Nucleotides with terminal phosphate capping

Abstract

Embodiments of the present disclosure relate to nucleotides with modified terminal phosphate capping groups. Also provided herein are methods and kits for sequencing applications using such nucleotides having modified terminal phosphate capping groups.

Description

NUCLEOTIDES WITH TERMINAL PHOSPHATE CAPPINGBACKGROUNDField

[0001] The present disclosure generally relates to polynucleotide sequencing methods, compositions, and kits for sequencing. The present disclosure also relates to nucleotides with terminal phosphate capping groups.Description of the Related Art

[0002] Advances in the study of molecules have been led, in part, by improvement in technologies used to characterize the molecules or their biological reactions. In particular, the study of the nucleic acids DNA and RNA has benefited from developing technologies used for sequence analysis and the study of hybridization events.

[0003] An example of the technologies that have improved the study of nucleic acids is the development of fabricated arrays of immobilized nucleic acids. These arrays consist typically of a high-density matrix of polynucleotides immobilized onto a solid support material. See, e.g., Fodor et al., Trends Biotech. 12: 19-26, 1994, which describes ways of assembling the nucleic acids using a chemically sensitized glass surface protected by a mask, but exposed at defined areas to allow attachment of suitably modified nucleotide phosphoramidites. Fabricated arrays can also be manufactured by the technique of “spotting” known polynucleotides onto a solid support at predetermined positions (e.g., Stimpson et al., Proc. Natl. Acad. Sei. 92: 6379- 6383, 1995).

[0004] One way of determining the nucleotide sequence of a nucleic acid bound to an array is called “sequencing by synthesis” or “SBS.” This technique for determining the sequence of DNA ideally requires the controlled (i.e., one at a time) incorporation of the correct complementary nucleotide opposite the nucleic acid being sequenced. This allows for accurate sequencing by adding nucleotides in multiple cycles as each nucleotide residue is sequenced one at a time, thus preventing an uncontrolled series of incorporations from occurring. The incorporated nucleotide is read using an appropriate label attached thereto before removal of the label moiety and the subsequent next round of sequencing.

[0005] Typically, fully functionalized nucleoside triphosphates (ffNs) are used in SBS. Cold-chain workflows may be used to synthesize, store, and ship such ffNs because they are unstable under heat stress. It is desirable to develop modified ffNs with improved stabilities at ambient conditions.SUMMARY

[0006] One aspect of the present disclosure relates to a nucleotide comprising a nucleobase, a ribose or 2' deoxyribose having a 3' hydroxy blocking group, and a terminally modified triphosphate group, wherein at least one hydroxy group of the terminal phosphate moiety of the terminally modified triphosphate group is replaced by:(a) a substituent RTPCselected from the group consisting of a halo, unsubstituted or substituted (C1-C6alkyl)amino, unsubstituted or substituted (C6-C10aryl)amino, unsubstituted or substituted (5 to 10 membered heteroaryl)amino, unsubstituted or substituted C1-C6alkyl, unsubstituted or substituted C1-C6alkoxy, unsubstituted or substituted C6-C10aryloxy, unsubstituted or substituted (C1-C6alkoxy)C1-C6alkyl, unsubstituted or substituted -O-(C1-C6alkoxy)C1-C6alkyl, unsubstituted or substituted -0-(C6-Cio aryl)C1-C6alkyl, unsubstituted or substituted -O-(5 to 10 membered heteroaryl)C1-C6alkyl, (unsubstituted or substituted amino)(C1-C6alkylene)-O-, and (unsubstituted or substituted amino)(2 to 20 heteroalkylene)-O-;(b) a monophosphate, diphosphate, or triphosphate group having at least one terminal hydroxy group independently substituted with the substituent RTPC: or(c) a second nucleotide comprising a second nucleobase, a ribose or 2' deoxyribose, and a monophosphate, diphosphate or triphosphate group, wherein the second nucleotide is connected to the nucleotide via the monophosphate, diphosphate or triphosphate group of the second nucleotide.

[0007] Another aspect of the present disclosure relates to an oligonucleotide or polynucleotide comprising a nucleotide as described herein incorporated thereto.

[0008] Another aspect of the present disclosure relates to a kit comprising a first type of nucleotide as described in the present disclosure.

