers), wherein at least one of the hydrophilic polymer layers comprises a branched polymer molecule (e.g., a branched PEG molecule comprising 4, 8, 16, or 32 branches).

[0129] The solid support comprises oligonucleotide adapters or primers bound to at least one hydrophilic polymer layer with a surface density in the range of about 1,000 primer molecules per µm 2 up to about 1,000,000 primer molecules per µm 2In some cases, the surface density of oligonucleotide primers can be at least 1,000, at least 10,000, at least 100,000 or at least 1,000,000 molecules per µm 2 In some cases, the surface density of oligonucleotide primers may be at most 1,000,000, at most 100,000, at most 10,000, or at most 1,000 molecules per µm 2 Each of the lower and upper values ​​described in this paragraph can be combined to form a range encompassed by the present disclosure, for example, in some cases the surface density of primers can be in the range of about 10,000 molecules per µm 2 up to about 100,000 molecules per µm 2 Those skilled in the art will recognize that the surface density of primer molecules can be any value within this range, e.g., approximately 455,000 molecules per µm 2 can have.

[0130] One of skill in the art would recognize that in a series of iterative sequencing reactions, occasionally one or more sites fail to incorporate a nucleotide during a given cycle, resulting in one or more sites being out of synchronization with the bulk of the extending nucleic acid chains. Under conditions where sequencing signals are derived from reactions occurring on individual copies of a target nucleic acid, these incorporation errors result in discrete errors in the output sequence. It is an object of the present invention to provide methods for reducing such errors in sequencing reactions.For example, the use of multivalent substrates capable of being incorporated into the extending strand, by providing increased probabilities of rebinding upon premature dissociation of a ternary polymerase complex, may reduce the frequency of "skipped" cycles in which no base is incorporated. Thus, in some embodiments, the present disclosure contemplates the use of multivalent substrates disclosed herein in which 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 the particle or polymer, as disclosed herein, by a labile or cleavable bond.In some embodiments, the present disclosure contemplates a reduction in the intrinsic error rate due to skipped incorporation operations as a result of using the multivalent substrates disclosed herein.

[0131] The present disclosure also contemplates sequencing reactions in which sequencing signals originating from or relating to a given sequence are derived from or originate from definable regions containing multiple copies of the target sequence. Sequencing methods using multiple copies of a target sequence have the advantage that the signals can be amplified by the presence of multiple simultaneous sequencing reactions within the defined region, each providing its own signal. The presence of multiple signals within a defined region also reduces the impact of a single skipped cycle, since the signal from a large number of correct base calls can overwhelm the signal from a smaller number of skipped or incorrect base calls.The present disclosure further contemplates the inclusion of free, unlabeled nucleotides during extension reactions or during a separate portion of the extension cycle to provide for incorporation at sites skipped in previous cycles. For example, unlabeled blocked nucleotides can be added during or after an 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 nucleotide bound to the multivalent binding substrate(s) that are or were present during a particular cycle, or a mixture of 1, 2, 3, 4, or more types of unlabeled blocked nucleotides can be included.

[0132] If each sequencing cycle runs perfectly, each reaction within the defined region will yield an identical signal. However, as mentioned elsewhere herein, in a series of iterative sequencing reactions, occasionally one or more sites will fail to incorporate a nucleotide during a given cycle, resulting in one or more sites being out of sync with the bulk of the extending nucleic acid chains. This problem, referred to as "phasing," leads to a degradation of the sequencing signal because the signal is contaminated with noise from sites that have skipped one or more cycles. This, in turn, creates the potential for error in baseline identification. The progressive accumulation of skipped cycles over multiple cycles also decreases the effective read length due to progressive degradation of the sequencing signal with each cycle.It is a further object of this disclosure to provide methods for reducing phase errors and / or improving read length in sequencing reactions.

[0133] The sequencing method may include contacting one or more target nucleic acids comprising multiple linked or unlinked copies of a target sequence with the multivalent binding compositions described herein. Contacting the target nucleic acid or multiple target nucleic acids comprising multiple linked or unlinked copies of a target sequence with one or more particle-nucleotide conjugates may provide a substantially increased local concentration of the correct nucleotide interrogated in a given sequencing cycle, thereby suppressing signals from misincorporations or phased nucleic acid chains (i.e., those extending nucleic acid chains that had one or more skipped cycles).

