JP7542000B2 - Multivalent binding compositions for nucleic acid analysis - Patents.com - Google Patents

Description

CROSS-REFERENCE 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. Provisional Application No. 62/897,172, filed September 6, 2019, and U.S. Provisional Application No. 62/852,876, filed May 24, 2019, each of which is incorporated herein in its entirety.

The present disclosure relates generally to multivalent binding compositions and their use in the analysis of nucleic acid molecules. In particular, the inventive concept relates to multivalent binding compositions having multiple copies of a nucleotide attached to a particle or polymer core, thereby effectively increasing the local concentration of the nucleotide to enhance the binding signal. Multivalent binding compositions can be applied, for example, in the fields of sequencing and biosensor microarrays.

Nucleic acid sequencing can be used to obtain information in a wide variety of biomedical contexts, including diagnostics, prognosis, biotechnology, and forensic biology. A variety of sequencing methods have been developed, including de novo sequencing methods including Maxam-Gilbert and chain termination reaction, shotgun sequencing, and bridge PCR, or next-generation methods including polony sequencing, 454 pyrosequencing, Illumina sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, HeliScope single molecule sequencing, SMRT® sequencing, and others. Despite advances in DNA sequencing, many challenges remain for cost-effective high-throughput sequencing. The present disclosure provides novel solutions and approaches that address many of the shortcomings of existing technologies.

Disclosed herein is a method for determining the identity of a nucleotide in a target nucleic acid sequence, the method 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 that are 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 form 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 moiety 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 composition is performed in the absence of unbound or solution-derived polymer-nucleotide conjugates. In some embodiments, the target nucleic acid sequence is replicated or amplified or 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, the contacting comprises the use of one polymer-nucleotide conjugate. In some embodiments, the contacting comprises the use of two or more polymer-nucleotide conjugates. In some embodiments, each of the two or more polymer-nucleotide conjugates comprises a different species of nucleotide moiety. In some embodiments, the contacting comprises the use of three polymer-nucleotide conjugates, and wherein each of the three polymer-nucleotide conjugates comprises a different species of nucleotide moiety. In some embodiments, the polymer-nucleotide conjugate comprises a blocked nucleotide moiety. In some embodiments, the blocked nucleotide is a 3'-O-azidomethyl nucleotide, a 3'-O-methyl nucleotide, or a 3'-O-alkylhydroxylamine nucleotide. In some embodiments, the contacting is performed in the presence of an ion that stabilizes the multivalent binding composition. In some embodiments, the contacting is performed in the presence of strontium ions, magnesium ions, calcium ions, or any combination thereof. In some embodiments, the polymerase molecule is catalytically inactive. In some embodiments, the polymerase molecule is rendered catalytically inactive by mutation or chemical modification. In some embodiments, the polymerase molecule is rendered catalytically inactive by the absence of a required ion or cofactor. In some embodiments, the polymerase molecule is catalytically active. In some embodiments, the polymer-nucleotide conjugate does not include a blocked nucleotide moiety. In some embodiments, the duration of the multivalent binding composition is greater than 2 seconds. In some embodiments, the method can be carried out at a temperature in the range of 25° C. to 62° C. In some embodiments, the polymer-nucleotide conjugate further comprises one or more fluorescent labels, and two or more copies of the target nucleic acid sequence are deposited on, attached to, or hybridized to a surface, where a fluorescent image of the multivalent binding complex on the surface has a contrast to noise ratio of greater than 20 in the detecting step. In some embodiments, the composition of (a) is deposited on the surface using a buffer incorporating a polar aprotic solvent. In some embodiments, the contacting step is performed under conditions that stabilize the multivalent binding complex when the nucleotide moiety is complementary to the next base of the target nucleic acid sequence and destabilize the multivalent binding complex when the nucleotide moiety is not complementary to the next base of the target nucleic acid sequence. In some embodiments, the polymer-nucleotide conjugate comprises a polymer having multiple branches, and the two or more nucleotide moieties are attached to the branches. In some embodiments, the polymer configuration is star-shaped, comb-shaped, cross-shaped, bottle-brush-shaped, or dendrimer-shaped. In some embodiments, the polymer-nucleotide conjugate comprises one or more binding groups selected from the group consisting of avidin, biotin, affinity tags, and combinations thereof. In some embodiments, the method further comprises a dissociation step of destabilizing the multivalent binding complex formed between the composition of (a) and the polymer-nucleotide conjugate, the dissociation step allowing for removal of the polymer-nucleotide conjugate. In some embodiments, the method further comprises an extension step to incorporate a nucleotide complementary to the next base of the target nucleic acid sequence into the two or more primer nucleic acid molecules. In some embodiments, the extension step is performed simultaneously with or after the dissociation step.

Disclosed herein is a method for determining the identity of a nucleotide in a target nucleic acid sequence, the method 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 that are 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 form 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 moiety and, optionally, one or more cleavable 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, the method further comprises, after detecting the multivalent binding complex, contacting the composition of (a) with a polymer-nucleotide conjugate comprising a reversibly terminated nucleotide or two or more copies of a reversibly terminated nucleotide. In some embodiments, the target nucleic acid sequence has been replicated or amplified or 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, the contacting comprises the use of one polymer-nucleotide conjugate. In some embodiments, the contacting comprises the use of two or more polymer-nucleotide conjugates. In some embodiments, each of the two or more polymer-nucleotide conjugates comprises a different species of nucleotide moiety. In some embodiments, the contacting comprises the use of three polymer-nucleotide conjugates, and wherein each of the three polymer-nucleotide conjugates comprises a different species of nucleotide moiety. In some embodiments, the polymer-nucleotide conjugate comprises a blocked nucleotide moiety. In some embodiments, the blocked nucleotide is 3'-O-azidomethyl, 3'-O-methyl, or 3'-O-alkylhydroxylamine. In some embodiments, the contacting is performed in the presence of an ion that stabilizes the multivalent binding composition. In some embodiments, the polymerase molecule is catalytically inactive. In some embodiments, the polymerase molecule is rendered catalytically inactive by mutation or chemical modification. In some embodiments, the polymerase molecule is catalytically active. In some embodiments, the polymer-nucleotide conjugate does not include a blocked nucleotide moiety. In some embodiments, the method can be performed at a temperature in the range of 25°C to 80°C. In some embodiments, the polymer-nucleotide conjugate further comprises one or more fluorescent labels, and two or more copies of the target nucleic acid sequence are deposited on, attached to, or hybridized to a surface, where a fluorescent image of the multivalent binding complex on the surface has a contrast to noise ratio of greater than 20 in the detection step.

Further disclosed herein is a system, the system comprising one or more computer processors individually or collectively programmed to: a) execute a method comprising: i) contacting a substrate comprising multiple copies of a target nucleic acid sequence tethered to its surface with a polymerase and a reagent comprising one or more primer nucleic acid sequences complementary to one or more regions of the target nucleic acid sequence to form a primed target nucleic acid sequence; ii) contacting the substrate surface with a reagent comprising a polymer-nucleotide conjugate under conditions sufficient to allow formation of a multivalent binding complex between the polymer-nucleotide conjugate and two or more copies of the primed target nucleic acid sequence, wherein the polymer-nucleotide conjugate comprises two or more copies of a known nucleotide moiety and a detectable label; and iii) acquiring and processing an image of the substrate surface to detect the multivalent binding complex, thereby determining the identity of nucleotides 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 defined order and at defined time intervals. In some embodiments, the system further comprises an imaging module configured to acquire an image of the substrate surface. In some embodiments, (ii) and (iii) are repeated two or more times, thereby determining the identity of a series of two or more nucleotides in a target nucleic acid sequence. In some embodiments, the series of steps further comprises a dissociation step that destabilizes the multivalent binding complex, allowing the polymer-nucleotide conjugate to be removed. In some embodiments, the series of steps further comprises an extension step to incorporate a nucleotide complementary to a next base in the target nucleic acid sequence into the two or more primer nucleic acid molecules. In some embodiments, the extension step is performed simultaneously with or after the dissociation step. In some embodiments, the detectable label comprises a fluorophore, and the image comprises a fluorescent image. In some embodiments, the fluorophore is cyanine dye 3 (Cy3), and images are acquired under non-signal saturating conditions 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 cyanine dye-3 emission, and a camera, while the surface is immersed in 25 mM ACES, pH 7.4 buffer, and the fluorescence image of the multivalent binding complex on the surface has a contrast to noise ratio of greater than 20 in the detection step. In some embodiments, the sequence of steps is completed in less than 60 minutes. In some embodiments, the sequence of steps is completed in less than 30 minutes. In some embodiments, the sequence of steps is completed in less than 10 minutes. In some embodiments, the accuracy of the base calling is characterized by a Q score of greater than 25 for at least 80% of the determined nucleotide identities. In some embodiments, the accuracy of the base calling is characterized by a Q score of greater than 30 for at least 80% of the determined nucleotide identities. In some embodiments, the accuracy of the base call is characterized by a Q score of greater than 40 for at least 80% of the determined nucleotide identities.

Disclosed herein are compositions comprising: a) a polymer core; and b) two or more nucleotide, nucleotide analog, nucleoside, or nucleoside analog moieties attached to the polymer core, where the length of the linker is dependent on the nucleotide, nucleotide analog, nucleoside, or nucleoside analog moieties attached to the polymer core. Also disclosed herein are compositions comprising: a) a mixture of polymer-nucleotide conjugates, each of which comprises: i) a polymer core; and ii) two or more nucleotide, nucleotide analog, nucleoside, or nucleoside analog moieties attached to the polymer core, where the length of the linker is dependent on the nucleotide, nucleotide analog, nucleoside, or nucleoside analog moieties attached to the polymer core, and the mixture comprises polymer-nucleotide conjugates having at least two different attached nucleotide, nucleotide analog, nucleoside, or nucleoside analog moieties. In some embodiments, the polymer core comprises a polymer having multiple branches, and two or more nucleotides, nucleotide analogs, nucleosides, or nucleoside analog moieties are attached to said branches. In some embodiments, the polymer configuration is star-shaped, comb-shaped, cross-shaped, bottle-brush-shaped, or dendrimer-shaped. In some embodiments, the polymer-nucleotide conjugate comprises one or more linking groups selected from the group consisting of avidin, biotin, affinity tags, 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.

In some embodiments, the disclosure provides a method for determining the identity of a nucleotide in a target nucleic acid, the method comprising, in any order, 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 a binding or incorporation complex to form between the polymer-nucleotide conjugate and the composition of step (a), where 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 establishing the identity of the nucleotide in the target nucleic acid polymer. In some further embodiments, the disclosure provides the method, wherein the target nucleic acid is DNA and/or the target nucleic acid is 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 disclosure provides the method, wherein the detectable label is a fluorescent label and/or the detection of the complex comprises fluorescence measurement. In some further embodiments, the disclosure provides the method, wherein the multivalent binding composition comprises one type of polymer-nucleotide conjugate, the multivalent binding composition comprises two or more types of polymer-nucleotide conjugates, and/or each of the two or more types of polymer-nucleotide conjugates comprises a different type of nucleotide. In some embodiments, the disclosure provides the method wherein the bound or incorporated complex further comprises a blocked nucleotide, in particular, the blocked nucleotide is a 3'-O-azidomethyl nucleotide, a 3'-O-alkylhydroxylamino nucleotide, or a 3'-O-methyl nucleotide. In some further embodiments, the disclosure provides the method wherein the contacting is performed in the presence of strontium ions, barium, magnesium ions, and/or calcium ions. In some embodiments, the disclosure provides the method wherein the polymerase molecule is catalytically inactive, such as rendered catalytically inactive by mutation, chemical modification, or the absence of a required ion or cofactor. In some embodiments, the disclosure also provides the method wherein the polymerase molecule is catalytically active and/or the bound complex does not comprise a blocked nucleotide. In some embodiments, the disclosure provides the method, wherein the binding complex has a duration of more than 2 seconds, and/or the method is performed 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, 72° C. or more, or may be performed within a range defined in any of the above. In some embodiments, the disclosure provides the method, wherein the binding composition is deposited, attached, or hybridized to a surface that provides a contrast to noise ratio of greater than 20 in the detection step. In some embodiments, the disclosure provides the method, wherein the composition is deposited under buffer conditions that incorporate a polar aprotic solvent. In some embodiments, the disclosure provides the method, wherein the contacting is performed under conditions that stabilize the binding composition when the nucleotide is complementary to the next base of the target nucleic acid and destabilize the binding composition when the nucleotide is not complementary to the next base of the target nucleic acid. In some embodiments, the disclosure provides the method, wherein the polymer-nucleotide conjugate comprises a polymer having multiple branches, and the multiple copies of the first nucleotide are attached to the branches, in particular, the first polymer has a star-shaped, comb-shaped, bridged, bottle-brush, or dendrimer-shaped configuration. In some embodiments, the disclosure provides the method, wherein the polymer-nucleotide conjugate comprises one or more binding groups selected from the group consisting of avidin, biotin, affinity tags, and combinations thereof. In some embodiments, the disclosure provides the method, further comprising a dissociation step to destabilize the binding complex formed between the composition of (a) and the polymer-nucleotide conjugate and remove the polymer-nucleotide conjugate. In some embodiments, the disclosure further comprises an extension step to incorporate a nucleotide complementary to the next base of the target nucleic acid into the primer nucleic acid, and optionally, the extension step is performed simultaneously with or after the dissociation step.

In some embodiments, the disclosure provides a composition comprising a branched polymer having two or more branches and two or more copies of a nucleotide, the nucleotide attached to a first plurality of the branches or arms, and optionally one or more interacting moieties attached to a second plurality of the branches or arms. In some embodiments, the composition may comprise one or more labels on the polymer. In some embodiments, the disclosure provides the composition, wherein the nucleosides have a surface density of at least four nucleotides per polymer. In some embodiments, the disclosure provides the composition, wherein the nucleosides comprise or incorporate a nucleotide or nucleotide analogue modified to prevent incorporation into a growing nucleic acid strand during a polymerase reaction. In some embodiments, the composition may comprise or incorporate a nucleotide or nucleotide analogue reversibly modified to prevent incorporation into a growing nucleic acid strand during a polymerase reaction. In some embodiments, the disclosure provides the composition, wherein the one or more labels comprise a fluorescent label, a FRET donor, and/or a FRET acceptor. In some embodiments, the composition may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 or more branches or arms, or 2, 4, 8, 16, 32, or 64 or more branches or arms. In some embodiments, the branches or arms may radiate from a central portion. In some embodiments, the composition may include one or more interacting moieties, which may include avidin or streptavidin, biotin moieties, affinity tags, enzymes, antibodies, minibodies, receptors, or other proteins, non-proteinaceous tags, metal affinity tags, or any combination thereof. In some embodiments, the disclosure provides the composition, wherein the polymer comprises polyethylene glycol, polypropylene glycol, polyvinyl acetate, polylactic acid, or polyglycolic acid. In some embodiments, the disclosure provides said compositions wherein the nucleotide or nucleotide analog is attached to a branch or arm by a linker, and in particular, the linker comprises PEG and the PEG linker moiety has an average molecular weight of about 1K Da, about 2K Da, about 3K Da, about 4K Da, about 5K Da, about 10K Da, about 15K Da, about 20K Da, about 50K Da, about 100K Da, about 150K Da, or about 200K Da or more. In some embodiments, the disclosure provides said compositions wherein the linker comprises PEG and the PEG linker moiety has an average molecular weight of about 5K Da to about 20K Da. In some embodiments, the disclosure provides the composition wherein at least one nucleotide or nucleotide analog comprises a deoxyribonucleotide, ribonucleotide, deoxyribonucleoside, or ribonucleoside, and/or the nucleotide or nucleotide analog is conjugated to a linker via the 5' end of the nucleotide or nucleotide analog. In some embodiments, the disclosure provides the composition wherein one of the nucleotides or nucleotide analogs comprises deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, deoxycytidine, adenosine, guanosine, 5-methyl-uridine, and/or cytidine, and the length of the linker is between 1 nm and 1,000 nm. In some embodiments, the disclosure provides the composition, wherein at least one nucleotide or nucleotide analog is a nucleotide modified to inhibit extension during a polymerase reaction or a sequencing reaction, such as, for example, a nucleotide lacking a 3' hydroxyl group, a nucleotide modified to include a blocking group at the 3' position, and/or a nucleotide modified with a 3'-O-azido, 3'-O-azidomethyl, 3'-O-alkylhydroxylamino, 3'-phosphorothioate, 3'-O-malonyl, or 3-O-benzyl group. In some embodiments, the disclosure provides the composition, wherein at least one nucleotide or nucleotide analog is a nucleotide that is not modified at the 3' position.