[0009] Another aspect of the present disclosure relates to a method of determining the sequences of a plurality of different target polynucleotides, comprising:(a) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides;(b) contacting the solid support with an aqueous incorporation mixture comprising DNA polymerase and one or more of four different types of nucleotides under conditions suitable for DNA polymerase-mediated primer extension, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides,wherein at least one type of nucleotide is a nucleotide in accordance with the present disclosure carrying a detectable label through a cleavable linker, and wherein each of the one or more of four different type of nucleotides comprises a 3' blocking group covalently attached to the 2' deoxyribose of the nucleotide;(c) imaging and performing one or more fluorescent measurements of the extended copy polynucleotides;(d) removing the 3' blocking group from the nucleotides incorporated into the extended copy polynucleotides;(e) washing the solid support after the removal of the 3' blocking group from the incorporated nucleotides; and(f) repeating steps (b) to (e) until the sequences of at least a portion of the target polynucleotides are determined.

[0010] Another aspect of the present disclosure relates to a method of determining the sequences of a plurality of different target polynucleotides, comprising:(a) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides;(b) contacting the solid support with an aqueous incorporation mixture comprising DNA polymerase and one or more of four different types of nucleotides under conditions suitable for DNA polymerase-mediated primer extension, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides, wherein at least two types of nucleotides are unlabeled and at least one type of unlabeled nucleotide is a unlabeled nucleotide in accordance with the present disclosure having a first functional moiety, and wherein each of the one or more of four different type of nucleotides comprises a 3' blocking group covalently attached to the 2' deoxyribose of the nucleotide;(c) contacting the extended copy polynucleotides with an aqueous labeling mixture comprising a first labeling reagent, wherein the first labeling reagent comprises one or more first detectable labels and a first binding moiety that is capable of specific binding to the first functional moiety of the unlabeled nucleotide;(d) imaging the solid support and performing one or more fluorescent measurements;(e) removing the 3' blocking group from the nucleotides incorporated into the extended copy polynucleotides;(f) washing the solid support after the removal of the 3' blocking group from the incorporated nucleotides; and(g) repeating steps (b) to (f) until the sequences of at least a portion of the target polynucleotides are determined.

[0011] A further aspect of the present disclosure relates to a method of preparing a growing polynucleotide complementary to a target single-stranded polynucleotide (e.g., in a sequencing application), comprising incorporating a nucleotide described herein into the growing complementary polynucleotide, wherein the incorporation of the nucleotide prevents the introduction of any subsequent nucleotide into the growing complementary polynucleotide. In some embodiments, the incorporation of the nucleotide is accomplished by a polymerase, a terminal deoxy nucleotidyl transferase (TdT), or a reverse transcriptase. In one embodiment, the incorporation is accomplished by a polymerase (e.g., a DNA polymerase).BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 plots the proportion (%) of degradation products and remaining modified nucleosides with tri-, tetra-, and hexa-phosphates after incubation at 60°C for 14 days.

[0013] FIG. 2 illustrates the31P NMR comparison between unmodified dATP and the yF-modified dATP.

[0014] FIG. 3 plots relative incorporation activity of the modified nucleosides with tri-, tetra-, and hexa-phosphate using a DNA polymerase.

[0015] FIGs. 4A and 4B plot the proportion (%) of remaining fully functionalized C nucleoside triphosphate (FFC) over time at four different storage temperatures. FIG. 4A plots remaining standard unmodified FFC, whereas FIG. 4B plots remaining modified ffC with a fluoro substitution at the terminal phosphate group.

[0016] FIG. 5 plots the percentage of incorporation of various A nucleoside triphosphates over time at two different concentrations, comparing the standard unmodified fully functionalized A nucleoside triphosphate (FFA) with dATP with a fluoro substituted terminal phosphate, and a ffA having a fluoro substituted terminal phosphate.DETAILED DESCRIPTION

[0017] Embodiments of the present disclosure relate to nucleotides with terminally modified phosphate groups. Such groups may improve stability of the nucleotide’s phosphate groups and / or protect the phosphate groups from degradation. Nucleotides having terminally modified triphosphate groups may be suitable for longer term storage (e.g., on the order of years).Nucleotides having terminally modified phosphate groups may be suitable for exposure to ambient conditions (e.g., room temperature).Definitions

[0018] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0019] It is noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless expressly and unequivocally limited to one referent. It will be apparent to those skilled in the art that various modifications and variations can be made to various embodiments described herein without departing from the spirit or scope of the present teachings. Thus, it is intended that the various embodiments described herein cover other modifications and variations within the scope of the appended claims and their equivalents.