[0134] Methods for obtaining nucleic acid sequence information may include contacting one or more target nucleic acids with one or more particle-nucleotide conjugates, wherein the target nucleic acid or nucleic acids comprise multiple, linked or unlinked copies of a target sequence. This method results in a reduction in the sequencing error rate, as evidenced by fewer bases being misidentified, fewer missing bases being reported, or fewer correct bases not being reported.In some embodiments, the reduction in sequencing error rate may comprise a reduction of 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, or more compared to the error rate observed using monovalent ligands, including free nucleotides, labeled free nucleotides, protein- or peptide-bound nucleotides, or labeled protein- or peptide-bound nucleotides.

[0135] The method for obtaining nucleic acid sequence information may include contacting one or more target nucleic acids with one or more particle-nucleotide conjugates, wherein the template nucleic acid or the multiple target nucleic acids comprise multiple linked or unlinked copies of a target sequence. This 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 monovalent ligands, including free nucleotides, labeled free nucleotides, protein- or peptide-bound nucleotides, or labeled protein- or peptide-bound nucleotides.

[0136] Disclosed herein are methods for obtaining nucleic acid sequence information, the methods comprising contacting one or more target nucleic acids with one or more particle-nucleotide conjugates, wherein the target nucleic acid or the multiple target nucleic acids comprise multiple linked or unlinked copies of a target sequence. This procedure results in an increase in the average read length of 10 nucleotides (NT), 20 NT, 25 NT, 30 NT, 50 NT, 75 NT, 100 NT, 125 NT, 150 NT, 200 NT, 250 NT, 300 NT, 350 NT, 400 NT, 500 NT or more compared to the average read length observed using monovalent ligands, including free nucleotides, labeled free nucleotides, protein- or peptide-bound nucleotides, or labeled protein- or peptide-bound nucleotides.

[0137] In some cases, the disclosed compositions and methods may result in average read lengths for sequencing applications that range from 100 nucleotides to 1,000 nucleotides.In some cases, the average read length may 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 1000 nucleotides.In some cases, the average read length may be a range bounded by any two of the values ​​within this range, e.g., an average read length in the range of 375 nucleotides to 825 nucleotides. Those skilled in the art will recognize that in some cases, the average read length may be any value within the range specified in this paragraph, e.g., 523 nucleotides.

[0138] The use of a multivalent binding composition for sequencing effectively shortens the sequencing time. The sequencing reaction cycle, comprising the steps of contacting, detecting, and incorporating, is performed in a total time ranging from about 5 minutes to about 60 minutes. In some cases, the sequencing reaction cycle is performed in 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.Each of the lower and upper values ​​described in this paragraph can be combined to form a range encompassed by the present disclosure. For example, in some cases, the sequencing reaction cycle can be performed in a total time ranging from about 10 minutes to about 30 minutes. Those skilled in the art will recognize that the sequencing cycle time can be any value within this range, e.g., about 16 minutes.

[0139] In some cases, the disclosed nucleic acid sequencing compositions and methods provide an average base calling accuracy of 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 values ​​over the course of a sequencing run. In some cases, the disclosed nucleic acid sequencing compositions and methods provide an average base calling accuracy of 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% of correctly called bases per every 1,000 bases, 10,000 bases, 25,000 bases, 50,000 bases, 75,000 bases, or 100,000 bases.

[0140] The use of a multivalent binding composition for sequencing provides a more accurate base reading. The disclosed nucleic acid sequencing compositions and methods provide an average Q-value for base calling accuracy over a sequencing run that ranges from about 20 to about 50. In some cases, the average Q-value is at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50. Those skilled in the art will recognize that the average Q-value can be any value within this range, e.g., about 32.

[0141] In some cases, the disclosed nucleic acid sequencing compositions and methods provide a Q-value 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 identified terminal (or N+1) nucleotides. In some cases, the disclosed nucleic acid sequencing compositions and methods provide a Q-value 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 identified terminal (or N+1) nucleotides.In some cases, the disclosed nucleic acid sequencing compositions and methods provide a Q-value 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 identified terminal (or N+1) nucleotides. In some cases, the disclosed nucleic acid sequencing compositions and methods provide a Q-value 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 identified terminal (or N+1) nucleotides.In some cases, the disclosed nucleic acid sequencing compositions and methods provide a Q-value of 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 identified terminal (or N+1) nucleotides.