In some embodiments, the disclosure provides a method for determining the sequence of a nucleic acid molecule, comprising, without regard to any particular order, 1) providing a nucleic acid molecule comprising a template strand and a complementary strand at least partially complementary to the template strand, 2) contacting the nucleic acid molecule with 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 the identity of a terminal nucleotide incorporated into the complementary strand of the nucleic acid molecule. In some embodiments, the disclosure provides a method for determining the sequence of a nucleic acid molecule, comprising, without regard to any particular order, 1) providing a nucleic acid molecule comprising a template strand and a complementary strand at least partially complementary to the template strand, 2) contacting the nucleic acid molecule with 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 the identity of a terminal nucleotide incorporated into the complementary strand of the nucleic acid molecule of the fully or partially incorporated embodiment described herein. In some embodiments, the present disclosure provides the method further comprising incorporating the terminal nucleotide into the complementary strand, and repeating the contacting, detecting, and incorporating steps for one or more additional iterations, thereby determining the sequence of the template strand of the nucleic acid molecule. In some embodiments, the present disclosure provides the method, wherein the nucleic acid molecule is tethered to a solid support, in particular, the solid support comprises a glass or polymer substrate, at least one hydrophilic polymer coating layer, and a plurality of oligonucleotide molecules attached to at least one hydrophilic polymer coating layer. In some embodiments, the present disclosure provides the method, further comprising an embodiment in which at least one hydrophilic polymer coating layer comprises PEG, and/or at least one hydrophilic polymer layer comprises a branched hydrophilic polymer having at least eight branches. In some embodiments, the disclosure provides the method, wherein the plurality of oligonucleotide molecules is present at a surface density of at least 500 molecules/ ^mm2 , at least 1,000 molecules/ ^mm2 , at least 5,000 molecules/ ^mm2 , at least 10,000 molecules/ ^mm2 , at least 20,000 molecules/ ^mm2 , at least 50,000 molecules/ ^mm2 , at least 100,000 molecules/ ^mm2 , or at least 500,000 molecules/ ^mm2 . In some embodiments, the disclosure provides the method, wherein the nucleic acid molecules are clonally amplified on a solid support. In some embodiments, the present disclosure provides the method, wherein the clonal amplification comprises the use of polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, single-stranded binding (SSB) protein-dependent amplification, or any combination thereof. In some embodiments, the present disclosure provides the method, wherein one or more nucleic acid binding compositions are labeled with a fluorophore, and the detecting step comprises the use of fluorescence imaging, in particular, the fluorescence imaging comprises two-wavelength excitation/four-wavelength emission 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 analog, are used to determine the identity of the terminal nucleotide, and the four different nucleic acid binding compositions are labeled with separate respective fluorophores, and the detecting step comprises simultaneous excitation at wavelengths sufficient to excite all four fluorophores and fluorescence emission imaging at wavelengths sufficient to detect each fluorophore. In some embodiments, the disclosure provides the method, wherein four different nucleic acid binding compositions, each comprising a different nucleotide or nucleotide analog, are used to determine the identity of the terminal nucleotide, the four different nucleic acid binding compositions being 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 the detecting step comprises simultaneous excitation at any two of 532 nm, 568 nm, and 633 nm, and fluorescence emission imaging at about 570 nm, 592 nm, 670 nm, and 702 nm, respectively, and/or the fluorescence imaging comprises dual-wavelength excitation/dual-wavelength emission fluorescence imaging. In some embodiments, the disclosure provides a method wherein four different nucleic acid binding compositions, each comprising a different nucleotide or nucleotide analog, are used to determine the identity of a terminal nucleotide, wherein the one, two, three, or four different nucleic acid binding compositions are each labeled with a separate fluorophore or set of fluorophores, and the detecting step comprises simultaneous excitation at wavelengths sufficient to excite the one, two, three, or four fluorophores or sets of fluorophores, and fluorescence emission imaging at wavelengths sufficient to detect each fluorophore. In some embodiments, the disclosure provides the method, wherein three different nucleic acid binding or incorporation compositions, each comprising a different nucleotide or nucleotide analog, 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 distinct fluorophore or set of fluorophore compositions, and the detecting step comprises simultaneous excitation at wavelengths sufficient to excite one, two, or three fluorophores or sets of fluorophores, and fluorescence emission imaging at wavelengths sufficient to detect each fluorophore, and the detection of the fourth nucleotide is determined or determinable with reference to a "dark" or unlabeled spot or location of the target nucleotide. In some embodiments, the disclosure provides the method, wherein the multivalent binding or incorporation composition can comprise three polymer-nucleotide conjugates, and wherein each of the three polymer-nucleotide conjugates comprises a different type of nucleotide. In some embodiments, the disclosure provides the method, wherein the step of detecting the binding or incorporation complexes is performed in the absence of unbound or solution-derived polymer-nucleotide conjugates.

In some embodiments, the disclosure provides a method in which four different nucleic acid binding compositions, each containing a different nucleotide or nucleotide analog, or three different nucleic acid binding or incorporation compositions are used to determine the identity of the terminal nucleotide, one of the four or three different nucleotide 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 fluorophores, and one is unlabeled or absent, and the detecting step includes simultaneous excitation at a first excitation wavelength and a second excitation wavelength, and the image is acquired at a first fluorescent emission wavelength and a second fluorescent emission wavelength. In some embodiments, the disclosure provides a method in which 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 fluorescent emission wavelength is about 570 nm, and the second fluorescent emission wavelength is about 670 nm. In some embodiments, the present disclosure provides the method, wherein the detection label may comprise one or more moieties of a fluorescence resonance energy transfer (FRET) pair, such that multiple classifications may be performed under a single excitation and imaging step. In some embodiments, the present disclosure provides the method, wherein a sequencing reaction cycle, including contacting, detecting, and incorporating/extending steps, is performed in less than 30 minutes, less than 20 minutes, or less than 10 minutes. In some embodiments, the present disclosure provides the method, wherein the average Q-score of base calling accuracy across a sequencing run is 30 or greater, and/or 40 or greater. In some embodiments, the present disclosure provides the method, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the identified terminal nucleotides have a Q-score greater than 30 and/or 40 or greater. In some embodiments, the present disclosure provides the method, wherein at least 95% of the identified terminal nucleotides have a Q-score greater than 30.

In some embodiments, the disclosure provides a reagent comprising one or more nucleic acid binding compositions and a buffer as disclosed herein. For example, in some embodiments, the disclosure provides a reagent comprising one, two, three, four or more nucleic acid binding or incorporation compositions, each of which comprises a single type of nucleotide. In some embodiments, the reagents of the disclosure include one, two, three, four, or more nucleic acid binding or incorporation compositions, each of which includes a single type of nucleotide or nucleotide analog, the nucleotide or nucleotide analog being 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); thymidine triphosphate (TTP), thymidine diphosphate (TDP), thymidine monophosphate (TMP), deoxythymidine triphosphate (dTTP), deoxythymidine diphosphate (dTDP), deoxythymidine monophosphate (dTMP), uridine triphosphate (UTP), or uridine diphosphate (UTP). (UTP), uridine diphosphate (UDP), uridine monophosphate (UMP), deoxyuridine triphosphate (dUTP), deoxyuridine diphosphate (dUDP), and deoxyuridine monophosphate (dUMP); one or more 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 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 or further examples, the disclosure provides a reagent that includes or further includes one, two, three, four or more nucleic acid binding or incorporation compositions, each of which includes a single type of nucleotide or nucleotide analog, and the nucleotide or nucleotide analog may correspond to one or more of the following: 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.

Disclosed herein are kits that include any of the nucleic acid binding or incorporation compositions of the embodiments disclosed herein, and/or any of the reagents of the embodiments disclosed herein, and/or one or more buffers, and instructions for use thereof.

Disclosed herein is a system for carrying out the method of any of the embodiments disclosed herein, the system comprising a nucleic acid binding or incorporating composition of any of the embodiments disclosed herein, and/or a reagent of any of the embodiments disclosed herein. In some embodiments, the system is configured for repeated sequential contacting of a tethered primed nucleic acid molecule with the nucleic acid binding or incorporating composition and/or the reagent, and for detection of binding or incorporation of the disclosed nucleic acid binding or incorporating composition into one or more primed nucleic acid molecules.

In some embodiments, the present disclosure provides compositions comprising particles (e.g., nanoparticles or polymer cores), the particles comprising a plurality of enzyme or protein binding or incorporation substrates, where the enzyme or protein binding or incorporation substrates are bound to one or more enzymes or proteins to form one or more binding or incorporation complexes (e.g., multivalent binding or incorporation complexes), and the binding or incorporation can be observed or identified by observing the location, presence, or duration of the one or more binding or incorporation complexes. In some embodiments, the particle may comprise a polymer, a branched polymer, a dendrimer, a liposome, a micelle, a nanoparticle, or a quantum dot. In some embodiments, the substrate may comprise a nucleotide, 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 to a polymerase. In some embodiments, the enzyme or protein may comprise a polymerase. In some embodiments, observing the location, presence, or duration of the 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, where each particle comprises a single type of nucleoside or nucleoside analog, and each nucleoside or nucleoside analog is associated with a fluorescent label with a detectably different emission or excitation wavelength. In some embodiments, the present disclosure provides a composition further comprising one or more labels, e.g., fluorescent labels, on the particle. In some embodiments, the present disclosure provides a composition comprising at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or more than 20 tethered nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs tethered to the particle. In some embodiments, the disclosure provides said compositions wherein the nucleoside or nucleoside analog is present at an areal density of 0.001-1,000,000/μm ² , 0.01-1,000,000/μm ² , 0.1-1,000,000/μm ² , 1-1,000,000/μm ² , 10-1,000,000/μm ² , 100-1,000,000/μm ² , 1,000-1,000,000/μm ^{2 , 1,000-100,000/μm 2 ,} 10,000-100,000/μm ² , or 50,000-100,000/μm ² , or within a range defined by any two of the foregoing values. In some embodiments, the present disclosure provides the composition, wherein the nucleoside or nucleoside analog is present within the nucleotide or nucleotide analog. In some embodiments, the present disclosure provides the composition, wherein the composition comprises or incorporates a nucleotide or nucleotide analog that is modified to prevent incorporation into a growing nucleic acid strand during a polymerase reaction. In some embodiments, the present disclosure provides the composition, wherein the composition comprises or incorporates a nucleotide or nucleotide analog that is reversibly modified to prevent incorporation into a growing nucleic acid strand during a polymerase reaction. In some embodiments, the present disclosure provides the composition, wherein the one or more labels comprise a fluorescent label, a FRET donor, and/or a FRET acceptor. In some embodiments, the present disclosure provides the composition, wherein the substrate (e.g., a nucleotide, a nucleotide analog, a nucleoside, or a nucleoside analog) is attached to a particle by a linker. In some embodiments, the disclosure provides such compositions, wherein at least one nucleotide or nucleotide analog is a nucleotide modified to inhibit extension during a polymerase or sequencing reaction, such as, for example, a nucleotide absent a 3' hydroxyl group, a nucleotide modified to include a blocking group at the 3' position, a nucleotide modified with a 3'-O-azido, 3'-O-azidomethyl, 3'-O-alkylhydroxylamino, 3'-phosphorothioate, 3'-O-malonyl, or 3'-O-benzyl group, and/or a nucleotide that is not modified at the 3' position.

In some embodiments, the disclosure provides a method for determining the sequence of a nucleic acid molecule, comprising, in any order, 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 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 the identity of a terminal nucleotide 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 incorporation steps for one or more additional iterations, thereby determining the sequence of the template strand of the nucleic acid molecule. In some embodiments, the disclosure provides the method, wherein the nucleic acid molecule is clonally amplified on a solid support. In some embodiments, the present disclosure provides the method, wherein the clonal amplification comprises the use of polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification, circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, single-stranded binding (SSB) protein-dependent amplification, or any combination thereof. In some embodiments, the present disclosure provides the method, wherein the sequencing reaction cycle, including the steps of contacting, detecting, and incorporating, is performed in less than 30 minutes, less than 20 minutes, or less than 10 minutes. In some embodiments, the present disclosure provides the method, wherein the average Q-score of the base call accuracy across the sequencing run is 30 or greater, or 40 or greater. In some embodiments, the present disclosure provides the method, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the identified terminal nucleotides have a Q-score greater than 30, 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 score greater than 30.

In some embodiments, the disclosure provides a reagent comprising one or more nucleic acid binding or incorporation compositions and a buffer as disclosed herein. In some embodiments, the disclosure provides a reagent comprising one, two, three, four or more nucleic acid binding or incorporation compositions, each of which comprises a single type of nucleotide or nucleotide analog, and the nucleotide or nucleotide analog comprises a nucleotide, nucleotide analog, nucleoside, or nucleoside analog. In some embodiments, the disclosure provides the method, wherein the reagent comprises one, two, three, four or more nucleic acid binding or incorporation compositions, each of which comprises a single type of nucleotide, or nucleotide analog, and the nucleotide, or nucleotide analog may 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, respectively. In some embodiments, the disclosure provides the method, wherein the reagent comprises one, two, three, four or more nucleic acid binding or incorporation compositions, each of which comprises a single type of nucleotide or nucleotide analog, and the nucleotide or nucleotide analog may 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, a, and dGMP.

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 use thereof.

In some embodiments, the disclosure provides a system for carrying out any of the methods disclosed herein, where the method may include the use of any of the compositions as disclosed herein, and/or any of the reagents as disclosed herein, one or more buffers, and optionally one or more nucleic acid molecules tethered or attached to a solid support, and the system is configured to perform repeated operations for sequential contacting of the nucleic acid molecules with the compositions and/or the reagents, and for detection of binding or incorporation of the nucleic acid binding or incorporation composition to the one or more nucleic acid molecules.

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.

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

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

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

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

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.

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

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

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

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

INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term in this specification and a term in a cited document, the term in this specification shall control.