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. The use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting. The use of the term “having” as well as other forms, such as “have,” “has,” and “had,” is not limiting. As used in this specification, whether in a transitional phrase or in the body of the claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the above terms are to be interpreted synonymously with the phrases “having at least” or “including at least.” For example, when used in the context of a process, the term “comprising” means that the process includes at least the recited steps but may include additional steps. When used in the context of a compound, composition, or device, the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.

[0021] As used herein, common organic abbreviations are defined as follows:°C Temperature in degrees Centigrade dATP Deoxyadenosine triphosphate dCTP Deoxycytidine triphosphate dGTP Deoxyguanosine triphosphate dTTP Deoxythymidine triphosphate ddNTP Dideoxynucleotide triphosphate ffA or FFA Fully functionalized A nucleotide or fully functionalized A nucleoside triphosphate ffC or FFC Fully functionalized C nucleotide or fully functionalized C nucleoside triphosphate ffG or FFG Fully functionalized G nucleotide or fully functionalized G nucleoside triphosphate ffN or FFN Fully functionalized nucleotide or fully functionalized nucleoside triphosphateffT or FFT Fully functionalized T nucleotide or fully functionalized T nucleoside triphosphate h Hour(s)RT Room temperatureSBS Sequencing by SynthesisUSM Universal scan mix

[0022] As used herein, the term “array” refers to a population of different probe molecules that are attached to one or more substrates such that the different probe molecules can be differentiated from each other according to relative location. An array can include different probe molecules that are each located at a different addressable location on a substrate. Alternatively, or additionally, an array can include separate substrates each bearing a different probe molecule, wherein the different probe molecules can be identified according to the locations of the substrates on a surface to which the substrates are attached or according to the locations of the substrates in a liquid. Exemplary arrays in which separate substrates are located on a surface include, without limitation, those including beads in wells as described, for example, in U.S. Patent No. 6,355,431 Bl, US 2002 / 0102578 and PCT Publication No. WO 00 / 63437. Exemplary formats that can be used in the invention to distinguish beads in a liquid array, for example, using a microfluidic device, such as a fluorescent activated cell sorter (FACS), are described, for example, in US Pat. No. 6,524,793. Further examples of arrays that can be used in the invention include, without limitation, those described in U.S. Pat Nos. 5,429,807; 5,436,327; 5,561,071; 5,583,211; 5,658,734; 5,837,858; 5,874,219; 5,919,523; 6,136,269; 6,287,768; 6,287,776; 6,288,220; 6,297,006; 6,291,193; 6,346,413; 6,416,949; 6,482,591; 6,514,751 and 6,610,482; and WO 93 / 17126; WO 95 / 11995; WO 95 / 35505; EP 742 287; and EP 799 897.

[0023] As used herein, the term “covalently attached” or “covalently bonded” refers to the forming of a chemical bonding that is characterized by the sharing of pairs of electrons between atoms. For example, a covalently attached polymer coating refers to a polymer coating that forms chemical bonds with a functionalized surface of a substrate, as compared to attachment to the surface via other means, for example, adhesion or electrostatic interaction. It will be appreciated that polymers that are attached covalently to a surface can also be bonded via other means in addition to covalent attachment.

[0024] The term “halogen” or “halo,” as used herein, means any one of the radio-stable atoms of column 7 of the Periodic Table of the Elements, e.g., fluorine, chlorine, bromine, or iodine, with fluorine and chlorine being preferred.