[0142] The disclosed weak nonspecific binding supports and the associated nucleic acid hybridization and amplification methods can be used for the analysis of nucleic acid molecules derived from a variety of different cell, tissue, or sample types known to those skilled in the art. For example, nucleic acids can be extracted from cells or tissue samples comprising one or more cell types derived from eukaryotes (such as animals, plants, fungi, protista), archaebacteria, or eubacteria. In some cases, nucleic acids can be extracted from prokaryotic or eukaryotic cells, such as adherent or non-adherent eukaryotic cells. For example, nucleic acids are extracted differently from 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., white blood cells, red blood cells, 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, nucleic acids are extracted from diseased cells, such as cancer cells, or from pathogenic cells that infect a host. Some nucleic acids can be extracted from a different subset of cell types, e.g.,Immune cells (such as T cells, cytotoxic T cells (killer T cells), helper T cells, alpha-beta T cells, gamma-delta T cells, T cell precursors, B cells, B cell precursors, lymphoid stem cells, myeloid precursor cells, 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 induced to differentiate, rare cells (e.g., circulating tumor cells (CTCs), circulating epithelial cells, circulating endothelial cells, circulating endometrial cells, bone marrow cells, precursor cells, foam cells, mesenchymal cells, or trophoblasts). Nucleic acids may further include nucleic acids derived from viral samples and from subviral pathogens such as viroids and infectious RNAs.Nucleic acids may be derived from clinical or other samples, such as sputum, saliva, ocular fluid, synovial fluid, blood, feces, urine, tissue exudate, sweat, pus, drainage fluid, or the like. Nucleic acids may also be derived from plant or fungal samples, such as leaf, cambium, root, meristem, pollen, ovule, seed, spore, inflorescence, mycelium, or the like. Nucleic acids may also be derived from environmental or industrial samples, such as water, air, dust, food, or the like. Other cells, tissues, and samples are contemplated and consistent with the disclosure herein.

[0143] Nucleic acid extraction from cells or other biological samples can be performed using a variety of techniques known to those skilled in the art. For example, a DNA extraction procedure may involve: (i) collecting the cell sample or tissue sample from which DNA is to be extracted, (ii) disrupting cell membranes (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 DNA from the supernatant to remove detergents, proteins, salts, or other reagents used during the cell membrane lysis step.

[0144] A variety of suitable commercial nucleic acid extraction and purification kits are consistent with the disclosure herein. Examples include, but are not limited to, the QIAamp kits (for isolating genomic DNA from human samples) and DNAeasy kits (for isolating genomic DNA from animal or plant samples) from Qiagen (Germantown, MD) or the Maxwell® and ReliaPrep™ series of kits from Promega (Madison, WI). VII. Systems

[0145] System Modules: As mentioned above, systems configured to perform any of the disclosed nucleic acid sequencing or nucleic acid detection and analysis methods are also disclosed herein. In some cases, the disclosed systems may comprise one or more of the multivalent binding compositions described herein, one or more buffers, and / or one or more nucleic acid molecules bound to a solid support.

[0146] In some cases, the system may further comprise a fluid flow controller and / or a fluid delivery system configured to sequentially and iteratively contact template nucleic acid molecules hybridized to nucleic acid molecules (e.g., adapters or primers) bound to a solid support with the disclosed multivalent binding compositions and / or reagents. In some cases, the contacting may be performed within one or more flow cells. In some cases, these flow cells may be fixed components of the system. In some cases, the flow cells may be removable and / or disposable components of the system.

[0147] In some cases, the system may further comprise an imaging module, wherein the imaging module may comprise, for example, one or more light sources, one or more optical components (e.g., lenses, mirrors, prisms, optical filters, colored glass filters, narrowband interference filters, broadband interference filters, dichroic reflectors, diffraction gratings, apertures, optical fibers or optical waveguides, and the like), 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 the binding of the disclosed multivalent binding compositions to target nucleic acid molecules (or template nucleic acid molecules) bound to a solid support or the interior of a flow cell.

[0148] Processors and Computing Systems: One or more processors may be used to implement the nucleic acid sequencing systems or other nucleic acid detection and analysis methods disclosed herein. The one or more processors may comprise 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 a variety of suitable integrated circuits (e.g., application-specific integrated circuits (ASICs) specifically designed to implement deep learning network architectures, or field-programmable gate arrays (FPGAs) to accelerate computation time, etc., and / or facilitate deployment), microprocessors, emerging next-generation microprocessor designs (e.g., memristor-based processors), logic devices, and the like.Although the disclosure is described with reference to a processor, other types of integrated circuits and logic devices may also be applicable. The processor may have any suitable data processing 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 a plurality of processors configured for parallel processing.