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

The novel features of the inventive concepts disclosed herein are set forth with particularity in the appended claims. A better understanding of the features and advantages of the compositions, methods, and systems of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the inventive concepts are utilized, and the accompanying drawings in which:
[0023] Figure 1 shows steps for utilizing non-limiting examples of multivalent binding compositions for sequencing a target nucleic acid. Figure 1A shows non-limiting example 4 of attaching a target nucleic acid to a surface. 1A-1C show steps of utilizing non-limiting examples of multivalent binding compositions for sequencing a target nucleic acid. FIG. 1B shows a clonally representative target nucleic acid for forming a cluster of amplified target nucleic acid molecules. Figure 1C shows a non-limiting example of a process for utilizing a multivalent binding composition for sequencing a target nucleic acid. Figure 1C shows a non-limiting example of priming a target nucleic acid to generate a primed target nucleic acid. FIG. 1D shows a non-limiting example of a process for utilizing a multivalent binding composition for sequencing a target nucleic acid. FIG. 1D shows a non-limiting example of contacting a primed target nucleic acid with a multivalent binding composition and a polymerase to form a binding complex. Figure 1A shows a process for utilizing non-limiting examples of multivalent binding compositions for sequencing a target nucleic acid. Figure 1E shows a non-limiting example image of a binding complex captured on a surface. Figure 1F shows a non-limiting example of a process for utilizing a multivalent binding composition for sequencing a target nucleic acid. Figure 1F shows a non-limiting example of extending a primer strand by one nucleotide. FIG. 1G shows a non-limiting example of a process for utilizing a multivalent binding composition to sequence a target nucleic acid. FIG. 1G shows another non-limiting example of a cycle in which a primed target nucleic acid is contacted with a multivalent binding composition and a polymerase to form a binding complex. FIG. 1C shows a process for utilizing non-limiting examples of multivalent binding compositions for sequencing a target nucleic acid. FIG. 1H shows a non-limiting example image of a binding complex captured on a surface in a subsequent sequencing cycle. FIG. 2 shows a flow chart outlining the steps for sequencing a target nucleic acid and extending the primer strand by single base addition. FIG. 3 shows a flow chart outlining the steps for sequencing a target nucleic acid and extending the primer strand by incorporating nucleotides onto a particle-nucleotide conjugate. A non-limiting example of detecting a target nucleic acid using a polymer-nucleotide conjugate is shown in Figure 4A, which shows the steps of contacting a polymerase and a polymer-nucleotide conjugate with several nucleic acid molecules. A non-limiting example of detecting a target nucleic acid using a polymer-nucleotide conjugate is shown in Figure 4B, which shows a binding complex formed between a polymerase, a polymer-nucleotide conjugate, and a target nucleic acid molecule. 5A-5C show schematic diagrams of non-limiting examples of various configurations of polymer-nucleotide conjugates: Figure 5A shows polymer-nucleotide conjugates having various multi-arm configurations; Schematic diagrams of non-limiting examples of various configurations of polymer-nucleotide conjugates are shown: Figure 5B shows a polymer-nucleotide conjugate with polymer branches radiating from a center. 5A-5C show schematic diagrams of non-limiting examples of various configurations of polymer-nucleotide conjugates: Figure 5C shows a polymer-nucleotide conjugate with a binding moiety, biotin; FIG. 6 shows a generalized graphical representation of the binding, retention, and increase in signal intensity observed during washing and removal of the multivalent substrate. 7A-7J show fluorescence images of steps in a sequencing reaction using a multivalent PEG substrate composition. FIG. 7A shows red and green fluorescence images of a DNA RCA template (G and A first bases) after exposure to 500 nM base-labeled nucleotides (A-Cy3 and G-Cy5) in an exposure buffer containing 20 nM Klenow polymerase and 2.5 mM Sr ⁺² . Images were collected after washing with an imaging buffer having the same composition as the exposure buffer but containing no nucleotides or polymerase. Contrast was scaled to maximize visualization of the faintest signals, although no signals persisted after washing with imaging buffer (FIG. 7A, inset). 7B-7E are fluorescence images showing multivalent PEG-nucleotide (base-labeled) ligands PB1 (FIG. 7B), PB2 (FIG. 7C), PB3 (FIG. 7D), and PB5 (FIG. 7E) with an effective nucleotide concentration of 500 nM after mixing in exposure buffer and imaging in imaging buffer as described above. FIG. 7F is a fluorescence image showing multivalent PEG-nucleotide (base-labeled) ligand PB5 at 2.5 uM after mixing in exposure buffer and imaging in imaging buffer as described above. FIG. 7G-7I are fluorescence images showing further base discrimination by exposing the multivalent binding composition to inactive mutants of Klenow polymerase (FIG. 7G), D882H (FIG. 7H), and D882E (FIG. 7I). FIG. 7J is a fluorescence image showing further base discrimination by exposing the multivalent binding composition to inactive mutants of wild-type Klenow (control) enzyme. The effectiveness of the multivalent reporter composition in determining the base sequence of a DNA sequence over five sequencing cycles is shown in Figure 8. Figure 8A shows images of the template and predicted sequence obtained after each sequencing cycle. Figure 8B shows the effectiveness of the multivalent reporter composition in determining the base sequence of a DNA sequence over five sequencing cycles, and shows aligned sequencing results utilizing the images taken in Figure 8A. 9A-J show fluorescence images of multivalent polyethylene glycol (PEG) polymer-nucleotide (base-labeled) conjugates with an effective ^nucleotide concentration of 500 nM and various PEG branch lengths after contacting a support surface containing a DNA template (containing G or A at the first base and prepared using rolling circle amplification (RCA)) in an exposure buffer containing 20 nM Klenow polymerase and 2.5 mM Sr+2. Images were acquired after washing with an imaging buffer having the same composition as the exposure buffer but lacking nucleotides and polymerase. The panels show images obtained using multivalent PEG-nucleotide ligands with arm lengths as follows: FIG. 9A: 1-arm KPEG; FIG. 9B: 2-arm KPEG; FIG. 9C: 3-arm KPEG; FIG. 9D: 5-arm KPEG; FIG. 9E: 10-arm KPEG; FIG. 9F: 20-arm KPEG. Figure 9G shows an image obtained using 10K PEG and inactive Klenow polymerase with mutation D882H. Figure 7H shows an image obtained using 10K PEG and inactive Klenow polymerase with mutation D882E. Figure 7I shows an image obtained using 10K PEG and inactive Klenow polymerase with mutation D882A. Figure 7J shows an image obtained using 10K PEG and active wild-type Klenow polymerase. FIG. 10 shows a quantitative representation of the fluorescence intensity separated by color values in the images shown in FIG. 9 A-F, where the orange trace corresponds to the red label (Cy3 label; A base) and the blue trace corresponds to the green label (Cy5 label; G base). FIG. 11 shows normalized fluorescence from multivalent substrate bound to DNA clusters as described in FIG. 7A-J, where substrate complexes are formed in the presence (condition B) and absence (condition A) of Triton-X100 (0.016%). Plots of normalized fluorescence intensity measured for multivalent polymer-nucleotide conjugates and free nucleotides are shown: Figure 12A is a two replicate of multivalent polymer-nucleotide conjugates bound to binding compositions vs. DNA clusters (conditions A and B) formed using labeled free nucleotides (condition C) after 1 minute. Figure 12A shows a plot of normalized fluorescence intensity measured for multivalent polymer-nucleotide conjugates and free nucleotides. Figure 12B shows a plot of normalized fluorescence intensity measured for multivalent polymer-nucleotide conjugates and free nucleotides. Figure 12B is a time course of fluorescence of multivalent substrate complexes over a 60 minute period.

I. Definitions As used herein, a "nucleic acid" (also referred to as a "polynucleotide", "oligonucleotide", "ribonucleic acid (RNA)" or "deoxyribonucleic acid (DNA)") is a linear polymer of two or more nucleotides, or variants or functional fragments thereof, linked by covalent internucleoside linkages. In naturally occurring examples of nucleic acids, the internucleoside linkages are phosphodiester linkages. However, other examples optionally include other internucleoside linkages, such as phosphorothiolate linkages, which may or may not include phosphate groups. Nucleic acids include double- and single-stranded DNA, as well as double- and single-stranded RNA, DNA/RNA hybrids, peptide-nucleic acid (PNA), hybrids of PNA with DNA or RNA, and may also include other types of nucleic acid modifications.

As used herein, "nucleotide" refers to a nucleotide, nucleoside, or analog thereof. Nucleotide refers to both naturally occurring and chemically modified nucleotides and may include, but is not limited to, a nucleoside, ribonucleotide, deoxyribonucleotide, protein nucleic acid residue, or derivative. Examples of nucleotides include adenine, thymine, uracil, cytosine, guanine, or residues thereof, deoxyadenine, deoxythymine, deoxyuracil, deoxycytosine, deoxyguanine, or residues thereof, adenine PNA, thymine PNA, uracil PNA, cytosine PNA, guanine PNA, or residues thereof or equivalents, N-glycosides or C-glycosides of purine or pyrimidine bases (e.g., deoxyribonucleosides containing 2-deoxy D-ribose, or ribonucleosides containing D-ribose).

As used herein, "complementary" refers to the topological compatibility or matching of the interacting surfaces of a ligand molecule and its receptor. Thus, a receptor and its ligand may be described as being complementary, and further, the contact surface properties are complementary to each other.

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

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

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

II. Methods for Analyzing Target Nucleic Acids Disclosed herein are multivalent binding or incorporation compositions and their use in the analysis of nucleic acid molecules involved in sequencing or other bioassay applications. Increased binding or incorporation of nucleotides to an enzyme (e.g., polymerase) or enzyme complex can be affected by increasing the effective concentration of the nucleotide. The increase can be achieved by increasing the concentration of the nucleotide in free solution or by increasing the amount of nucleotides in close proximity to the relevant binding or incorporation site. Increase can also be achieved by physically restricting the number of nucleotides to a limited volume, which results in a local increase in concentration, and thus such structures can bind or incorporate into the binding or incorporation site with a higher apparent avidity than can be observed for individual nucleotides that are unconjugated, tethered, or otherwise unconstrained. A non-limiting means for achieving such restriction is by providing 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.

The multivalent binding or incorporation compositions disclosed herein may include at least one particle-nucleotide conjugate, which has multiple copies of the same nucleotide attached to a particle. When the nucleotide is complementary to the target nucleic acid, the particle-nucleotide conjugate forms a binding or incorporation complex with a polymerase and the target nucleic acid, which exhibits greater stability and longer duration than binding or incorporation complexes formed using a single unconjugated or untethered nucleotide. Each of the nucleotide moieties of the multivalent binding composition may bind to a complementary N+1 nucleotide of a primed target nucleic acid molecule, thereby forming a multivalent binding complex that includes two or more target nucleic acid molecules, two or more polymerase (or other enzyme) molecules, and a multivalent binding composition (e.g., a polymer-nucleotide conjugate). Each of the nucleotide moieties 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 that includes two or more target nucleic acid molecules, two or more polymerase (or other enzyme) molecules, and a multivalent binding composition (e.g., a polymer-nucleotide conjugate). From this bound complex, the nucleotide can probe the complementary base before incorporating a modified reversibly blocked nucleotide that extends the replicative strand by one base. Furthermore, one can envision probing the N nucleotide by the bound complex, moving forward with the reversibly terminated nucleotide and probing the subsequent N+1 base before and after deblocking. In this way, by reading twice what has been probed, error checking can be performed and the overall accuracy of the base call can be improved. The key distinguishing factor from conventional methods is that the binding is used to probe the matched base, whereas the stepping or incorporation step is only used to move forward on the extending strand.

The multivalent binding or incorporation compositions can be used to localize detectable signals to active regions of biochemical interactions, such as the sites of protein-nucleic acid interactions, nucleic acid hybridization reactions, or enzymatic reactions such as polymerase reactions. For example, the multivalent binding or incorporation compositions described herein can be utilized to identify sites of base incorporation in an extending nucleic acid strand during a polymerase reaction and provide base discrimination for sequencing and array-based applications. When the nucleotides are complementary to the target nucleic acid, increased binding or incorporation between the target nucleic acid and the nucleotides in the multivalent binding or incorporation composition provides an enhanced signal that significantly improves the accuracy of base calling and reduces imaging times.

Furthermore, the use of multivalent binding compositions allows for sequencing signals from a given sequence to occur within cluster regions that contain multiple copies of the target sequence. Sequencing methods that incorporate multiple copies of the target sequence have the advantage that the signal can be amplified since there are multiple simultaneous sequencing reactions within a defined region, each providing its own signal. The presence of multiple signals within a defined region also mitigates the impact of a single skipped cycle due to the fact that the signal from a large number of correct base calls will overwhelm the signal from a small number of skipped or incorrect base calls, thus providing a method for reducing phasing errors and/or improving the read length of the sequencing reaction.

The multivalent binding compositions disclosed herein and their uses provide one or more of the following: (i) stronger signals for better base calling accuracy compared to conventional nucleic acid amplification and sequencing methods; (ii) allowing better discrimination of sequence-specific signals from background signals; (iii) reduced requirements for the amount of starting material needed; (iv) increased sequencing speed and reduced sequencing time; (v) reduced phase errors; and (vi) improved read length during sequencing reactions.

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

A. Sequencing of Target Nucleic Acids Figures 1A-1H show one exemplary method in which multivalent binding compositions are used to sequence a target nucleic acid. As shown in Figure 1A, the target nucleic acid (102) can be tethered to a solid support surface (101). The target nucleic acid can be directly or indirectly attached to the surface. Although not shown in Figure 1A, the target nucleic acid (102) can be hybridized to an adaptor that is covalently or non-covalently attached to the surface. When one or more adaptors are used to attach the target nucleic acid to the surface, the target surface can include fragments that are complementary to the adaptors and thus hybridize to the adaptors. In some instances, one adaptor sequence can be tethered to the surface. In some instances, multiple adaptor sequences can be tethered to the surface. In some instances, the target nucleic acid (102) can be directly attached to the solid support surface without the use of adaptors. The solid support can be a low non-specific binding surface.

In FIG. 1B, after the initial step of attaching the target nucleic acid to the surface of a solid support surface (e.g., via hybridization to an adapter), the target nucleic acid is clonally amplified to form clusters of amplified nucleic acid. 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 adapters on the support surface can range from the same as the surface density of the tethered adapters (or primers). Clonal amplification may 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-stranded binding (SSB), protein-dependent amplification, or any combination thereof.

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

As shown in FIG. 1D, the primed target nucleic acid (104) is combined with a multivalent binding or incorporation composition and a polymerase (106) to form a binding or incorporation complex. A non-limiting example of the multivalent binding or incorporation composition of FIG. 1D includes four particle-nucleotide conjugates (105a), (105b), (105c), and (105d). Each particle-nucleotide conjugate has multiple copies of a nucleotide attached to the particle, and the four particle-nucleotide conjugates each cover four types of nucleotides. The particle-nucleotide conjugate that has a nucleotide complementary to the next base on the primed target nucleic acid forms a binding or incorporation complex with the polymerase and the target nucleic acid. In some examples, 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 can be labeled with a distinct label. In some embodiments, three of the four nucleotide conjugates may be labeled and the fourth is unlabeled or conjugated to an undetectable label. In some embodiments, one, two, three, or four particle-nucleotide conjugates may be labeled with the same label, or with three, two, one, or zero particle-nucleotide conjugates that may each be labeled with a label corresponding to the identity of the conjugated nucleotide, or may remain unlabeled or be conjugated to an undetectable label. In some embodiments, detection of polymerase complexes incorporating particle-nucleotide conjugates may be performed using four-color detection, where 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 may be exposed to or contacted with the target nucleic acid simultaneously, while in some other embodiments, the four particle-nucleotide conjugates may be exposed to or contacted with the target nucleic acid sequentially, either individually or in groups of two or three. In some embodiments, the step of detecting polymerase complexes incorporating particle-nucleotide conjugates can be performed using three-color detection, where conjugates corresponding to three of the four nucleotides are present in the sample, three conjugates have distinct labels corresponding to the nucleotides conjugated thereto, and one conjugate is unlabeled or conjugated to an undetectable label. In some embodiments, only three conjugates are provided, such that conjugates corresponding to three of the four nucleotides are present in the sample, three conjugates have distinct labels corresponding to the nucleotides conjugated thereto, and one conjugate is absent. In some embodiments, the identity of the nucleotide corresponding to the unlabeled or absent nucleotide conjugate can be determined with respect to the location and/or identity of a "dark" spot or location of the known target nucleic acid that does not exhibit a fluorescent signal. In some embodiments, the present disclosure provides the method, where detection of the bound or incorporated complexes is performed in the absence of unbound or solution-derived polymer-nucleotide conjugates.

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 may be established by monitoring the absence of a signal or the presence of an unlabeled complex, such as identifying a "dark" spot or an unlabeled region in a sequencing reaction. In some embodiments, detection of a polymerase complex incorporating a particle-nucleotide conjugate may be performed using two-color detection, where conjugates corresponding to two of the four nucleotides are present in a sample, two conjugates have separate labels corresponding to the nucleotides conjugated thereto, and two conjugates are unlabeled or 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-nucleotides are labeled, the identity of the nucleotide corresponding to the unlabeled conjugate may be established by monitoring the absence of a signal or the presence of an unlabeled complex, such as identifying a "dark" spot or an unlabeled region in a sequencing reaction. In some embodiments where two of the four particle-nucleotides are labeled, the four particle-nucleotide conjugates can be exposed or contacted sequentially to the target nucleic acid, either individually or in groups of two or three. In some embodiments, two of the four particle-nucleotide conjugates can share a common label, and the four particle-nucleotide conjugates can be exposed or contacted sequentially, individually, or in groups of two or three to the target nucleic acid, where each contacting step indicates 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.