[0025] As used herein, “Cato Ct>” in which “a” and “b” are integers refer to the number of carbon atoms in an alkyl, alkenyl or alkynyl group, or the number of ring atoms of a cycloalkyl or aryl group. That is, the alkyl, the alkenyl, the alkynyl, the ring of the cycloalkyl, and ring of the aryl can contain from “a” to “b”, inclusive, carbon atoms. For example, a “Ci to C4 alkyl”group refers to all alkyl groups having from 1 to 4 carbons, that is, CH3-, CH3CH2-, CH3CH2CH2- , (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)- and (CH3)3C-; a C3to C4cycloalkyl group refers to all cycloalkyl groups having from 3 to 4 carbon atoms, that is, cyclopropyl and cyclobutyl. Similarly, a “4 to 6 membered heterocyclyl” group refers to all heterocyclyl groups with 4 to 6 total ring atoms, for example, azetidine, oxetane, oxazoline, pyrrolidine, piperidine, piperazine, morpholine, and the like. If no “a” and “b” are designated with regard to an alkyl, alkenyl, alkynyl, cycloalkyl, or aryl group, the broadest range described in these definitions is to be assumed. As used herein, the term “C1-C6” includes Ci, C2, C3, C4, C5 and Ce, and a range defined by any of the two numbers. For example, C1-C6alkyl includes Ci, C2, C3, C4, C5 and Ce alkyl, C2-C6alkyl, Ci-C3alkyl, etc. Similarly, C2-C6alkenyl includes C2, C3, C4, C5 and Ce alkenyl, C2-C5 alkenyl, C3-C4alkenyl, etc.; and C2-C6alkynyl includes C2, C3, C4, C5 and Ce alkynyl, C2- C5 alkynyl, C3-C4alkynyl, etc. C3-Cs cycloalkyl each includes hydrocarbon ring containing 3, 4, 5, 6, 7 and 8 carbon atoms, or a range defined by any of the two numbers, such as C3-C7 cycloalkyl or Cs-Ce cycloalkyl.

[0026] As used herein, “alkyl” refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). The alkyl group may have 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as “1 to 20” refers to each integer in the given range; e.g., “1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated). The alkyl group may also be a medium size alkyl having 1 to 9 carbon atoms. The alkyl group could also be a lower alkyl having 1 to 6 carbon atoms. By way of example only, “C1-6 alkyl” or “C1-C6alkyl” indicates that there are one to six carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n- butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like.

[0027] As used herein, “alkoxy” refers to the formula -OR wherein R is an alkyl as is defined above, such as “C1-9 alkoxy” or “C1-C9 alkoxy”, including but not limited to methoxy, ethoxy, n-propoxy, 1 -methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, and tertbutoxy, and the like.

[0028] As used herein, “alkenyl” refers to a straight or branched hydrocarbon chain containing one or more double bonds. The alkenyl group may have 2 to 20 carbon atoms, although the present definition also covers the occurrence of the term “alkenyl” where no numerical range is designated. The alkenyl group may also be a medium size alkenyl having 2 to 9 carbon atoms. The alkenyl group could also be a lower alkenyl having 2 to 6 carbon atoms. By way of exampleonly, “C2-C6alkenyl” or “C2-6 alkenyl” indicates that there are two to six carbon atoms in the alkenyl chain, i.e., the alkenyl chain is selected from the group consisting of ethenyl, propen- 1-yl, propen-2-yl, propen-3-yl, buten-l-yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methyl-propen-l-yl, 2-methyl-propen-l-yl, 1-ethyl-ethen-l-yl, 2-methyl-propen-3-yl, buta- 1,3-dienyl, buta-1,2,- dienyl, and buta-l,2-dien-4-yl. Typical alkenyl groups include, but are in no way limited to, ethenyl, propenyl, butenyl, pentenyl, and hexenyl, and the like.

[0029] As used herein, “alkynyl” refers to a straight or branched hydrocarbon chain containing one or more triple bonds. The alkynyl group may have 2 to 20 carbon atoms, although the present definition also covers the occurrence of the term “alkynyl” where no numerical range is designated. The alkynyl group may also be a medium size alkynyl having 2 to 9 carbon atoms. The alkynyl group could also be a lower alkynyl having 2 to 6 carbon atoms. By way of example only, “C2-6 alkynyl” or “C2-C6alkenyl” indicates that there are two to six carbon atoms in the alkynyl chain, i.e., the alkynyl chain is selected from the group consisting of ethynyl, propyn-1- yl, propyn-2-yl, butyn-l-yl, butyn-3-yl, butyn-4-yl, and 2-butynyl. Typical alkynyl groups include, but are in no way limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl, and the like.