[0149] The one or more processors or computers used to implement the disclosed methods may be part of a larger computer system and / or may be operatively coupled to a computer network (a "network") via a communications interface to facilitate the transfer and sharing of data. The network may be a local area network, an intranet and / or extranet, an intranet and / or extranet communicating with the Internet, or the Internet. The network is, in part, a telecommunications and / or data network. The network may include one or more computer servers, which in some cases enables distributed computing, such as cloud computing.In some cases, the network may implement a peer-to-peer network with the help of the computer system, which may allow devices coupled to the computer system to behave like a client or a server.

[0150] The computer system may also include memory or storage locations (e.g., random-access memory, read-only memory, flash memory, Intel® Optane™ technology), electronic storage devices (e.g., hard drives), communication interfaces (e.g., network adapters) for communicating with one or more other systems and peripheral devices, such as cache, other memories, data storage, and / or electronic display adapters. The memory, storage devices, interfaces, and peripheral devices may communicate with the one or more processors, e.g., a CPU, via a communication bus, e.g., as found on a motherboard. The memory device(s) may be data storage devices (or data warehouses) for storing data.

[0151] The one or more processors, e.g., a CPU, execute a sequence of machine-readable instructions embodied in a program (or software). The instructions are stored in a memory location. The instructions are passed to the CPU, which subsequently programs or otherwise configures the CPU to implement the methods of the present disclosure. Examples of operations performed by the CPU include fetching, decoding, executing, and writing 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).

[0152] The storage unit stores files such as drivers, libraries, and stored 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 additional data storage units located external to the computer system, such as on a remote server that communicates with the computer system over an intranet or the Internet.

[0153] Some aspects of the methods and systems provided herein may be implemented with machine-executable code (e.g., processor) stored at an electronic location of the computer system, such as in memory or electronic storage device. The machine-executable or machine-readable code may be provided in the form of software. During use, the code is executed by the one or more processors. In some cases, the code is retrieved from the storage device and stored in memory for easy access by the one or more processors. In some cases, the electronic storage device is omitted, and the machine-executable instructions are 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 it may be compiled at runtime. The code may be provided in a programming language selected so that the code can be precompiled or executed as compiled.

[0154] Various aspects of the technology may be considered "products" or "articles of manufacture," e.g., "computer program or software products," often in the form of machine-executable (or processor-executable) code and / or associated data stored on some type of machine-readable medium, wherein the executable code comprises a plurality of instructions for controlling a computer or computer system in performing one or more of the methods disclosed herein. Machine-executable code may be stored in an optical storage device comprising an optically readable medium such as an optical disk, CD-ROM, DVD, or Blu-ray disk. Machine-executable code may be stored in an electronic storage device, such as memory (e.g., read-only memory, random access memory, flash memory) or on a hard disk."Storage media" includes any or all of the tangible memory of the computers, processors, or the like, or associated modules thereof, such as various semiconductor memory chips, optical drives, tape drives, disk drives, and the like, which may at any time provide non-volatile storage for the software encoding the methods and algorithms disclosed herein.

[0155] The software code may, at times, be transmitted, in whole or in part, over the Internet or various other telecommunications networks. Such communications enable, for example, the loading of the software from one computer or processor to another, such as from a management server or host computer to the computing platform of an application server. Thus, other types of media used to transmit the software-encoded instructions include optical, electrical, and electromagnetic waves, such as those used over physical interfaces between local devices, over wired and optical fixed networks, and over various atmospheric connections.The physical elements that carry such waves, such as wired or wireless connections, optical links, or the like, are also considered media that carry the software-encoded instructions for performing the methods disclosed herein. As used herein, unless limited to non-transitory, tangible "storage" media, terms such as computer or "machine-readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0156] The computer system often includes or is capable of communicating with an electronic display for providing, for example, images captured by a machine vision system. The display is often also capable of providing a user interface (UI). Examples of user interfaces include, but are not limited to, graphical user interfaces (GUIs), web-based user interfaces, and the like.

[0157] System Control Software: In some cases, the disclosed systems may include a computer (or processor) and computer-readable media containing code for providing a user interface and manual, semi-automatic, or fully automated control of all system functions, e.g., control of a fluid flow control and / or fluid dispensing system (or subsystem), a temperature control system (or subsystem), an imaging system (or subsystem), etc. 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 standalone module, for example, a personal computer or laptop computer.Examples of fluid flow control functions that may be provided by the instrument control software include, but are not limited to, volumetric fluid flow rates, fluid flow velocities, the timing and duration of sample and reagent additions, purge steps, and the like. Examples of temperature control functions that may be provided by the instrument control software include, but are not limited to, specifying the temperature setpoint(s) and controlling the timing, duration, and ramp rates for temperature changes.Examples of imaging system control functions that may be provided by the instrument control software include, but are not limited to, autofocus capability, control of exposure times and intensities of illumination or excitation light, control of image acquisition rate, exposure time, data storage options, and the like.