Figure 1E shows an image taken on a surface after binding or incorporation complexes have formed between a polymerase, a target nucleic acid, and a particle-nucleotide conjugate having a nucleotide complementary to the next base of the primed target nucleic acid. The image taken includes four binding or incorporation complexes (107a), (107b), (107c), and (107d) formed on the surface, each having a different nucleotide that can be distinguished based on the label (e.g., fluorescent emission color) on the particle-nucleotide conjugate. Using particle-nucleotide conjugates, binding or incorporation signals from specific sequences can occur within cluster regions that contain multiple copies of the target sequence, thus greatly enhancing the sequencing signal. Although Figure 1E includes four particle-nucleotide conjugates, each having a different type of nucleotide, some methods can 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 can be labeled with the same label, or with a label that corresponds to the identity of its conjugated nucleotide. In some embodiments, three of the four nucleotide conjugates may be labeled, with the fourth being unlabeled or conjugated to an undetectable label. In some embodiments, one, two, three, or four particle-nucleotide conjugates may be labeled with individual labels, with three, two, one, or zero particle-nucleotide conjugates, respectively, being unlabeled or conjugated to an undetectable label. In some embodiments, the detection step may include simultaneous and/or sequential excitation of up to four different excitation wavelengths, e.g., fluorescence imaging is performed by detecting single and/or multiple fluorescent emission bands that uniquely classify each of the possible base pairs (A, G, C, or T). In some embodiments, the identity of the terminal nucleotide can be determined using four different nucleic acid binding or incorporation compositions, each containing a different nucleotide or nucleotide analog, 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 fluorophores, and one is unlabeled, the detection step includes 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.

When a multivalent binding or incorporation composition is used in place of a single unbound or untethered nucleotide to form a binding or incorporation complex with the polymerase and the primed target nucleic acid, the local concentration of the nucleotide increases many-fold, thereby enhancing the signal intensity. The formed binding or incorporation complex also has a longer duration, which in turn helps to shorten the imaging step. The high signal intensity is due to the high binding or incorporation avidity of the polymer-nucleotide conjugate (which may contain multiple fluorophores or other labels), thus forming a complex that remains stable throughout the binding or incorporation and imaging steps. The strong binding or incorporation between the polymerase, the primed target strand, and the polymer-nucleotide or nucleotide analog conjugate further means that the multivalent binding or incorporation complex thus formed remains stable during the washing step, and the signal intensity remains high when the nucleotide analog that was not matched with the other reaction mixture components is 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 be extended by one base.

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

After the extension step, a contacting step as shown in FIG. 1G can be performed again to form a binding or incorporation complex and mimic the next sequencing cycle. The contacting, detecting and extension steps can be repeated for one or more cycles, thereby determining the sequence of the target nucleic acid molecule. For example, FIG. 1H shows a surface image obtained after performing multiple sequencing cycles, which can then be processed to determine the sequence of the target nucleic acid molecule.

Extension of a primed target nucleic acid can be prevented or inhibited due to the use of a blocked nucleotide on the strand or a catalytically inactive polymerase. If the nucleotide in the polymer-nucleotide conjugate has a blocking group that prevents extension of the nucleic acid, incorporation of the nucleotide can be achieved by removal of the blocking group from the nucleotide (e.g., by detachment of the nucleotide from the polymer, branched polymer, dendrimer, particle, etc.). If extension of a primed target nucleic acid is inhibited due to the use of a catalytically inactive polymerase, incorporation of the nucleotide can be achieved by the provision of a cofactor or activator, such as a metal ion.

Further disclosed herein is a system configured to perform any of the disclosed nucleic acid sequencing or nucleic acid analysis methods. The system may include a fluid flow controller and/or a fluid distribution system configured to continuously and repeatedly contact a primed target nucleic acid molecule attached to a solid support with the disclosed polymerase and multivalent binding or incorporation compositions and/or reagents. The contacting steps may be performed in one or more flow cells. In some examples, the flow cell may be a fixed component of the system. In some examples, the flow cell may be a removable and/or disposable component of the system.

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 tethered inside a solid support or 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.

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

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

Referring again to FIG. 2, if the nucleotide on the particle-nucleotide conjugate is complementary to the next base of the primed target nucleic acid (205), the particle-nucleotide conjugate, polymerase, and primed target nucleic acid form a ternary binding or incorporation complex, which can be detected by a detection method compatible with the label of the particle-nucleotide conjugate (e.g., fluorescent imaging). (205) may further include measuring the duration 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 under conditions that change the conformation of the polymerase and destabilize the binding or incorporation (e.g., addition of strontium ions). 206 may further include washing or removing the dissociated particle-nucleotide conjugate and/or polymerase. (207) describes a 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) can be repeated for multiple cycles to determine the sequence of the target nucleic acid.

Figure 3 is another flow chart outlining steps for sequencing a target nucleic acid, including cleaving nucleotides from particle-nucleotide conjugates and incorporating the cleaved nucleotides. (301) describes attaching target library sequences to a solid support surface by hybridizing the target nucleic acid molecules to complementary adaptors on the substrate surface. The target nucleic acid molecules can be single-stranded or partially double-stranded. (Prior to (301), the nucleic acid molecules of the target library may have been prepared to include fragments complementary to the adapter sequences by ligation or other methods. (302) describes a step of clonal amplification to generate clusters of target nucleic acid molecules on a surface. (303) describes hybridizing a sequencing primer to a complementary primer binding or incorporation sequence on the target nucleic acid to form a primed target nucleic acid. (304) describes combining the primed target nucleic acid with a multivalent binding or incorporation composition comprising a polymerase, a labeled (e.g., fluorescently labeled) particle-nucleotide conjugate. In the particle-nucleotide conjugate, the nucleotide is attached to the particle via a chemical bond or interaction that can be later cleaved. (404) may further include a step of washing or removing unbound reagents including the polymerase and the particle-nucleotide conjugate.

Referring again to FIG. 3, if the nucleotide on the particle-nucleotide conjugate is complementary to the next base of the primed target nucleic acid (305), the particle-nucleotide conjugate, polymerase, and primed target nucleic acid form a ternary binding or incorporation complex, which can be detected by a detection method (e.g., fluorescent imaging) compatible with the label of the particle-nucleotide conjugate. (305) may further include measuring the duration of the ternary binding or incorporation complex. In (306), the polymerase is placed under conditions that make it catalytically active to incorporate the nucleotide. The conditions may include exposing the polymerase to Mg ions 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 becomes unstable. (306) may further include washing or removing the dissociated particle-nucleotide conjugate and/or polymerase. After extension, steps (304), (305) and (306) can be repeated for multiple cycles to determine the sequence of the target nucleic acid.

B. Detection of Target Nucleic Acid Molecules Figures 4A-4B show one exemplary method in which a multivalent binding or incorporation composition is used to detect a target nucleic acid. As shown in Figure 4A, a polymer-nucleotide conjugate (401) is placed in contact with a polymerase (406), a first nucleic acid molecule (404) and a second nucleic acid molecule (405). The polymer-nucleotide conjugate (401) has multiple polymer branches radiating from a core, some of which are attached to nucleotides or oligonucleotides (402) and some of which are attached to labels (403). When the nucleotides or oligonucleotides (402) on the polymer-nucleotide conjugate (401) are complementary to at least a portion of the first nucleic acid (404), a multivalent binding or incorporation complex is formed as shown in Figure B, and a strong binding or incorporation signal serves to detect a target nucleic acid having a sequence complementary or partially complementary to the nucleotides or oligonucleotides on the polymer-nucleotide conjugate. In some examples, at least one of the polymerase, the nucleic acid molecule, and the polymer-nucleotide conjugate is attached to a solid support.

The multivalent binding or incorporation compositions described herein may be used in methods of detecting target nucleic acids in a sample. Additionally disclosed herein is a system configured for performing any of the disclosed nucleic acid analysis methods. The system may include a fluid flow controller and/or fluid distribution system configured to continuously and repeatedly contact a nucleic acid molecule with the disclosed polymerase and multivalent binding or incorporation compositions and/or reagents. The contacting step may be performed in one or more flow cells. In some examples, the flow cell may be a fixed component of the system. In some examples, the flow cell may be a removable and/or disposable component of the system. The system may further include a cartridge including a sample collection unit and an assay assembly, the sample collection unit configured to collect a sample, the assay assembly including at least one reaction site including a multivalent binding or incorporation composition adapted to interact with the analyte, which allows a predetermined portion of the sample to react with an assay reagent included in the assay assembly to generate a signal indicative of the presence of the analyte in the sample, and detecting the signal generated from the analyte.

III. Multivalent Binding or Incorporation Compositions The present disclosure relates to multivalent binding or incorporation compositions having multiple nucleotides conjugated to a particle (e.g., a polymer, a branched polymer, a dendrimer, or an equivalent structure). When the multivalent binding or incorporation composition is contacted with a polymerase and multiple copies of a primed target nucleic acid, a ternary complex is formed that can be detected, which in turn can achieve a more accurate determination of the bases of the target nucleic acid.

When a multivalent binding or incorporation composition is used to replace a single unconjugated or untethered nucleotide to form a complex with a polymerase and one or more copies of a target nucleic acid, the local concentration of the nucleotide and the binding strength of the complex (when a complex containing two or more target nucleic acid molecules is formed) increases many-fold, which in turn enhances the signal strength, particularly the correct signal versus a mismatch. The multivalent binding or incorporation compositions described herein can include at least one particle-nucleotide conjugate (each particle-nucleotide conjugate containing multiple copies of a single nucleotide moiety) to interact with the target nucleic acid. The multivalent composition can further include two, three, or four different particle-nucleotide conjugates, each with a different nucleotide conjugated to the particle.

A multivalent binding composition or incorporation composition may include one, two, three, four, or more species of particle-nucleotide conjugates, where each particle-nucleotide conjugate includes a different species of nucleotide. A first type of particle-nucleotide conjugate may include a nucleotide selected from the group consisting of ATP, ADP, AMP, dATP, dADP, and dAMP. A second type of particle-nucleotide conjugate may include 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 include a nucleotide selected from the group consisting of CTP, CDP, CMP, dCTP, dCDP, and dCMP. A fourth type of particle-nucleotide conjugate may include 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 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 composition or incorporation composition may further comprise one or more labels corresponding to the specific nucleotides conjugated to each of the conjugates. Non-limiting examples of labels include fluorescent labels, colorimetric labels, electrochemical labels (e.g., glucose or other reducing sugars, or thiols or other redox active moieties, etc.), luminescent labels, chemiluminescent labels, spin labels, radioactive labels, stereolabels, affinity tags, etc.

A. Particle-Nucleotide Conjugates In particle-nucleotide conjugates, multiple copies of the same nucleotide may be covalently or non-covalently attached to the particle. Examples of particles may include branched polymers; dendrimers; cross-linked polymer particles such as agarose, polyacrylamide, acrylate, methacrylate, cyanoacrylate, methyl methacrylate particles; glass particles; ceramic particles; metal particles; quantum dots; liposomes; emulsion particles, or other particles known in the art (e.g., nanoparticles, microparticles, etc.). In a preferred embodiment, the particle is a branched polymer.

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

The nucleotide may be linked to the particle by a linker, and the nucleotide may be attached to one end or position of the polymer. The nucleotide may be conjugated to the particle through the 5' end of the nucleotide. In some particle-nucleotide conjugates, one nucleotide is attached to one end or position of the polymer. In some particle-nucleotide conjugates, multiple nucleotides are attached to one end or position of the polymer. The conjugated nucleotide is sterically accessible to one or more proteins, one or more enzymes, and a nucleotide binding or incorporating moiety. In some embodiments, the nucleotide may be provided separately from the nucleotide binding or incorporating moiety, such as a polymerase. In some embodiments, the linker does not include a light releasing or absorbing group.

The particles may further have binding or incorporation moieties. In some embodiments, the particles may self-assemble without the use of separate interacting moieties. In some embodiments, the particles may self-assemble due to buffer or salt conditions, as in the case of, for example, calcium-mediated interactions of hydroxyapatite particles, lipid- or polymer-mediated interactions of micelles or liposomes, or salt-mediated aggregation of metal (such as iron or gold) nanoparticles.

The particle-nucleotide conjugate may have one or more labels. Examples of labels include, but are not limited to, fluorophores, spin labels, metals or metal ions, colorimetric labels, nanoparticles, PET labels, radioactive labels, or polymers, or other such labels that can render the composition detectable by methods known in the art of detecting macromolecules or molecular interactions. Labels may be attached to the nucleotide (e.g., by attachment to the 5' phosphate moiety of the nucleotide), the particle itself (e.g., a PEG subunit), the terminus of the polymer, the central portion, or any other location within the polymer-nucleotide conjugate that will be recognized by those skilled in the art as being sufficient to render 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.

In some embodiments, the label is a fluorophore. Non-limiting examples of fluorescent moieties include fluorescein and derivatives of fluorescein such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-aminofluorescein, rhodamine and TRITC, TMR, Derivatives of rhodamine such as Lissamine rhodamine, Texas Red, Rhodamine B, Rhodamine 6G, Rhodamine 10, NHS-Rhodamine, TMR-Iodoacetamide, Lissamine rhodamine B sulfonyl chloride, Lissamine rhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarins and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE hydrazide, BODIPY and BODIPY BODIPY FL C3-SE, BODIPY C3 530/550, 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, derivatives such as Br-BODIPY 493/503, cascade blue, cascade blue acetyl azide, cascade blue cadaverine, cascade blue blue) ethylenediamine, cascade blue hydrazide, Lucifer Yellow and derivatives such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanine and indolium-based cyanine dyes, benzo-indolium-based cyanine dyes, pyridium-based cyanine dyes, thiozolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy3, Cy5 and derivatives, lanthanide chelates, BCPDA, TBP, TMT, BHHCT, BCOT, europium chelates, terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCycler Red dyes, derivatives such as CALFluor dyes, JOE and its derivatives, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, stilbene, DEG dyes, NR dyes, near infrared dyes Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed. , Plenum Press New York (1999), or Hermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof, or any combination thereof. The cyanine dyes may exist in either sulfonated or non-sulfonated form and may be composed of two indolenine, benzo-indolium, pyridinium, thiozolium, and/or quinolinium groups separated by a polymethine bridge between the 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-indole, or 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]- Cy5 (which may include 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-1,3-diene) 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 include 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 the two indolenine groups. Cy2, which is an oxazole derivative rather than an indolenine, and the benzo-derivatized Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule.

In some embodiments, the detection labels can be FRET-paired, so that multiple classifications can be performed in a single excitation and imaging step. As used herein, FRET can include excitation exchange (Förster) transfer, or electron exchange (Dexter) transfer.

B. Polymer-Nucleotide Conjugates One example of a particle-nucleotide conjugate is a polymer-nucleotide conjugate. Some non-limiting examples of polymer-nucleotide conjugates are shown in Figures 5A-5C. For example, Figure 5A shows polymer-nucleotide conjugates with various configurations, e.g., a "starburst" configuration, including a fluorescently labeled streptavidin core and four nucleotides attached to the core via biotinylated linear PEG linkers of molecular weight ranging from 1K to 10K daltons; Figure 5B shows polymer-nucleotide conjugates with, e.g., a 12, 24, 48, or 96-arm dendrimer core and linear PEG linkers of molecular weight ranging from 1K to 10K daltons radiating from the center; and Figure 5C shows examples of polymer-nucleotide conjugates including, e.g., a network of streptavidin cores linked together by branched PEG linkers that include binding or incorporating moieties such as biotin.

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

Suitable polymers may feature repeating units that incorporate functional groups suitable for derivatization, such as amine, hydroxyl, carbonyl, or allyl groups. Polymers may further have one or more pre-derivatized substituents, such that one or more particular subunits incorporate a derivatization or branching site, regardless of whether other subunits incorporate the same site, substituent, or moiety. Pre-derivatized substituents may include, or may further include, for example, nucleotides, nucleosides, nucleotide analogs, labels such as fluorescent labels, radioactive labels, or spin labels, interactive moieties, additional polymer moieties, etc., or any combination of the foregoing.

In polymer-nucleotide conjugates, the polymer may have multiple branches. Branched polymers can have a variety of configurations, including, but not limited to, star-like ("starburst") morphology, aggregated star-like ("helter skelter") morphology, bottle brush, or dendrimer. Branched polymers can emanate from or incorporate multiple branch points, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more branch points, emanating from a central attachment point or central portion. In some embodiments, each subunit of the polymer may optionally constitute a separate branch point.