[0030] The term “aromatic” refers to a ring or ring system having a conjugated pi electron system and includes both carbocyclic aromatic (e.g., phenyl) and heterocyclic aromatic groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of atoms) groups provided that the entire ring system is aromatic.

[0031] As used herein, “aryl” refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent carbon atoms) containing only carbon in the ring backbone. When the aryl is a ring system, every ring in the system is aromatic. The aryl group may have 6 to 18 carbon atoms, although the present definition also covers the occurrence of the term “aryl” where no numerical range is designated. In some embodiments, the aryl group has 6 to 10 carbon atoms. The aryl group may be designated as “C6-C10aryl,” “Ce or C10 aryl,” or similar designations. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl.

[0032] An “aralkyl” or “arylalkyl” is an aryl group connected, as a substituent, via an alkylene group, such as “C7-14 aralkyl” and the like, including but not limited to benzyl, 2- phenylethyl, 3 -phenylpropyl, and naphthylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., a C1-6 alkylene group).

[0033] As used herein, “heteroaryl” refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent atoms) that contain(s) one or more heteroatoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur, in thering backbone. When the heteroaryl is a ring system, every ring in the system is aromatic. The heteroaryl group may have 5-18 ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms), although the present definition also covers the occurrence of the term “heteroaryl” where no numerical range is designated. In some embodiments, the heteroaryl group has 5 to 10 ring members or 5 to 7 ring members. The heteroaryl group may be designated as “5-7 membered heteroaryl,” “5-10 membered heteroaryl,” or similar designations. Examples of heteroaryl rings include, but are not limited to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzo thienyl.

[0034] A “heteroaralkyl” or “heteroarylalkyl” is heteroaryl group connected, as a substituent, via an alkylene group. Examples include but are not limited to 2-thienylmethyl, 3- thienylmethyl, furylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., a Ci-6 alkylene group).

[0035] As used herein, “carbocyclyl” means a non-aromatic cyclic ring or ring system containing only carbon atoms in the ring system backbone. When the carbocyclyl is a ring system, two or more rings may be joined together in a fused, bridged or spiro-connected fashion. Carbocyclyls may have any degree of saturation provided that at least one ring in a ring system is not aromatic. Thus, carbocyclyls include cycloalkyls, cycloalkenyls, and cycloalkynyls. The carbocyclyl group may have 3 to 20 carbon atoms, although the present definition also covers the occurrence of the term “carbocyclyl” where no numerical range is designated. The carbocyclyl group may also be a medium size carbocyclyl having 3 to 10 carbon atoms. The carbocyclyl group could also be a carbocyclyl having 3 to 6 carbon atoms. The carbocyclyl group may be designated as “C3-6 carbocyclyl”, “C3 C6 carbocyclyl” or similar designations. Examples of carbocyclyl rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicycle[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl.

[0036] As used herein, “cycloalkyl” means a fully saturated carbocyclyl ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0037] As used herein, “heterocyclyl” means a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring backbone. Heterocyclyls may be joined together in a fused, bridged or spiro-connected fashion. Heterocyclyls may have any degree of saturation provided that at least one ring in the ring system is not aromatic. The heteroatom(s) may be present in either a non-aromatic or aromatic ring in the ring system. The heterocyclyl group may have 3to 20 ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms), although the present definition also covers the occurrence of the term “heterocyclyl” where no numerical range is designated. The heterocyclyl group may also be a medium size heterocyclyl having 3 to 10 ring members. The heterocyclyl group could also be a heterocyclyl having 3 to 6 ring members. The heterocyclyl group may be designated as “3-6 membered heterocyclyl” or similar designations. In preferred six membered monocyclic heterocyclyls, the heteroatom(s) are selected from one up to three of O, N or S, and in preferred five membered monocyclic heterocyclyls, the heteroatom(s) are selected from one or two heteroatoms selected from O, N, or S. Examples of heterocyclyl rings include, but are not limited to, aziridinyl, azetidinyl, azepanyl, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl, pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathianyl, 1,4- oxathiinyl, 1,4-oxathianyl, 2H- 1.2-oxazinyl, trioxanyl, hexahydro-1, 3, 5-triazinyl, 1,3-dioxolyl,1.3-dioxolanyl, 1 ,3-dithiolyl, 1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1.4-thiazinyl, thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, and tetrahydroquinoline.