[0158] Image processing software: In some cases of the disclosed systems, the system may further comprise computer-readable media including code for providing image processing and analysis capability. Examples of image processing and analysis capability that may be provided by the software 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 capability, etc.), manual, semi-automatic, or fully automatic edge detection and object identification (e.g., for identifying clusters of amplified template nucleic acid molecules on a substrate surface), manual, semi-automatic, or fully automatic signal intensity measurements and / or thresholding in one or more detection channels (e.g.,one or more fluorescence emission channels), manual, semi-automatic, or fully automated statistical analysis (e.g., to compare signal intensities with a reference value for baseline calling purposes).

[0159] In some cases, the system software may provide integrated real-time image analysis and instrument control so that sample loading, reagent addition, rinsing, and / or imaging / base calling steps can be extended, modified, or repeated as needed, e.g., until optimal base calling results are achieved. A variety of image processing and analysis algorithms known to those skilled in the art may be used to implement real-time or post-processing image analysis capabilities. Examples include, but are not limited to, the Canny edge detection method, the Canny-Deriche edge detection method, first-order gradient edge detection methods (e.g., the Sobel operator), second-order differential edge detection methods, phase congruent edge detection methods (phase coherent edge detection methods), other image segmentation algorithms (e.g.,Intensity thresholding, intensity clustering, intensity histogram-based methods, etc.), feature and pattern recognition algorithms (e.g., the generalized Hough transform for recognizing arbitrary shapes, the circular Hough transform, etc.), and mathematical analysis algorithms (e.g., Fourier transform, fast Fourier transform, wavelet analysis, autocorrelation, etc.) or combinations thereof.

[0160] In some cases, the system control software and the image processing / analysis software may be written as separate software modules. In some cases, the system control software and the image processing / analysis software may be incorporated into an integrated software package. VIII. Examples 1. Preparation of a multivalent binding composition

[0161] A type of multi-armed substrate, as in Fig. The PEG-dNTP shown in Figure 5A was prepared by reacting propargylamine dNTPs with biotin-PEG-NHS. This aqueous reaction was completed and purified, resulting in a pure biotin-PEG-dNTP species. Several different PEG lengths were used in separate reactions, corresponding to average molecular weights ranging from 1 kDa to 20 kDa. The biotin-PEG-dNTP species were mixed with either freshly prepared or commercially available dye-labeled streptavidin (SA) using a dye:SA ratio of 3-5:1. Mixing of biotin-PEG-dNTP with dye-labeled streptavidin was performed in the presence of excess biotin-PEG-dNTP to ensure saturation of the biotin binding sites on each streptavidin tetramer. Complete complexes were purified from excess biotin-PEG-dNTP by size exclusion chromatography.Each nucleotide type was conjugated and purified separately, then mixed together to create a four-base mixture for sequencing.

[0162] Another type of multi-arm substrate, as in Fig. The reaction mixture, shown in Figure 5A, was prepared in a single pot by reacting multi-arm PEG-NHS with excess dye-NH2 and propargylamine-dNTP. Various multi-arm PEG-NHS variants with 4–16 arms and molecular weights ranging from 5 kDa to 40 kDa were used. After reaction, excess small-molecule dye and dNTP were removed by size-exclusion chromatography.

[0163] Each nucleotide type was independently conjugated and purified and then mixed together to create a four-base mixture for sequencing. Class II substrates, as described in Fig. 5B, were prepared using one-pot reactions to simultaneously conjugate dye and dNTP. Alkyne-PEG-NHS was reacted with excess propargylamine dNTP. This product (alkyne-PEG-dNTP) was subsequently purified to homogeneity by chromatography. Several PEG lengths were used, with average molecular weights ranging from 1 kDa to 20 kDa. Dendrimer cores containing a variable, discrete number (12, 24, 48, 96) of azide conjugation sites were employed. Conjugation of alkyne-dye and alkyne-PEG-dNTP to the dendrimer core occurred in a one-pot reaction with excess dye and dNTP species via copper-mediated click chemistry. After the reaction, excess small-molecule dye and dNTP were removed by size-exclusion chromatography.Each nucleotide type was independently conjugated and purified and then mixed together to create a four-base mixture for sequencing. We note that this scheme allows for the easy substitution of alternative cores, such as dextrans, other polymers, proteins, etc.