The length and size of the branches can vary depending on the type of polymer. In some branched polymers, the length of the branches can be 1-1,000 nm, 1-100 nm, 1-200 nm, 1-300 nm, 1-400 nm, 1-500 nm, 1-600 nm, 1-700 nm, 1-800 nm, or 1-900 nm, or longer, or can be within or between any of the values disclosed herein.

In some polymer-nucleotide conjugates, the polymer core may have a size corresponding to an apparent molecular weight of 1K Da, 2K Da, 3K Da, 4K Da, 5K Da, 10K Da, 15K Da, 20K Da, 30K Da, 50KDa, 80K Da, 100K Da, or any value within the range defined by any two of the foregoing. The apparent molecular weight of the 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 by other methods known in the art.

In some branched polymers, the branches may have a size corresponding to an apparent molecular weight of 1K Da, 2K Da, 3K Da, 4K Da, 5K Da, 10K Da, 15K Da, 20K Da, 30K Da, 50K Da, 80K Da, 100K Da, or any value within the range defined by any two of the foregoing. The apparent molecular weight of the 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 by other methods known in the art. The polymer may have multiple branches. The number of branches in the polymer may be 2, 3, 4, 5, 6, 7, 8, 12, 16, 24, 32, 64, 128 or more, or a number that spans the range defined by any two of these values.

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

In some examples, the length of the linker (e.g., a PEG linker) can range from about 1 nm to about 1,000 nm. In some examples, the length of the linker can 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 examples, the length of the linker can range between any two values in this paragraph. For example, in some examples, the length of the linker can range from about 75 nm to about 400 nm. One of skill in the art will recognize that in some examples, the length of the linker can be any value within the range of this paragraph, for example, 834 nm.

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

In a polymer-nucleotide conjugate, each branch or subset of branches of the polymer has attached thereto a moiety that includes a nucleotide (e.g., an adenine, thymine, uracil, cytosine, or guanine residue or a derivative or mimetic thereof) that is capable of binding to or being incorporated into a polymerase, reverse transcriptase, or other nucleotide binding or incorporation domain. Optionally, the moiety may be capable of incorporation into a growing nucleic acid strand during a polymerase reaction. In some instances, the moiety may be blocked such that it cannot be incorporated into a growing nucleic acid strand during a polymerase reaction. In other instances, the moiety may be reversibly blocked such that it cannot be incorporated into a growing nucleic acid strand during a polymerase reaction until such block is removed, after which the moiety can be incorporated into a growing nucleic acid strand during a polymerase reaction.

The nucleotide may be conjugated to the polymer branch through the 5' end of the nucleotide. In some examples, the nucleotide may be modified to inhibit or prevent incorporation of the nucleotide into a growing nucleic acid chain during a polymerase reaction. By way of example, the nucleotide may comprise a 3' deoxyribonucleotide, a 3' azido nucleotide, a 3'-methyl azido nucleotide, or another such nucleotide known or that may be known in the art, such that it cannot be incorporated into a growing nucleic acid chain during a polymerase reaction. In some embodiments, the nucleotide may comprise a 3'-O-azido group, a 3'-O-azidomethyl group, a 3'-phosphorothioate group, a 3'-O-malonyl group, a 3'-O-alkylhydroxylamino group, or a 3'-O-benzyl group. In some embodiments, the nucleotide lacks a 3' hydroxyl group.

The polymer may further have a binding or incorporation moiety on each branch or a subset of branches. Some examples of binding or incorporation moieties include, but are not limited to, biotin, avidin, streptavidin, etc., polyhistidine domains, complementary pair 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 its derivatives, benzylcysteine and its derivatives, antibodies, epitopes, protein A, protein G. The binding or incorporation moiety may be any interacting molecule or fragment thereof known in the art to promote binding or interaction between proteins, between proteins and ligands, between proteins and nucleic acids, between nucleic acids, or between small molecule interacting domains or moieties.

In some embodiments, the compositions provided herein may include one or more elements of complementary interaction moieties. Non-limiting examples of complementary interaction moieties include, for example, biotin and avidin; SNAP-benzylguanosine; antibodies, or FABs and epitopes; IgG FC and protein A, protein G, protein A/G, or protein L; maltose binding protein and maltose; lectins and cognate polysaccharides; ion chelating moieties, complementary nucleic acids, nucleic acids capable of forming triple helical or triple helical interactions; nucleic acids capable of forming G-quartets, and the like. Those skilled in the art will recognize that many pairs of moieties exist and are commonly used due to their property of interacting strongly and specifically with each other. Thus, it will be readily recognized that such complementary pairs or sets are deemed suitable for this purpose in constructing or envisioning the compositions of the present disclosure. In some embodiments, the compositions disclosed herein may include compositions in which one element of the complementary interaction moiety is attached to one molecule or multivalent ligand and another element of the complementary interaction moiety is attached to a separate molecule or multivalent ligand. In some embodiments, the compositions disclosed herein may include compositions in which both or all elements of the complementary interaction moiety are attached to a single molecule or multivalent ligand. In some embodiments, the compositions disclosed herein may include compositions in which both or all elements of the complementary interaction moiety are attached to separate arms or positions on a single molecule or multivalent ligand. In some embodiments, the compositions disclosed herein may include compositions in which both or all elements of the complementary interaction moiety are attached to the same arm or positions on a single molecule or multivalent ligand. In some embodiments, a composition comprising one element of the complementary interaction moiety and a composition comprising another element of the complementary interaction moiety may be mixed simultaneously or sequentially. In some embodiments, the interaction between the molecules or particles disclosed herein allows for the association or aggregation of multiple molecules or particles, for example, such that the detectable signal is increased. In some embodiments, the fluorescent, colorimetric, or radioactive signal is enhanced. In other embodiments, other interacting moieties disclosed herein or known in the art are contemplated. In some embodiments, the compositions provided herein may be provided in which one or more molecules comprising a first interacting moiety, e.g., one or more imidazole or pyridine moieties, and one or more additional molecules comprising a second interacting moiety, e.g., a histidine residue, are mixed simultaneously or sequentially. In some embodiments, the compositions include one, two, three, four, five, six, or more imidazole or pyridine moieties. In some embodiments, the compositions include one, two, three, four, five, six, or more histidine residues. In such embodiments, the interaction between the molecules or particles provided may be facilitated by the presence of a divalent cation, such as nickel, manganese, magnesium, calcium, strontium, etc. In some embodiments, for example, a (His)3 group may interact with a (His)3 group on another molecule or particle through the coordination of a nickel or manganese ion.

The multivalent binding composition or incorporation composition may include one or more buffers, salts, ions, or additives. In some embodiments, representative additives may include, but are not limited to, betaine, spermidine, surfactants such as Triton X-100, Tween 20, SDS, or NP-40, ethylene glycol, polyethylene glycol, dextran, polyvinyl alcohol, vinyl alcohol, methylcellulose, heparin, heparan sulfate, glycerol, 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 that has the effect of altering the DNA's persistence length so that sites within the chain are more accessible to DNA binding or incorporation moieties, altering the number of conjugated or cross-linked moieties within a polymer, or altering the conformational dynamics of a DNA molecule).

The multivalent binding or incorporation compositions may include zwitterionic compounds as additives. Further representative additives can be found in Lorenz, T. C. J. Vis. Exp. (63), e3998, doi:10.3791/3998 (2012), which is incorporated herein by reference for its disclosure of additives for promoting nucleic acid binding or kinetics, or for promoting the 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 promote nucleic acid interactions, such as self-association, secondary or tertiary structure formation, base pairing, surface association, peptide association, protein binding, etc.

IV. Binding of target nucleic acid to multivalent binding or incorporation compositions When a multivalent binding or incorporation composition is used to replace a single unconjugated or untethered nucleotide to form a complex with a polymerase and one or more copies of a target nucleic acid, the local concentration of the nucleotide as well as the binding strength of the complex (when a complex containing two or more target nucleic acid molecules is formed) increases many-fold, thereby enhancing the signal strength, especially the correct signal versus a mismatch. The present disclosure contemplates contacting a 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.

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

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

Without intending to be bound by a particular theory, the multivalent binding compositions disclosed herein have been observed to associate with polymerase-nucleotide complexes to form ternary binding complexes at a time-dependent rate that is substantially slower than the association rate known to be obtained with nucleotides in free solution. Thus, the on-rate (K _on ) is substantially and surprisingly slower than the on-rate of a single nucleotide, or of a nucleotide not attached to a multivalent ligand complex. Importantly, however, the off-rate (K _off ) of the multivalent ligand complex is substantially slower than that observed for nucleotides in free solution. Thus, the multivalent ligand complexes of the present disclosure provide a surprising and beneficial improvement in the persistence of ternary polymerase-polynucleotide-nucleotide complexes (especially relative to such 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 allows for the controllable formation of visible ternary complexes, such that subsequent visualization, modification, or processing steps can be performed essentially independent of dissociation of the complex; i.e., the complex can be formed, imaged, altered, or otherwise used as desired, and the complex will remain stable until the user performs a positive dissociation step, such as exposing the complex to a dissociation buffer.

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

In various embodiments, polymerases suitable for binding or incorporating interactions as described herein may include any polymerase as such or as may be known in the art. For example, it is known that each organism encodes one or more DNA polymerases within its genome. Examples of suitable polymerases include 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 T4, RB69, and phi29 DNA polymerases, Pyrococcus furiosus DNA polymerase, and the like. These may include, but are not limited to, bacteriophage polymerases such as DNA polymerase (Pfu polymerase); Bacillus subtilis DNA polymerase III, and E. coli DNA polymerase III alpha and epsilon; reverse transcriptases, such as 9th degree N polymerase, HIV type M or O reverse transcriptase, avian myeloblastosis virus reverse transcriptase, or Moloney murine leukemia virus (MMLV) reverse transcriptase, or telomerase. Further non-limiting examples of DNA polymerases may include those from various archaeal genera such as Aeropyrum, Archaeglobus, Desulfurococcus, Pyrobaculum, Pyrococcus, Pyrolobus, Pyrodictium, Staphylothermus, Stetteria, Sulfolobus, Thermococcus, and Vulcanisaeta, or variants thereof, including those polymerases known in the art, such as Vent™, Deep Vent™, Pfu, KOD, Pfx, Therminator™, and Tgo polymerases. In some cases, the polymerase is Klenow polymerase.

Ternary complexes have a longer duration when the nucleotides on the polymer-nucleotide conjugate are complementary to the target nucleic acid than when they are non-complementary nucleotides. Ternary complexes also have a longer duration when the nucleotides on the polymer-nucleotide conjugate are complementary to the target nucleic acid than when they are non-conjugated or tethered complementary nucleotides. For example, in some embodiments, the ternary complexes can have a duration of less than 1 second, more than 1 second, more than 2 seconds, more than 3 seconds, more than 5 seconds, more than 10 seconds, more than 15 seconds, more than 20 seconds, more than 30 seconds, more than 60 seconds, more than 120 seconds, more than 360 seconds, more than 3600 seconds, or more than 50 seconds, or more than 120 seconds, more than 360 seconds, more than 3600 seconds, or more than 120 seconds, or more than 36 ...

The duration can be measured by observing the onset and/or duration of the 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 that includes one or more nucleotides can be present in the binding complex, thus allowing for detection of a signal from the label during the duration of the binding complex.

It has been observed that different ranges of durations are achievable with different salts or ions, for example, complexes formed in the presence of magnesium ions (Mg ²⁺ ) have been shown to form faster than complexes formed with other ions. Furthermore, it has been observed that complexes formed in the presence of strontium ions (Sr ²⁺ ), for example, readily form and completely or substantially completely dissociate upon ion recovery or washing, such as with a buffer lacking one or more components of the composition of the polymer and/or one or more nucleotides and/or one or more interacting moieties, or with a buffer containing a chelating agent that may cause or accelerate removal of divalent cations from the multivalent complex containing the reagent. Thus, in some embodiments, the composition of the present disclosure comprises Mg ²⁺ . In some embodiments, the composition of the present disclosure comprises Ca ²⁺ . In some embodiments, the composition of the present disclosure comprises Sr ²⁺ . In some embodiments, the composition of the present disclosure comprises cobalt ions (Co ²⁺ ). In some embodiments, the composition of the present disclosure comprises MgCl _2. In some embodiments, the composition of the present disclosure comprises CaCl ₂ . In some embodiments, the compositions of the present disclosure include _SrCl2 . In some embodiments, the compositions of the present disclosure include _CoCl2 . In some embodiments, the compositions are free or substantially free of magnesium. In some embodiments, the compositions are free or substantially free of calcium. In some embodiments, the methods of the present disclosure provide for contacting one or more nucleic acids with one or more of the compositions disclosed herein that lack either calcium or magnesium, or that lack both calcium and magnesium.

Dissociation of the ternary complex can be controlled by changing the buffer conditions. After the imaging step, a buffer with increased salt content is used to cause dissociation of the ternary complex so that the labeled polymer-nucleotide conjugate can be washed away, providing a means by which the signal can be attenuated or terminated, such as in the transition between one sequencing cycle and the next. In some embodiments, this dissociation can be affected by washing the complex with a buffer lacking the necessary metal or cofactor. In some embodiments, the wash buffer may include one or more compositions for the purpose of maintaining pH control. In some embodiments, the wash buffer may include one or more monovalent cations, such as sodium. In some embodiments, the wash buffer lacks or is substantially lacking divalent cations, e.g., free or substantially free of strontium, calcium, magnesium, or manganese. In some embodiments, the wash buffer further includes a chelating agent, e.g., EDTA, EGTA, nitrilotriacetic acid, polyhistidine, imidazole, etc. In some embodiments, the wash buffer may maintain the pH of the environment at the same level as for the bound complex. In some embodiments, the wash buffer may raise or lower the pH of the environment relative to the level found in the bound complex. In some embodiments, the pH may be within a range from 2-4, 2-7, 5-8, 7-9, 7-10, or less than 2, or more than 10, or within a range defined by any two of the values provided herein.

The addition of certain ions may affect the binding of the polymerase to a primed target nucleic acid, the formation of a ternary complex, the dissociation of the ternary complex, or the incorporation of one or more nucleotides into the nucleic acid during extension during the polymerase reaction, etc. In some embodiments, the relevant anions may include chloride, acetate, gluconate, sulfate, phosphate, etc. In some embodiments, ions may be incorporated into the compositions of the present disclosure by adding one or more acids, bases, or salts, such as _NiCl2 , _CoCl2 , _MgCl2 , MnCl2, _SrCl2 , _CaCl2 , _CaSO4 , _SrCO3 , _BaCl2 , etc. Representative salts, ions, solutions, and conditions are described in Remington: The Science and Practice of Pharmacy, 20th. Edition, Gennaro, A. R., "The Science and Practice of Pharmacy," _vol . 13, no. 1, pp. 1171-1175, 1999. , Ed. (2000), which is incorporated by reference in its entirety, particularly Chapter 17 and related disclosures of salts, ions, salt solutions, and ionic solutions.

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

Binding of the target nucleic acid to the multivalent binding composition may be provided in the presence of a catalytically inactive polymerase. In one embodiment, the polymerase may be catalytically inactive by mutation. In one embodiment, the polymerase may be catalytically inactive by chemical modification. In some embodiments, the polymerase may be catalytically inactive by the absence of a required substrate, ion, or cofactor. In some embodiments, the polymerase enzyme may be catalytically inactive by the absence of magnesium ions.

The binding of the target nucleic acid to the multivalent binding composition is carried out in the presence of a polymerase where the binding solution, reaction solution, or buffer lacks magnesium or manganese. Alternatively, the binding of the target nucleic acid to the multivalent binding composition is carried out in the presence of a polymerase where the binding solution, reaction solution, or buffer contains calcium or strontium.