[0038] As used herein, (cycloalkyl) alkyl refers to a cycloalkyl group connected via an alkylene group, such as (C3-C7 cycloalkyl)C1-C6alkyl. Non-limiting examples include cyclohexyl-(CH2)i-6-, cyclopentyl-(CH2)i-6-, or cyclopropyl-(CH2)i-6-.

[0039] As used herein, (aryl)alkyl refers to an aryl group connected via an alkylene group, such as (C6-C10aryl)C1-C6alkyl. Non-limiting example includes phenyl-(CH2)i-6-

[0040] As used herein, (heteroaryl)alkyl refers to a heteroaryl group connected via an alkylene group, such as (5 to 10 membered heteroaryl)C1-C6alkyl. Non-limiting examples include pyridyl-(CH2)i-6-, pyrimidinyl-(CH2)i-6-, or pyrrolyl-(CH2)i-6-.

[0041] As used herein, (heterocyclyl)alkyl refers to a heterocyclyl group connected via an alkylene group, such as (3 to 10 membered heterocyclyl)C1-C6alkyl. Non-limiting examples include morpholinyl- (CH2)I -6-, piperidinyl- (CH2)I -6-, piperazinyl- (CH2)I -6-, pyrrolidinyl-(CH2)i-6-, or azetidinyl-(CH2)i-6-.

[0042] As used herein, “alkoxyalkyl” or “(alkoxy)alkyl” refers to an alkoxy group connected via an alkylene group, such as C2-C8 alkoxyalkyl, or (C1-C6alkoxy)C1-C6alkyl, for example, -(CH2)I-3-OCH3.

[0043] An “O-carboxy” group refers to a “-OC(=O)R” group in which R is selected from hydrogen, Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, Ce-io aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

[0044] A “C-carboxy” group refers to a “-C(=O)OR” group in which R is selected from the group consisting of hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, Ce- 10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein. A nonlimiting example includes carboxyl (i.e., -C(=O)OH).

[0045] A “sulfonyl” group refers to an “-SO2R” group in which R is selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, Ce-io aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

[0046] A “sulfino” group refers to a “-S(=O)OH” group.

[0047] A “sulfo” group refers to a“-S(=O)2OH” or “-SO3H” group.

[0048] A “sulfonate” group refers to a “-SO3 ” group.

[0049] A “sulfate” group refers to “-SO4 ” group.

[0050] A “S-sulfonamido” group refers to a “-SO2NRARB” group in which RA and RB are each independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, Ce-io aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

[0051] An “N-sulfonamido” group refers to a “-N(RA)SO2RB” group in which RA and Rb are each independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, Ce-io aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

[0052] A “C-amido” group refers to a “-C(=O)NRARB” group in which RA and RB are each independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, Ce-io aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

[0053] An “N-amido” group refers to a “-N(RA)C(=O)RB” group in which RA and RB are each independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, Ce-io aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

[0054] An “amino” group refers to a “-NRARB” group in which RA and RB are each independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, Ce- 10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein. A nonlimiting example includes free amino (i.e., -NH2).

[0055] An “aminoalkyl” group refers to an amino group connected via an alkylene group.

[0056] An “alkoxyalkyl” group refers to an alkoxy group connected via an alkylene group, such as a “Cz-Cs alkoxyalkyl” and the like.

[0057] As used herein, “-OAc” or “-O-acyl” refers to acetyloxy with the structure - O-C(=O)CH3.