[0164] Class III polymer-nucleotide conjugates as in Fig. The PEG-NH2 fragments shown in Figure 5C were constructed by reacting 4- or 8-arm PEG-NH2 with a saturating mixture of biotin and propargylamine dNTP. This reaction was then purified by size-exclusion chromatography. The result of this reaction was a multi-arm PEG containing a discrete distribution of biotin and nucleotide. This heterogeneous population was then reacted with dye-labeled streptavidin and purified by size-exclusion chromatography. Each nucleotide type was conjugated and purified independently and then mixed together to generate a four-base mixture for sequencing. Note that the distribution of biotin and nucleotide is tunable by the input ratio of biotin-NH2 to propargylamine dNTP. 2. Detection of a ternary complex

[0165] Binding reactions using the multivalent binding composition with PEG-polymer nucleotide conjugates were analyzed to demonstrate possible formation of a ternary binding complex, and the fluorescence images of the different steps are shown in Fig. 7A-7J. In Fig. Figure 7A, red and green fluorescence images after exposure of DNA RCA templates (Rolling Circle Application) (G and A first base) to 500 nM base-labeled nucleotides (A-Cy3 and G-Cy5) in exposure buffer containing 20 nM Klenow polymerase and 2.5 mM Sr +2Multivalent PEG substrate compositions were prepared using varying ratios of 4-arm PEG-amine (4ArmPEG-NH), biotin-PEG-amine (Biotin-PEG-NH), and nucleotide (Nuc) as follows: Samples PB1 and PB5, 4ArmPEG-NH: Biotin-PEG-NH: Nuc = 0.25:1:0.5; Sample PB2, 4ArmPEG-NH: Biotin-PEG-NH: Nuc = 0.125:0.5:0.25; Sample PB3, 4ArmPEG-NH: Biotin-PEG-NH: Nuc = 0.25:1:0.5. Images were acquired after washing with imaging buffer with the same composition as the exposure buffer, but without nucleotides or polymerase.

[0166] The contrast was scaled to maximize visualization of the weakest signals, but no signals remained after washing with imaging buffer ( Fig. 7A, insert). In Fig. 7B-7E show the fluorescence images of multivalent PEG-nucleotide ligands (base-labeled) at 500 nM after mixing in the exposure buffer and imaging in the imaging buffer as above ( Fig. 7B: PB1; Fig. 7C: PB2; Fig. 7D: PB3; Fig. 7E: PB5; Fig. Figure 7F: Fluorescence image showing the multivalent (base-labeled) PEG-nucleotide ligand PB5 at 2.5 µM after mixing in exposure buffer and imaging in imaging buffer as above. In Fig. 7G-7I, the fluorescence images show further base discrimination by exposure of multivalent ligands to inactive mutants of Klenow polymerase ( Fig. 7G: D882H; Fig. 7H: D882E; Fig. 7I: D882A, and the wild-type Klenow enzyme (control enzyme) is in Fig. 7J).

[0167] Using multivalent ligand formulations, base discrimination can be enabled by providing polymerase-ligand interactions with increased avidity. Furthermore, it is shown that an increased concentration of multivalent ligands can generate higher signals and that various Klenow mutations that eliminate catalytic activity can be used for avidity-based sequencing. 3. Sequencing of target nucleic acid molecules using ternary complexes

[0168] To demonstrate sequencing based on multivalent ligand reporters, four known templates were amplified using RCA methods on a weakly binding substrate. Successive cycles were exposed to exposure buffer containing 20 nM Klenow polymerase and 2.5 mM Sr. +2and washed with imaging buffer and imaged. After imaging, the substrates were washed with wash buffer (EDTA and high salt), and blocked nucleotides were added to advance to the next base. The cycle was repeated for 5 cycles. Spots were detected using standard image processing and spot detection, and the sequences were called using a two-color green and red scheme (G-Cy3 and A-Cy5) to identify the templates being cycled. As in Fig. 8A and Fig. As shown in Figure 8B, multivalent ligands are able to provide base discrimination through all 5 sequencing cycles. 4. Control of nucleotide dissociation from the ternary complex