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

The step of contacting one or more nucleic acids with a polymer-nucleotide conjugate disclosed herein is carried out in a solution that contains either calcium or magnesium, or both calcium and magnesium. Alternatively, the step of contacting one or more nucleic acids with a polymer-nucleotide conjugate disclosed herein is carried out in a solution that lacks either calcium or magnesium, or both calcium or magnesium, and in a separate step, regardless of the order of steps, includes adding either calcium or magnesium, or both calcium and magnesium to the solution. In some embodiments, the step of contacting one or more nucleic acids with a polymer-nucleotide conjugate disclosed herein is carried out in a solution that lacks strontium, and in a separate step, regardless of the order of steps, includes adding strontium to the solution.

V. Use of Multivalent Binding or Incorporation Compositions in Combination with Low Non-Specific Binding Surfaces Disclosed herein are solid supports that include low non-specific binding surface compositions that allow improved nucleic acid hybridization and amplification performance. In general, the disclosed supports may include a substrate (or support structure), a covalently or non-covalently bound low-binding chemically modified layer, such as a silane layer, a polymer film, and one or more layers of one or more covalently or non-covalently bound primer sequences that can be used to tether single-stranded target nucleic acid to the support surface. In some examples, the formulation of the surface, such as the chemical composition of one or more layers, the coupling chemistry used to crosslink one or more layers to the support surface and/or to each other, and the total number of layers, can be varied to minimize or reduce non-specific binding of proteins, nucleic acid molecules, and other hybridization and amplification reaction components to the support surface compared to a comparable monolayer. In many cases, the formulation of the surface can be varied to minimize or reduce non-specific hybridization at the support surface compared to a comparable monolayer. The surface formulation may be altered so that non-specific amplification on the support surface is minimized or reduced compared to a comparable monolayer. The surface formulation may be altered so that specific amplification rate and/or yield on the support surface is maximized. Amplification levels suitable for detection are achieved at 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30 amplification cycles, depending on the examples disclosed herein.

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

The substrate or support structure may take on any of 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 examples, the substrate or support structure may be locally planar (e.g., including a microscope slide or the surface of a microscope slide). Overall, the substrate or support structure may be cylindrical (e.g., including a capillary or the interior surface of a capillary), spherical (e.g., including the exterior surface of a nonporous bead), or irregular (e.g., including the exterior surface of an irregularly shaped nonporous bead or particle). In some examples, the surface of the substrate or support structure used for nucleic acid hybridization and amplification may be a solid nonporous surface. In some examples, the surface of the substrate or support structure used for nucleic acid hybridization and amplification may be porous such that the coating described herein penetrates the porous surface and the nucleic acid hybridization and amplification reactions carried out therein occur within the pores.

The substrate or support structure including one or more chemically modified layers, e.g., layers of low non-specific binding polymers, may be freestanding or integrated into another structure or assembly. For example, in some instances, the substrate or support structure may include one or more surfaces within an integrated or assembled microfluidic flow cell. The substrate or support structure may include one or more surfaces within a microplate format, e.g., the bottom surface of a well within a microplate. As noted above, in some preferred embodiments, the substrate or support structure includes an interior surface (e.g., luminal surface) of a capillary. In alternative preferred embodiments, the substrate or support structure includes an interior surface (e.g., luminal surface) of a capillary etched into a planar chip.

As noted above, the non-specific low-binding supports of the present disclosure exhibit reduced non-specific binding of proteins, nucleic acids, and other components of hybridization and/or amplification formulations used in solid-phase nucleic acid amplification. The degree of non-specific binding exhibited by a given support surface may be assessed qualitatively or quantitatively. For example, in some instances, exposure of the surface to fluorescent dyes (e.g., cyanine dyes such as Cy3, or Cy5, fluorescein, coumarin, rhodamine, 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 washing protocol and fluorescent imaging may be used as a qualitative tool for comparison of non-specific binding on supports containing various surface formulations. In some examples, exposure of the surface to fluorescent dyes, Cy3, Cy5, etc., fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, and/or fluorescently labeled proteins (e.g., polymerases) under a standardized set of conditions, followed by a specified washing protocol and fluorescent imaging, may be used as a quantification tool for comparison of nonspecific binding on supports containing various surface formulations, provided that care is taken to ensure that the fluorescent signal is linearly related (or related in a predictable manner) to the number of fluorophores on the support surface (e.g., under conditions where signal saturation and/or self-quenching of the fluorophores is not an issue) and that appropriate calibration standards are used. In some examples, other techniques known to those of skill in the art, such as radioisotope labeling and counting, may be used to quantitatively assess the extent to which nonspecific binding is exhibited by the various support surface formulations of the present disclosure.

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

As noted above, in some examples, the degree of non-specific binding exhibited by the disclosed low binding supports may be assessed using a standardized protocol for contacting the surface with a labeled protein (e.g., bovine serum albumin (BSA), streptavidin, DNA polymerase, reverse transcriptase, helicase, single-stranded binding protein (SSB), etc., or any combination thereof) labeled nucleotide, labeled oligonucleotide, etc., under a standardized set of incubation and rinsing conditions, followed by detection of the amount of label remaining on the surface and comparison of the resulting signal therefrom to an appropriate calibration standard. In some examples, the label may include a fluorescent label. In some examples, the label may include a radioisotope. In some examples, the label may include other detectable labels known to those of skill in the art. In some examples, the degree of non-specific binding exhibited by a given support surface formulation may thus be assessed in terms of the number of non-specifically bound protein molecules (or other molecules) per unit area. In some examples, a low binding support of the present disclosure may exhibit ^non -specific protein binding (or non-specific binding of other molecules ^as specified ( ^e.g. , cyanine dyes such as Cy3, ^{or Cy5} , fluorescein, coumarin, rhodamine, ^etc. , or other dyes disclosed herein)) of less than ^0.001 molecules/ ^μm2 , less than 0.01 molecules/ ^μm2 , less than 0.1 molecules/ ^μm2 , less than 0.25 molecules/μm2, less than 0.5 molecules/μm2, less than 1 molecule/μm2, less than 10 molecules/μm2, less than 100 molecules/μm2, or less than 1,000 molecules/μm2. One of skill in the art will understand that a given support surface of the present disclosure may exhibit non-specific binding of any value within this range, for example, less than 86 molecules/ ^μm2 . For example, some modified surfaces disclosed herein exhibit non-specific binding of less than 0.5 molecules/ ^um2 of protein after 15 minutes of contact with a 1 uM solution of Cy3-labeled streptavidin (GE Amersham) in phosphate buffered saline (PBS) buffer, followed by three rinses with deionized water. Some modified surfaces disclosed herein exhibit non-specific binding of less than 0.25 molecules/ ^um2 of Cy3 dye molecules. In an independent nonspecific binding assay, 1 μM labeled Cy3SA (ThermoFisher), 1 μM Cy5SA dye (ThermoFisher), 10 μM aminoallyl-dUTP-ATTO-647N (JenaBiosciences), 10 μM aminoallyl-dUTP-ATTO-Rho11 (JenaBiosciences), 10 μM aminoallyl-dUT P-ATTO-Rho11 (JenaBiosciences), 10 μM 7-propargylamino-7-deaza-dGTP-Cy5 (JenaBiosciences), and 10 μM 7-propargylamino-7-deaza-dGTP-Cy3 (JenaBiosciences) were incubated on low binding substrates in a 384 well plate format for 15 minutes at 37° C. Each well was rinsed 2-3 times with 50 ul deionized RNase/DNase free water and 2-3 times with 25 mM ACES buffer pH 7.4. The 384-well plate was imaged on a GETyphoon instrument using Cy3, AF555, or Cy5 filter sets (depending on the dye test performed) at a PMT sensitivity adjustment of 800 and a resolution of 50-100 μm as specified by the manufacturer. For higher resolution imaging, images were collected with an Olympus IX83 microscope (Olympus Corp., Center Valley, PA) equipped with a total internal reflection fluorescence (TIRF) objective (100X, 1.5 NA, Olympus), a CCD camera (e.g., Olympus EM-CCD monochrome camera, Olympus XM-10 monochrome camera, or Olympus DP80 color and monochrome camera), an illumination source (Olympus 100W Hg lamp, Olympus 75W Xe lamp, Olympus U-HGLGPS fluorescent light source, etc.), and an excitation wavelength of 532 nm or 635 nm. Dichroic mirrors were purchased from Semrock (IDEX Health & Science, LLC, Rochester, New York), e.g., 405, 488, 532, or 633 nm dichroic reflectors/beamsplitters, and bandpass filters were chosen 532LP or 645LP matched to the appropriate excitation wavelength. Some modified surfaces disclosed herein exhibit nonspecific binding of dye molecules of less than 0.25 molecules/um2.

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

A low background surface consistent with the disclosure herein may exhibit a ratio of specific dye attachment (e.g., Cy3 attachment) to non-specific dye adsorption (e.g., Cy3 dye adsorption) 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 greater than 50 specific dye molecules per non-specifically adsorbed molecule. Similarly, a low background surface consistent with the disclosure herein having a fluorophore, e.g., Cy3, attached thereto when exposed to excitation energy may exhibit a ratio of specific fluorescent signal (e.g., arising from Cy3-labeled oligonucleotides attached to the surface) to non-specifically adsorbed dye fluorescent signal 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 greater than 50:1.

In some examples, the degree of hydrophilicity (or "wettability" by aqueous solutions) of the disclosed support surfaces may be assessed through water contact angle measurements, for example, where a small drop of water is placed on the surface and the contact angle with the surface is measured, for example, using an optical tensiometer. In some examples, a static contact angle may be determined. In some examples, an advancing or receding contact angle may be determined. In some examples, the water contact angle of a hydrophilic, low-binding support surface disclosed herein may range from about 0 degrees to about 30 degrees. In some examples, the water contact angle of a hydrophilic, low-binding support surface disclosed herein may be 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 or less. In many examples, the contact angle is 40 degrees or less. One of ordinary skill in the art will understand that a given hydrophilic, low-binding support surface of the present disclosure may exhibit a water contact angle having a value anywhere within this range.

In some examples, the hydrophilic surfaces disclosed herein facilitate reduced wash times for bioassays, often due to reduced non-specific binding of biomolecules to the low-binding surface. In some examples, a suitable wash step may be performed in less than 60, 50, 40, 30, 20, 15, 10, or less than 10 seconds. For example, in some examples, a suitable wash step may be performed in less than 30 seconds.

Some low-binding surfaces of the present disclosure exhibit significantly improved stability or durability to long-term exposure to solvents and elevated temperatures, or repeated cycles of solvent exposure or temperature changes. For example, in some examples, the stability of the disclosed surfaces may be tested by fluorescently labeling functional groups on the surface, or tethered biomolecules (e.g., oligonucleotide primers) on the surface, and monitoring the fluorescent signal before, during, and after long-term exposure to solvents and elevated temperatures, or repeated cycles of solvent exposure or temperature changes. In some examples, 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% (or any combination of these percentages measured over these time periods) over 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 the solvent and/or elevated temperature. In some examples, 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% (or any combination of these percentages measured over this range of cycles) over 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 1,000 cycles of repeated exposure to solvent changes and/or temperature changes.

In some examples, 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 100 times greater than the signal of adjacent unpopulated areas of the surface. Similarly, 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 100 times greater than the signal of adjacent increased nucleic acid population areas of the surface.

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

One or more primers may be attached or tethered to the support surface. In some examples, one or more adapters, or primers, may include a spacer sequence, an adapter sequence for hybridization to an adapter-linked target library nucleic acid sequence, a forward amplification primer, a reverse amplification primer, a sequencing primer, and/or a molecular barcode sequence, or any combination thereof. In some examples, one primer or adapter sequence may be tethered to at least one layer of the surface. In some examples, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different primer or adapter sequences may be tethered to at least one layer of the surface.

In some examples, the length of the tethered adaptor and/or primer sequence may range from about 10 nucleotides to about 100 nucleotides. In some examples, the length of the tethered adaptor and/or primer sequence 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. In some examples, the length of the tethered adaptor and/or primer sequence may be up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, or up to 10 nucleotides. Any of the lower and upper limits described in this paragraph may be combined to form a range included in the disclosure, for example, in some examples, the length of the tethered adaptor and/or primer sequence may range from about 20 nucleotides to about 80 nucleotides. One of skill in the art will recognize that the length of the tethered adaptor and/or primer sequence may have any value within this range, for example, about 24 nucleotides.

In some examples, the resulting surface density of primers on a low binding support surface of the present disclosure may range from about 100 primer molecules/μm ² to about 100,000 primer molecules/μm ^2. In some examples, the resulting surface density of primers on a low binding support surface of the present disclosure may range from about 1000 primer molecules/μm ² to about 1,000,000 primer molecules/μm ^2. In some examples, the surface density of primers may be at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000 molecules/μm ^2. In some examples, the surface density of primers may be up to 1,000,000, up to 100,000, up to 10,000, or up to 1,000 molecules/μm ² . Any of the lower and upper limits listed in this paragraph may be combined to form a range within the present disclosure, for example, in some examples, the surface density of primers may range from about 10,000 molecules/μm ² to about 100,000 molecules. One of skill in the art will recognize that the surface density of primer molecules may have any value within this range, for example, about 455,000 molecules/μm ^2. In some examples, the surface density of target library nucleic acid sequences initially hybridized to adapter or primer sequences on the support surface may be equal to or less than that indicated for the surface density of tethered primers. In some examples, the surface density of clonally amplified target library nucleic acid sequences hybridized to adapter or primer sequences on the support surface may range to the same range as that indicated for the surface density of tethered primers.

The above local densities do not exclude variations in density across a surface, so that a surface may include a region having, for example, an oligo density of 500,000/ ^μm2 , while also including at least a second region having a substantially different local density.

VI. Exemplary Alternative Embodiments The disclosed methods of determining the sequence of a target nucleic acid include a) contacting a double-stranded or partially double-stranded target nucleic acid molecule, comprising a template strand to be sequenced and a primer strand to be extended, with one or more of the disclosed nucleic acid binding compositions, and b) detecting binding of the nucleic acid binding compositions 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.

The sequencing method may further include 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. After detecting the ternary binding complex, the primed strand of the primed target nucleic acid is extended by one base before the next round of analysis is performed. The primed target nucleic acid may be extended using conjugated nucleotides attached to the polymer in the multivalent binding composition, or using unconjugated or untethered free nucleotides provided after the multivalent binding composition is removed.

Extension of a primed target nucleic acid can be prevented or inhibited due to the use of a blocked nucleotide on the strand or a catalytically inactive polymerase. If the nucleotide in the polymer-nucleotide conjugate has a blocking group that prevents extension of the nucleic acid, incorporation of the nucleotide can be achieved by removal of the blocking group from the nucleotide (e.g., by detachment of the nucleotide from the polymer, branched polymer, dendrimer, particle, etc.). If extension of a primed target nucleic acid is inhibited due to the use of a catalytically inactive polymerase, incorporation of the nucleotide can be achieved by the provision of a cofactor or activator, such as a metal ion.

Detection of the ternary complex is accomplished prior to, simultaneously with, or after incorporation of the nucleotide residue. In some embodiments, the primed target nucleic acid may include a target nucleic acid having multiple primed positions for attachment of a polymerase and/or a nucleic acid binding moiety. In some embodiments, multiple polymerases may attach to a single target nucleic acid molecule, such as multiple sites within a target nucleic acid molecule. In some embodiments, multiple polymerases may be bound to a multivalent binding composition disclosed herein that includes multiple nucleotides. In some embodiments, the target nucleic acid molecule may be the product of strand displacement synthesis, rolling circle amplification, ligation or fusion of multiple copies of a query sequence, or other methods for producing nucleic acid molecules that include multiple copies of the same sequence known in the art or disclosed elsewhere herein. Thus, in some embodiments, multiple polymerases may attach to multiple identical or substantially identical positions within a target nucleic acid that includes multiple identical or substantially identical copies of a query sequence. In some embodiments, the multiple polymerases may then engage in an interaction with one or more multivalent binding compositions. However, in preferred embodiments, the number of binding sites in the target nucleic acid is at least two, and the number of nucleotide or substrate moieties present on a particle-nucleotide conjugate, such as a polymer-nucleotide conjugate, is also two or more.