[0169] Ternary complexes are prepared and imaged as in Example 2. The complexes are imaged for different time periods to demonstrate the persistence of the ternary complex, e.g., up to 60 seconds. After a period of time, the complexes are washed with a buffer identical to the buffer used for complex formation that lacks only one divalent cation, e.g., 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 0.016% Triton X100 (without SrOAc), or alternatively, the complexes are washed with a buffer identical to the buffer used for complex formation that contains a chelating agent but otherwise lacks a divalent cation, e.g., B. 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 0.016% Triton X100 (without SrOAc), with 100 nm - 100 mM EDTA. The fluorescence of the complexes is monitored over time, allowing the dissociation of the ternary complexes to be observed and quantified.A representative temporal progression of this dissolution is shown in . Fig. 6 shown. 5. Extension of the complementary target nucleic acid sequence

[0170] After preparation, imaging, and dissociation of ternary complexes as in Example 4, a deblocking solution sufficient to remove the blocking moiety, such as an O-azidomethyl group, an O-alkylhydroxylamine group, or an O-amine group, from the 3' end of the extending DNA strand is introduced into the chamber containing the bound DNA molecules. Subsequently, or simultaneously, an extending solution is introduced into the chamber with the bound DNA molecules. The extending solution contains a buffer, a divalent cation sufficient to support polymerase activity, an active polymerase, and an appropriate amount of all four nucleotides, with the nucleotides blocked such that they are unable to support further extension following the addition of a single nucleotide to the extending DNA strand, such as B. by incorporating a 3'-O-azidomethyl group, a 3'-O-alkylhydroxylamine group or a 3'-O-amine group.The extension strand is thus extended by one and only one base, and the binding of catalytically inactive polymerase and multivalent binding substrate can be used to call the next base in the cycle.

[0171] Alternatively, the nucleotides bound to the multivalent substrate can be linked by a labile bond, allowing a buffer containing the bound DNA molecules to be introduced into the chamber. This buffer contains a divalent cation or other cofactor sufficient to render the polymerase catalytically active. Conditions sufficient to cleave the base from the multivalent substrate so that it can be incorporated into the extending strand can be provided before, after, or simultaneously. This cleavage and incorporation results in the dissociation of the label and the polymer backbone of the multivalent substrate, extending the extending DNA strand by exactly one base.A wash is performed to remove spent polymer backbone, and new multivalent substrate is introduced into the chamber containing the bound DNA molecules, allowing the new base to be invoked as in Example 1. 6. Use of polymer-nucleotide conjugates with different lengths of the PEG branch

[0172] The polymer-nucleotide conjugates with varying PEG arm lengths described in Example 3 were subjected to a single sequencing cycle and imaged as described in Example 1. As described in Fig. As shown in Figures 9A-9J, increasing the length of the PEG branches resulted in an increased signal up to a length corresponding to an apparent mean PEG MW of 5 kDa ( Fig. 9A-9D). The use of longer PEG arms than these resulted in decreases in the fluorescence signal for both Cy3-A and Cy5-G ( Fig. 9E-9G). Quantitative measurements of signal intensity are available in Fig. 10 shown graphically. 7. Enhancement of the binding of multivalent substrates by adding detergents

[0173] Multivalent substrates were prepared and assembled into binding complexes in the presence and absence of detergents: one set using 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 5 mM SroAc, 0% Triton X100 (condition A) and one set using 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 5 mM SroAc, 0.016% Triton X100. Fig. Figure 11 shows normalized fluorescence from these multivalent substrates bound to DNA clusters, with the formed substrate complexes showing significantly increased fluorescence intensity in the presence (condition B) of Triton-X100 (0.016%). 8. Evaluation of the time courses of binding of multivalent substrates

[0174] Multivalent substrates were prepared as in Example 2 and assembled into binding complexes. Complexes with free labeled nucleotides were also formed under identical buffer conditions. To characterize the persistence time of the complexes, complexes were imaged over a period of 60 minutes. Fig. 12A-12B show representative results. Multivalent binding complexes are stable over timescales of > 60 minutes ( Fig. 12B), while labeled free nucleotides dissociate in less than one minute ( Fig. 12A). VIII. Conclusion

[0175] The present disclosure provides greatly improved methods and compositions for DNA sequencing and biosensor applications. It is understood that the foregoing description is intended to be illustrative and not limiting. Many embodiments will be apparent to those skilled in the art upon reviewing the foregoing description. By way of example, the inventive concepts have been described primarily with reference to the use of polymer-nucleotide conjugates; however, it will be readily apparent to those skilled in the art that other types of particle-nucleotide conjugates could also be used. For example, in some embodiments, it may be desirable to use particle-nucleotide conjugates that include a quantum dot; a liposome; or an emulsion particle. Alternatively, conjugation could be achieved through noncovalent bonding, such as hydrogen bonding or other interactions.The scope of the disclosed inventive concepts should therefore be determined not by reference to the foregoing description, but by reference to the appended claims, together with the full scope of equivalents to which such claims are entitled.

Claims

[1] A method for determining an 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; (ii) two or more primer nucleic acid molecules complementary to one or more regions of the target nucleic acid sequence; and (iii) two or more polymerase molecules; (b) contacting the composition with a polymer-nucleotide conjugate under conditions sufficient to allow a multivalent binding complex to be formed 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 two or more nucleotide residues; 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, wherein the detection of the binding complex is carried out in the absence of unbound or in solution polymer-nucleotide conjugates. [4] The method of claim 1, wherein the target nucleic acid sequence has been replicated or amplified or has been produced by replication or amplification. [5] The method of claim 1, wherein detecting the multivalent binding complex comprises a fluorescence measurement. [6] The method of claim 1, wherein the contacting comprises using a type of polymer-nucleotide conjugate. [7] The method of claim 1, wherein the contacting comprises using two or more types of polymer-nucleotide conjugates. [8] The method of claim 7, wherein each of the two or more types of polymer-nucleotide conjugates comprises a different type of nucleotide residue. [9] The method of claim 8, wherein the contacting comprises using three types of polymer-nucleotide conjugates, and wherein each type of the three types of polymer-nucleotide conjugates comprises a different type of nucleotide residue. [10] The method of claim 1, wherein the polymer-nucleotide conjugate comprises a blocked nucleotide residue. [11] The method of claim 10, wherein the blocked nucleotide is a 3'-O-azidomethyl, 3'-O-methyl or 3'-O-alkylhydroxylamine nucleotide. [12] The method of claim 1, wherein the contacting occurs in the presence of an ion that stabilizes the multivalent binding complex. [13] The method of claim 1, wherein the contacting is carried out in the presence of strontium ions, magnesium ions, calcium ions or any combination thereof. [14] The method of claim 1, wherein the polymerase molecules are catalytically inactive. [15] The method of claim 1, wherein the polymerase molecules have been rendered catalytically inactive by mutation or chemical modification. [16] The method of claim 1, wherein the polymerase molecules have been rendered catalytically inactive by the absence of a necessary ion or cofactor. [17] The method of claim 1, wherein the polymerase molecules are catalytically active. [18] The method of claim 1, wherein the polymer-nucleotide conjugate does not comprise a blocked nucleotide residue. [19] The method of claim 1, wherein the multivalent binding complex has a persistence time of more than 2 seconds. [20] The process of claim 1, wherein the process is carried out at a temperature within a range of 25°C to 42°C. [21] The method of claim 1, 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 on, attached to, or hybridized to a surface, wherein a fluorescent image of the multivalent binding complex on the surface has a contrast-to-noise ratio in the detection step of greater than 20. [22] The method of claim 1, wherein the composition of (a) is deposited on a surface using a buffer containing a polar aprotic solvent. [23] The method of claim 1, wherein the contacting is carried out under a condition that stabilizes the multivalent binding complex when the nucleotide residue is complementary to a nearest base of the target nucleic acid sequence and destabilizes the multivalent binding complex when the nucleotide residue is not complementary to the nearest base of the target nucleic acid sequence. [24] The method of claim 1, wherein the polymer-nucleotide conjugate comprises a polymer having a plurality of branches and the two or more nucleotide residues are attached to the branches. [25] The method of claim 24, wherein the first polymer has a star, comb, cross-linked, bottlebrush or dendrimer configuration. [26] The method of claim 1, wherein the polymer-nucleotide conjugate comprises one or more binding groups selected from the group consisting of an avidin, a biotin, an affinity tag, and combinations thereof. [27] The method of claim 1, further comprising a dissociation step that destabilizes the multivalent binding complex formed between the composition of (a) and the polymer-nucleotide conjugate, wherein the dissociation step enables the removal of the polymer-nucleotide conjugate. [28] The method of claim 27, further comprising an extension step of incorporating a nucleotide complementary to a nearest base of the target nucleic acid sequence into the two or more primer nucleic acid molecules. [29] The method of claim 28, wherein the extension step occurs simultaneously with or after the dissociation step. [30] The method of claim 1, wherein the polymer-nucleotide conjugate comprises one or more detectable labels.

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