It may be advantageous to provide the multivalent binding compositions in combination with other elements to provide an optimized signal, e.g., to provide identification of nucleotides at specific positions in a nucleic acid sequence. In some embodiments, the compositions disclosed herein are provided in combination with a surface that provides low background binding or low levels of protein binding, particularly a hydrophilic or polymer-coated surface. Exemplary surfaces can be found, for example, in U.S. Patent Application Serial No. 16/363,842, the contents of which are incorporated herein by reference in their entirety.

In some examples, the nucleic acid molecule is tethered to the surface of a solid support, e.g., via hybridization of the template strand to an adapter or primer nucleic acid sequence that is tethered to the solid support. In some examples, the solid support comprises a glass, fused silica, silicon, or polymer substrate. In some examples, the solid support comprises a low non-specific binding coating comprising one or more hydrophilic polymer layers (e.g., PEG layers), where 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).

The solid support comprises oligonucleotide adaptors or primers tethered to at least one hydrophilic polymer layer at a surface density ranging from about 1,000 primer molecules/μm ² to about 1,000,000 primer molecules/μm ^2. In some examples, the surface density of oligonucleotide primers may be at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000 molecules/μm ^2. In some examples, the surface density of oligonucleotide primers may be up to 1,000,000, up to 100,000, up to 10,000, or up to 1,000 molecules/μm ^2. Any of the lower and upper limits listed in this paragraph may be combined to form ranges within the disclosure, for example, in some examples, the surface density of primers may range from about 10,000 molecules/μm ² to about 100,000 molecules/μm ² . One of ordinary skill in the art will recognize that the areal density of primer molecules may have any value within this range, for example, about 455,000 molecules/ ^μm2 .

Those skilled in the art will recognize that in a series of iterative sequencing reactions, one or more sites will fail to incorporate a nucleotide during a given cycle, and thus one or more sites will be out of sync with the majority of the growing nucleic acid strand. Under conditions where the sequencing signal is derived from a reaction that occurs with a single copy of the target nucleic acid, these failures to incorporate will also produce individual errors in the output sequence. The purpose of this disclosure is to describe a method for reducing this type of error in a sequencing reaction. The use of multivalent substrates that can be incorporated into the growing strand by increasing the probability of reassociation upon premature dissociation of the ternary polymerase complex can 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 as 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 the nucleotide is connected to a particle or polymer as disclosed herein via a labile or linkage. In some embodiments, the present disclosure contemplates a reduction in the intrinsic error rate due to skipped incorporation as a result of the use of multivalent substrates disclosed herein. The present disclosure contemplates a reduction in the intrinsic error rate due to skipped incorporation as a result of the use of multivalent substrates disclosed herein.

The present disclosure further contemplates sequencing reactions in which the sequencing signal derived from or associated with a given sequence originates from or occurs within a definable region that contains multiple copies of the target sequence. Sequencing methods that incorporate multiple copies of the target sequence have the advantage that the signal can be amplified since there are multiple simultaneous sequencing reactions within a defined region, each providing its own signal. The presence of multiple signals within a defined region also mitigates the impact of a single skipped cycle, since the signal from a large number of correct base calls can overwhelm the signal from a small number of skipped or incorrect base calls. The present disclosure further contemplates the inclusion of free unlabeled nucleotides during the extension reaction, or during a separate portion of the extension cycle, to provide for incorporation at sites that may have been skipped in the previous cycle. For example, unlabeled blocked nucleotides can be added during or after the incorporation cycle so that they can be incorporated at skipped sites. The unlabeled blocked nucleotides may be of the same species as the multivalently bound substrate or nucleotides attached to the substrate that are or were present during a particular cycle, or may include a mixture of one, two, three, four or more species of unlabeled blocked nucleotides.

When each sequencing cycle has progressed to completion, each reaction within a defined region provides an identical signal. However, as described elsewhere herein, in a series of iterative sequencing reactions, occasionally one or more sites fail to incorporate a nucleotide during a given cycle, and thus one or more sites become out of sync with the majority of the growing nucleic acid strand. This problem, called "phasing," causes degradation of the sequencing signal because the signal is contaminated with spurious signals from sites that have skipped one or more cycles. This can result in errors in base identification. The gradual accumulation of skipped cycles over multiple cycles further reduces the effective read length due to the gradual degradation of the sequencing signal with each cycle. It is a further object of the present disclosure to provide a method for reducing phasing errors and/or improving read length in sequencing reactions.

A sequencing method may include contacting a target nucleic acid or a plurality of target nucleic acids, including multiple linked or unlinked copies of a target sequence, with a multivalent binding composition as described herein. Contacting the target nucleic acid or a plurality of target nucleic acids, including 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 being interrogated in a given sequencing cycle, thereby suppressing signal from improperly incorporated or phased nucleic acid strands (i.e., an elongating nucleic acid strand having one or more skipped cycles).

A method of obtaining nucleic acid sequence information can include contacting a target nucleic acid or a plurality of target nucleic acids with a particle-nucleotide conjugate, wherein the target nucleic acid or a plurality of target nucleic acids includes multiple linked or unlinked copies of a target sequence. In this method, the sequencing error rate is reduced, as indicated by a reduction in misidentification of bases, reporting of non-existent bases, or failure to report correct bases. In some embodiments, the reduction in the sequencing error rate can include a reduction of 5%, 10%, 15%, 20% 25%, 50%, 75%, 100%, 150%, 200%, or more compared to the error rate observed using a monovalent ligand including free nucleotides, labeled free nucleotides, protein or peptide bound nucleotides, or labeled protein or peptide bound nucleotides.

A method for obtaining nucleic acid sequence information can include contacting a target nucleic acid or a plurality of target nucleic acids with one or more particle-nucleotide conjugates, wherein the template nucleic acid or a plurality of target nucleic acids comprises a plurality of linked or unlinked copies of a target sequence. In this method, the average read length is increased by 5%, 10%, 15%, 20% 25%, 50%, 75%, 100%, 150%, 200%, 300%, or more compared to the average read length observed using a monovalent ligand comprising free nucleotides, labeled free nucleotides, protein or peptide-bound nucleotides, or labeled protein or peptide-bound nucleotides.

In this method, the average read length is increased by 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 a monovalent ligand comprising free nucleotides, labeled free nucleotides, protein or peptide-bound nucleotides, or labeled protein or peptide-bound nucleotides.

In some examples, the disclosed compositions and methods can provide average read lengths ranging from 100 nucleotides to 1,000 nucleotides for sequencing applications. In some examples, 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 1,000 nucleotides. In some instances, the average read length may be in a range bounded by any two values within this range, e.g., an average read length ranging from 375 nucleotides to 825 nucleotides. One of skill in the art will recognize that in some instances, the average read length may be any value within the range specified in this paragraph, e.g., 523 nucleotides.

The use of multivalent binding compositions for sequencing effectively reduces sequencing time. A sequencing reaction cycle, including the contacting, detecting, and incorporating steps, is carried out for a total time ranging from about 5 minutes to about 60 minutes. In some examples, the sequencing reaction cycle is carried out for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes. In some examples, the sequencing reaction cycle is carried out for up to 60 minutes, up to 50 minutes, up to 40 minutes, up to 30 minutes, up to 20 minutes, up to 10 minutes, or up to 5 minutes. Any of the lower and upper limits described in this paragraph may be combined to form ranges included in the present disclosure, for example, in some examples, the sequencing reaction cycle is carried out for a total time ranging from about 10 minutes to about 30 minutes. One of skill in the art will recognize that the sequencing cycle time may be any value within this range, for example, about 16 minutes.

In some examples, the disclosed compositions and methods for nucleic acid sequencing will provide an average base call 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% accurate over the course of a sequencing run. In some examples, the disclosed compositions and methods for nucleic acid sequencing will provide an average base call 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% accurate for every 1,000 bases, every 10,0000 bases, every 25,000 bases, every 50,000 bases, every 75,000 bases, or every 100,000 bases called.

The use of multivalent binding compositions for sequencing provides a more accurate base readout. The disclosed compositions and methods for nucleic acid sequencing will provide an average Q score ranging from about 20 to about 50 in base calling accuracy across a sequencing run. In some examples, the average Q score is at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50. One of skill in the art will recognize that the average Q score can be any value within this range, for example, about 32.

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

The disclosed non-specific low binding supports and related nucleic acid hybridization and amplification methods may be used to analyze nucleic acid molecules derived from any of a variety of cells, tissues, or samples known to those of skill in the art. For example, nucleic acids may be extracted from cells, or tissue samples containing one or more types of cells, derived from eukaryotes (such as animals, plants, fungi, protists), archaea, or eubacteria. In some cases, nucleic acids may be extracted from eukaryotic cells, such as prokaryotes or adherent or non-adherent eukaryotic cells. Nucleic acids may be variously extracted from, for example, primordial or immortalized rodent, porcine, feline, canine, bovine, equine, primate, or human cell lines. Nucleic acids may be extracted from any of a variety of cells, organs or tissues, such as 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 of the heart, lung, brain, liver, kidney, spleen, pancreas, thymus, bladder, stomach, colon or small intestine. Nucleic acids may be extracted from normal or healthy cells. Alternatively or in combination, nucleic acids are extracted from abnormal cells, such as cancer cells, or pathogenic cells infecting the host. Some nucleic acids may be extracted from distinct subsets of cell types, for example immune cells (such as T cells, cytotoxic (killer) T cells, helper T cells, αβ T cells, γδ T cells, T cell precursors, B cells, B cell precursors, lymphoid stem cells, myeloid precursors, 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, progenitor cells, foam cells, mesenchymal cells, or trophoblast cells). Nucleic acids may further include nucleic acids derived from viral samples and subviral pathogens such as viroids and infectious RNA. Nucleic acids may be derived from clinical or other samples such as those from saliva, sputum, ocular fluid, synovial fluid, blood, feces, urine, tissue exudates, sweat, pus, drainage fluids, and the like. Nucleic acids may further be derived from plant or fungal samples such as leaves, cambium, roots, meristems, pollen, ova, seeds, spores, inflorescences, mycelium, and the like. Nucleic acids may also be derived from environmental or industrial samples such as water, air, dust, food, and the like. Other cells, tissues, and samples are contemplated and consistent with the disclosure herein.

Nucleic acid extraction from cells or other biological samples may be performed using any of a number of techniques known to those of skill in the art. For example, a DNA extraction procedure may include (i) collection of a cell or tissue sample from which DNA is to be extracted, (ii) disruption of cell membranes to release DNA and other cytoplasmic components (i.e., cell lysis), (iii) treatment of 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) purification of the DNA from the supernatant to remove detergents, proteins, salts, or other reagents used during the cell membrane lysis step.

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

VII. Systems System Modules: As noted above, further disclosed herein are systems configured to perform any of the disclosed nucleic acid sequencing or nucleic acid detection and analysis methods. In some examples, the disclosed systems may include one or more multivalent binding compositions described herein, one or more buffers, and/or one or more nucleic acid molecules tethered to a solid support.

In some examples, the system may further include a fluid flow controller and/or a fluid distribution system configured to continuously and repeatedly contact a template nucleic acid molecule hybridized to a nucleic acid molecule (e.g., an adaptor or primer) tethered to a solid support with the disclosed multivalent binding compositions and/or reagents. In some examples, the contacting may be performed in one or more flow cells. In some examples, the flow cells may be a fixed component of the system. In some examples, the flow cells may be a removable and/or disposable component of the system.

In some examples, the system may further include an imaging module, where the imaging module includes, for example, one or more light sources, one or more optical components (e.g., lenses, mirrors, prisms, optical filters, colored glass filters, narrow band interference filters, broad band interference filters, dichroic reflectors, diffraction gratings, apertures, optical fibers, or optical waveguides, etc.), and one or more image sensors (e.g., a charge-coupled device (CCD) sensor or camera, a complementary metal-oxide-semiconductor (CMOS) image sensor or camera, or a negative channel metal oxide semiconductor (NMOS) image sensor or camera) for imaging and detecting binding of the disclosed multivalent binding compositions to target (or template) nucleic acid molecules tethered within a solid support or flow cell.

Processors and Computer Systems: One or more processors can be used to implement the systems for nucleic acid sequencing or other nucleic acid detection and analysis methods disclosed herein. The one or more processors may include hardware processors such as a central processing unit (CPU), a graphics processing unit (GPU), a general-purpose processing unit, or a computing platform. The one or more processors may be comprised of any of a variety of suitable integrated circuits (e.g., 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 to facilitate deployment), microprocessors, emerging next generation microprocessor designs (e.g., memristor-based processors), logic devices, and the like. Although the present disclosure is described with reference to processors, other types of integrated circuits and logic devices may also be applicable. The processor may have any suitable data manipulation capabilities. For example, the processor may perform 512-bit, 256-bit, 128-bit, 64-bit, 32-bit, or 16-bit data operations. The one or more processors may be single-core or multi-core processors, or multiple processors configured for parallel processing.

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

A computer system may also include memory or storage locations (e.g., random access memory, read-only memory, flash memory, Intel® Optane™ technology), electronic storage (e.g., hard disks), communications interfaces (e.g., network adapters) for communicating with one or more other systems, and peripherals, e.g., cache, other memory, data storage devices, and/or electronic display adapters. The memory, storage devices, interfaces, and peripherals may be in communication with one or more processors, e.g., CPUs, via a communications bus, e.g., as found on a motherboard. The storage devices may be data storage devices (or data repositories) for storing data.

One or more processors, e.g., a CPU, execute a sequence of machine-readable instructions that are embodied in a program (or software). The instructions are stored in a memory location. The instructions are targeted to the CPU, which then programs or otherwise configures the CPU to implement the methods of the present disclosure. Examples of operations performed by the CPU include fetch, decode, execute, and writeback. The CPU may be part of a circuit, such as an integrated circuit. One or more other components of the system may be included in the circuit. In some cases, the circuit is an application-specific integrated circuit (ASIC).

The storage device stores files such as drivers, libraries, and saved programs. The storage device stores user data, such as user-specific preferences and user-specific programs. The computer system may optionally include one or more additional data storage devices that are external to the computer system, such as located on a remote server in communication with the computer system over an intranet or the Internet.

Some aspects of the methods and systems described herein may be performed by machine (e.g., processor) executable code stored in an electronic storage location of a computer system, such as, for example, a 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 one or more processors. In some embodiments, the code is retrieved from storage and stored in memory for ready use by one or more processors. In some cases, electronic storage is omitted and machine readable instructions are stored in memory. The code may be pre-compiled and configured for use on a machine with one or more processors adapted to execute the code, or may be compiled at run-time. The code may be provided in a programming language that may be selected to enable the code to be executed in a pre-compiled or as-compiled fashion.

Various aspects of the technology can be considered as "products" or "articles of manufacture", e.g., "computer program or software products", often in the form of machine (or processor) executable code and/or associated data stored on some type of machine-readable medium, where 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. The machine-executable code may be stored on optical storage devices, including optically readable media such as optical disks, CD-ROMs, DVDs, or Blu-ray disks. The machine-executable code may be stored in electronic storage devices, such as memory (e.g., read-only memory, random access memory, flash memory) or on a hard disk. A "storage" type medium can include any or all of the tangible memory of a computer or processor, or associated modules thereof, such as various semiconductor memory chips, optical drives, tape drives, disk drives, etc., which may provide non-transitory storage at any time for the software encoding the methods and algorithms disclosed herein.

All or a portion of the software code is sometimes communicated over the Internet or various other telecommunications networks. Such communication allows, for example, loading of the software from one computer or processor to another, for example, from a management server or host computer to a computer platform of an application server. Thus, other types of media used to convey software-coded instructions include light waves, radio waves, and electromagnetic waves, such as those used at physical interfaces between local devices, through wired and optical landline networks, and by various atmospheric links. Physical elements involving such waves, such as wired or wireless links, optical links, etc., are also considered media that convey software-coded instructions for carrying out 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 media involved in providing instructions to a processor for execution.

The computer system may include or be in communication with, for example, an electronic display for providing images captured by the machine vision system. In many cases, the display may provide a user interface (UI). Examples of UIs include, but are not limited to, graphical user interfaces (GUIs), web-based user interfaces, etc.

System Control Software: In some examples, the disclosed systems may include a computer (or processor) and a computer readable medium containing code for providing a user interface for manual, semi-automated, or fully automated control of all system functions, e.g., fluid flow controllers and/or control of the fluid distribution system (or subsystem), temperature control system (or subsystem), imaging system (or subsystem), etc. In some examples, the system computer or processor may be an integrated component of the instrument system (e.g., a microprocessor or motherboard embedded within the instrument). In some examples, the system computer or processor may be a stand-alone module, e.g., 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 rates, timing and duration of sample and reagent additions, rinse steps, etc. Examples of temperature control functions that may be provided by the instrument control software include, but are not limited to, specification of temperature set points, and control of the timing, duration, and ramp rates of temperature changes. Examples of imaging system control functions that may be provided by the instrument control software include, but are not limited to, autofocus functions, control of illumination or excitation light exposure time and intensity, control of image acquisition speed, exposure times, data storage options, etc.

Image Processing Software: In some examples of the disclosed systems, the system may further include a computer readable medium containing code for providing image processing and analysis functions. Examples of image processing and analysis functions that may be provided by the software include, but are not limited to, manual, semi-automated, or fully automated image exposure adjustment (e.g., white balance, contrast adjustment, signal averaging and other noise reduction functions, etc.), manual, semi-automated, or fully automated edge detection and object identification (e.g., to identify clusters of amplified template nucleic acid molecules on the substrate surface), manual, semi-automated, or fully automated signal intensity measurement and/or thresholding in one or more detection channels (e.g., one or more fluorescent emission channels), and manual, semi-automated, or fully automated statistical analysis (e.g., to compare signal intensities to reference values for base calling purposes).

In some examples, 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 may be extended, modified, or repeated as necessary, for example, until optimal base calling results are obtained. Any of 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 functions. Examples include, but are not limited to, Canny edge detection methods, Canny-Deriche edge detection methods, first-order gradient edge detection methods (such as the Sobel operator), second-order derivative edge detection methods, phase-matching (phase coherence) edge detection methods, other image segmentation algorithms (e.g., intensity threshold, intensity clustering methods, intensity histogram-based methods, etc.), feature and pattern recognition algorithms (e.g., generalized Hough transform for detecting arbitrary shapes, circular Hough transform, etc.), and mathematical analysis algorithms (e.g., Fourier transform, fast Fourier transform, wavelet analysis, autocorrelation, etc.), or combinations thereof.

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

VIII. Example 1. Preparation of Multivalent Binding Compositions As shown in FIG. 5A, one type of multi-arm substrate was made by reacting propargylamine dNTP with biotin-PEG-NHS. This aqueous reaction was driven to completion and purified to yield pure biotin-PEG-dNTP species. In separate reactions, several different PEG lengths were used corresponding to average molecular weights ranging from 1K Da to 20K Da. 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. The complete complex was purified from excess biotin-PEG-dNTPs by size-exclusion chromatography. Each nucleotide species was varied and purified individually, then mixed together to create a four-base mixture for sequencing.

Another species of multi-arm substrate, as shown in Figure 5A, was made in a single pot by reacting multi-arm PEG NHS with excess Dye-NH2 and propargylamine dNTPs. Various multi-arm PEG NHS variants were used ranging from 4 to 16 arms and with molecular weights ranging from 5K Da to 40K Da. After the reaction, excess small molecule dyes and dNTPs were removed by size exclusion chromatography. Each nucleotide species was independently conjugated and purified, then mixed together to create a four-base mixture for sequencing.

Class II substrates as shown in Figure 5B were made using a one-pot reaction to conjugate dyes and dNTPs simultaneously. Alkyne-PEG-NHS was reacted with excess propargylamine dNTPs. This product (alkyne-PEG-dNTPs) was then purified to homogeneity by chromatography. Multiple PEG lengths were used with average molecular weights varying between 1K Da and 20K Da. Dendrimer cores containing variable and discrete numbers of azide conjugation sites (12, 24, 48, 96) were used. Conjugation of alkyne dyes and alkyne-PEG-dNTPs to the dendrimer cores occurred in a one-pot reaction containing excess dye and dNTP species via copper-mediated click chemistry. After reaction, excess small molecule dyes and dNTPs were removed by size exclusion chromatography. Each nucleotide species was independently conjugated and purified and then mixed together to create a four-base mixture for sequencing. Note that this scheme allows for ready substitution of alternative cores such as dextrans, other polymers, proteins, etc.

Class III polymer-nucleotide conjugates, as shown in FIG. 5C, were constructed by reacting 4- or 8-arm PEG NHS with a saturated mixture of biotin and propargylamine dNTPs. The 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 nucleotides. This heterogeneous population was then reacted with dye-labeled streptavidin and purified by size-exclusion chromatography. Each nucleotide type was independently conjugated and purified, then mixed together to create a 4-base mixture for sequencing. Note that the distribution of biotin can be tuned by the input ratio of biotin- _NH2 to propargylamine dNTPs.

2. Detection of Ternary Complexes Binding reactions using multivalent binding compositions with PEG polymer-nucleotide conjugates were analyzed to detect possible formation of ternary binding complexes, and fluorescence images of the various steps are illustrated in Figures 7A-7J. In Figure 7A, red and green fluorescence images are shown following exposure of a DNA rolling circle application (RCA) template (G and A first bases) to 500 nM base-labeled nucleotides (A-Cy3 and G-Cy5) in an exposure buffer containing 20 nM Klenow polymerase and 2.5 mM Sr ⁺² . Multivalent 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 collected after washing with imaging buffer of the same composition as the exposure buffer but without nucleotide or polymerase.

Contrast was scaled to maximize visualization of the faintest signal, but after washing with imaging buffer, the signal did not persist (inset in Figure 7). In Figures 7B-7E, fluorescence images show multivalent PEG-nucleotide (base-labeled) ligands at least 500 nM after mixing in exposure buffer and imaging in imaging buffer as above (Figure 7B: PB1, Figure 7C: PB2, Figure 7D: PB3, Figure 7E: PB5). Figure 7F: Fluorescence image shows multivalent PEG-nucleotide (base-labeled) ligand PB5 at 2.5 uM after mixing in exposure buffer and imaging in imaging buffer. In Figures 7G-7I, the fluorescence images further demonstrate base discrimination upon exposure of multivalent ligands to inactive mutants of Klenow polymerase (Figure 7G: D882H, Figure 7H: D882E, Figure 7I: D882A, and the wild-type Klenow (control) enzyme is shown in Figure 7J).

Using multivalent ligand formulations, base discrimination may be possible by providing polymerase-ligand interactions with increased avidity. Furthermore, it has been shown that increased concentrations of multivalent ligands produce higher signals, and various Klenow mutations that knock out catalytic activity can be used for avidity-based sequencing.

3. Sequencing of Target Nucleic Acid Molecules Using Ternary Complexes To demonstrate sequencing based on the multivalent ligand reporter, four known templates were amplified using the RCA method on a low binding substrate. Successive cycles were exposed to exposure buffer containing 20 nM Klenow polymerase and 2.5 mM Sr ⁺² , washed with imaging buffer, and imaged. After imaging, the substrate was washed with wash buffer (EDTA and high salt) and a blocked nucleotide was added to proceed to the next base. The cycle was repeated for five cycles. Spots were detected using standard imaging procedures and spot detection, and sequences were called using a red and green dual color scheme (G-Cy3 and A-Cy5) to identify the templates being cycled. As shown in Figures 8A and 8B, the multivalent ligand is able to provide base discrimination throughout all five sequencing cycles.

4. Control of Nucleotide Dissociation from the Ternary Complex Ternary complexes are prepared and imaged as in Example 2. The complexes are imaged for various times, e.g., as long as 60 seconds, to demonstrate the persistence of the ternary complex. After time, the complex is washed with the same buffer used to form it, only lacking any divalent cations, e.g., 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 0.016% TritonX100 (no SrOAc), or the complex is washed with the same buffer used to form it, containing the chelator but lacking any divalent cations, e.g., 100 nm-100 mM EDTA, 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 0.016% TritonX100 (no SrOAc). The fluorescence of the complex is observed over time allowing the observation and quantification of the dissociation of the ternary complex. A representative time course of this dissolution is shown in FIG. 6.

5. Extending the Complementary Sequence of the Target Nucleic Acid After preparing, imaging, and dissociating the ternary complex as in Example 4, a deblocking solution is flowed from the 3' end of the extending DNA strand into a chamber containing the bound DNA molecules, such as an O-azidomethyl group, an O-alkylhydroxylamino group, or an O-amino group, sufficient to remove the blocking moiety. Subsequently or simultaneously, an extension solution is flowed into the chamber containing the bound DNA molecules. The extension solution contains a buffer, sufficient divalent cations to support polymerase activity, active polymerase, and appropriate amounts of all four nucleotides, where the nucleotides are blocked such that they are unable to support further extension after a single nucleotide is added to the extending DNA strand, such as by incorporation of a 3'-O-azidomethyl group, a 3'-O-alkylhydroxylamino group, or a 3'-O-amino group. The growing strand is thus extended by one and only one base, the catalytically inactive polymerase, and the binding of the multivalent binding substrate can be used to call the next base in the cycle.

Alternatively, the nucleotides attached to the multivalent substrate can be attached through labile bonds, so that a buffer can flow into the chamber containing the bound DNA molecules containing enough divalent cations or other cofactors to render the polymerase catalytically active. Before, after, or simultaneously, conditions can be provided that are sufficient to cleave a base from the multivalent substrate so that it can be incorporated into the growing strand. This cleavage and incorporation results in labeling of the multivalent substrate and dissociation of the polymer backbone while extending the growing DNA strand by just one base. A wash is performed to remove the used polymer backbone, and a new multivalent substrate flows into the chamber containing the bound DNA molecules, allowing new bases to be called as in Example 1.

6. Use of Polymer-Nucleotide Conjugates with Varying PEG Branch Lengths Polymer-nucleotide conjugates with various PEG arm lengths as described in Example 3 were subjected to a single sequencing cycle and imaged as described in Example 1. As shown in Figures 9A-J, increasing the length of the PEG branches increased the signal up to a length corresponding to an apparent average PEG MW of 5K Da (Figures 9A-D). Use of longer PEG arms caused a decrease in the fluorescent signal of both Cy3-A and Cy5-G (Figures 9E-9G). Quantitative measures of signal intensity are shown graphically in Figure 10.

7. Enhancement of Binding of Multivalent Substrates by Addition of Detergent Multivalent substrates were prepared and assembled into binding complexes in the presence and absence of detergent. One set used 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 5 mM SroAc, 0% TritonX100 (condition A), and the other set used 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 5 mM SroAc, 0.016% TritonX100. Figure 11 shows the normalized fluorescence of these multivalent substrates bound to DNA clusters, and substrate complexes formed in the presence of Triton-X100 (0.016%) (condition B) clearly show enhanced fluorescence intensity.

8. Assessment of the Time Course of Multivalent Substrate Binding Multivalent substrates were prepared and assembled into binding complexes as in Example 2. Complexes were also formed under identical buffer conditions using free labeled nucleotides. The complexes were imaged over a 60 minute period to characterize the duration of the complexes. Figures 12A-B show representative results. The multivalent binding complexes are stable over time scales greater than 60 minutes (Figure 12B), while the labeled free nucleotides dissociate in less than 1 minute (Figure 12A).

VIII. CONCLUSION The present disclosure provides significantly improved methods and compositions for DNA sequencing and biosensor applications. It should be understood that the above features are intended to be illustrative and not limiting. Many embodiments will be apparent to those of skill in the art upon review of the above description. By way of example, while the inventive concepts have been described primarily with respect to the use of polymer-nucleotide conjugates, it will be readily recognized by those of skill in the art that other types of particle-nucleotide conjugates can be used. For example, in some embodiments it may be desirable to use particle-nucleotide conjugates, including quantum dots, liposomes, or emulsion particles. Alternatively, conjugation may be achieved by non-covalent bonds or other interactions, such as hydrogen bonds. Thus, the scope of the inventive concepts should not be determined with reference to the above description, but instead should be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims (30)

1. A method for determining the identity of a nucleotide in a target nucleic acid sequence, the method comprising:
( a ) a composition comprising:
( i ) two or more copies of the target nucleic acid sequence;
( ii ) two or more primer nucleic acid molecules that are complementary to one or more regions of the target nucleic acid sequence; and
( iii ) two or more polymerase molecules;
Providing
( b ) contacting the composition of (a) with a polymer-nucleotide conjugate under conditions sufficient to permit the formation of a multivalent binding complex between the polymer-nucleotide conjugate and two or more copies of the target nucleic acid sequence in the composition, wherein the polymer-nucleotide conjugate comprises two or more nucleotide moieties ;
(c) detecting said multivalent binding complexes, thereby determining the identity of said nucleotides in said target nucleic acid sequence. The method of claim 1, wherein the target nucleic acid sequence is DNA. The method of claim 1 , wherein the detection of the multivalent binding complex is performed in the absence of unbound or solution-borne polymer-nucleotide conjugate. The method of claim 1 , wherein the target nucleic acid sequence is replicated, amplified, or produced by replication or amplification. The method of claim 1 , wherein the step of detecting the multivalent binding complex comprises a fluorescent measurement. The method of claim 1, wherein the contacting step comprises the use of one polymer-nucleotide conjugate. The method of claim 1, wherein the contacting step comprises the use of two or more polymer-nucleotide conjugates. The method of claim 7, wherein each of the two or more polymer-nucleotide conjugates comprises a different type of nucleotide moiety. 9. The method of claim 8, wherein the contacting step comprises the use of three types of polymer-nucleotide conjugates, and each of the three types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety. The method of claim 1 , wherein the polymer-nucleotide conjugate comprises a blocked nucleotide moiety. 11. The method of claim 10, wherein the blocked nucleotide is a 3'-O-azidomethyl, 3'-O-methyl, or 3'-O-alkylhydroxylamine nucleotide. The method of claim 1 , wherein the contacting step is carried out in the presence of ions that stabilize the multivalent binding complex. 10. The method of claim 1, wherein the contacting step is carried out in the presence of strontium ions, magnesium ions, calcium ions, or any combination thereof. The method of claim 1 , wherein the polymerase molecule is catalytically inactive. The method of claim 1 , wherein the polymerase molecule is rendered catalytically inactive by mutation or chemical modification. The method of claim 1, wherein the polymerase molecule is rendered catalytically inactive by the absence of a required ion or cofactor. The method of claim 1 , wherein the polymerase molecule is catalytically active. The method of claim 1 , wherein the polymer-nucleotide conjugate does not contain a blocked nucleotide moiety. The method of claim 1 , wherein the duration of the multivalent binding complex is greater than 2 seconds. The method of claim 1, wherein the method is carried out at a temperature in the range of 25°C to 42°C. 2. The method of claim 1, wherein the polymer-nucleotide conjugate further comprises one or more fluorescent labels, and two or more copies of the target nucleic acid sequence are deposited on, attached to, or hybridized to a surface, and wherein a fluorescent image of the multivalent binding complex on the surface has a contrast to noise ratio of greater than 20 in the detecting step. 10. The method of claim 1, wherein the composition of (a) is deposited on the surface using a buffer that incorporates a polar aprotic solvent. 2. The method of claim 1, wherein the contacting step is performed under conditions that stabilize the multivalent binding complex when the nucleotide moiety is complementary to the next base of the target nucleic acid sequence and that destabilize the multivalent binding complex when the nucleotide moiety is not complementary to the next base of the target nucleic acid sequence. The method of claim 1, wherein the polymer-nucleotide conjugate comprises a polymer having multiple branches and the two or more nucleotide moieties are attached to the branches. 25. The method of claim 24, wherein the polymer configuration is star, comb, cross, bottle brush, or dendrimer. 2. The method of claim 1, 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. 2. The method of claim 1, further comprising a dissociation step of destabilizing the multivalent binding complex formed between the composition of (a) and a polymer-nucleotide conjugate, wherein the dissociation step allows for removal of the polymer-nucleotide conjugate. 28. The method of claim 27, further comprising an extension step to incorporate a nucleotide complementary to the next base of the target nucleic acid sequence into the two or more primer nucleic acid molecules. 29. The method of claim 28, wherein the extending step is performed simultaneously with or after the dissociating step. The method of claim 1, wherein the polymer-nucleotide conjugate comprises one or more detectable labels.