[0058] When a group is described as “optionally substituted” it may be either unsubstituted or substituted. Likewise, when a group is described as being “substituted”, the substituent may be selected from one or more of the indicated substituents. As used herein, a substituted group is derived from the unsubstituted parent group in which there has been an exchange of one or more hydrogen atoms for another atom or group. Unless otherwise indicated, when a group is deemed to be “substituted,” it is meant that the group is substituted with one or more substituents independently selected from C1-C6alkyl, C1-C6alkenyl, Ci-G, alkynyl, C3-C7 carbocyclyl (optionally substituted with halo, C1-C6alkyl, Ci-G, alkoxy, C1-C6haloalkyl, and Ci- Ce haloalkoxy), Cs-Cv-carbocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6alkyl, Ci- Ce alkoxy, C1-C6haloalkyl, and C1-C6haloalkoxy), 3-10 membered heterocyclyl (optionally substituted with halo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, and C1-C6haloalkoxy), 3-10 membered heterocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, and C1-C6haloalkoxy), aryl (optionally substituted with halo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, and C1-C6haloalkoxy), aryl(C1-C6)alkyl (optionally substituted with halo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, and C1-C6haloalkoxy), 5-10 membered heteroaryl (optionally substituted with halo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, and Ci- Ce haloalkoxy), 5-10 membered heteroaryl(C1-C6)alkyl (optionally substituted with halo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, and C1-C6haloalkoxy), halo, -CN, hydroxy, C1-C6alkoxy, C1-C6alkoxy (C1-C6)alkyl (i.e., ether), aryloxy, sulfhydryl (mercapto), halo(C1-C6)alkyl (e.g., - CF3), halo(C1-C6)alkoxy (e.g., -OCF3), C1-C6alkylthio, arylthio, amino, amino(C1-C6) alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato, isothiocyanate, sulfinyl, sulfonyl, -SO3H, sulfonate, sulfate, sulfino, -OSOzCi-CLalkyl. oxo (=0) and thioxo (=S). Wherever a group is described as “optionally substituted” that group can be substituted with the above substituents. In some embodiments, when an alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl or heterocyclyl group is substituted, each is independently substituted with one or more substituents selected from the group consisting of halo, -CN, -SO3 , -OSO3 , -SO3H, -SRA,are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl.

[0059] As understood by one of ordinary skill in the art, a compound described herein may exist in ionized form, e.g., -CO2 , -SO3 , or -O-SCF- . If a compound contains a positively or negatively charged substituent group, for example, -SO3 , it may also contain a negatively or positively charged counterion such that the compound as a whole is neutral. For example, a compound may contain both -CO2 and a quaternary ammonium cation, or both -SO3 and a quaternary ammonium cation. In other aspects, the compound may exist in a salt form, where the counterion is provided by a conjugate acid or base. It is understood that when the compound described herein is substituted with -SO3H, such substituent may exist in its anionic form -SO3 in aqueous solution.

[0060] It is to be understood that certain radical naming conventions can include either a mono-radical or a di-radical, depending on the context. For example, where a substituent requires two points of attachment to the rest of the molecule, it is understood that the substituent is a di-radical. For example, a substituent identified as alkyl that requires two points of attachment includes di-radicals such as -CH2-, -CH2CH2-, -CHzCHfCHsjCEh-, and the like. Other radical naming conventions clearly indicate that the radical is a di-radical such as “alkylene” or “alkenylene.”

[0061] When two “adjacent” R groups are said to form a ring “together with the atom to which they are attached,” it is meant that the collective unit of the atoms, intervening bonds, and the two R groups are the recited ring. For example, when the following substructure is present:and R1and R2are defined as selected from the group consisting of hydrogen and alkyl, or R1and R2together with the atoms to which they are attached form an aryl or carbocyclyl, it is meant that R1and R2can be selected from hydrogen or alkyl, or alternatively, the substructure has structure:where A is an aryl ring or a carbocyclyl containing the depicted double bond.

[0062] Wherever a substituent is depicted as a di-radical (i.e., has two points of attachment to the rest of the molecule), it is to be understood that the substituent can be attached in any directional configuration unless otherwise indicated. Thus, for example, a substituent depicted as -AE- orincludes the substituent being oriented such that the A is attachedat the leftmost attachment point of the molecule as well as the case in which A is attached at the rightmost attachment point of the molecule. In addition, if a group or substituent is depicted asand L is defined an optionally present linker moiety; when L is not present (orVAx A absent), such group or substituent is equivalent to 'A E

[0063] As used herein, a “nucleotide” includes a nitrogen containing heterocyclic base, a sugar, and one or more phosphate groups. They are monomeric units of a nucleic acid sequence. In RNA, the sugar is a ribose, and in DNA a deoxyribose, i.e., a sugar lacking a hydroxy group that is present in ribose. The nitrogen containing heterocyclic base can be purine, deazapurine, or pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof, such as 7-deaza adenine or 7-deaza guanine. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof. The C-l atom of deoxyribose is bonded to N-l of a pyrimidine or N-9 of a purine.

[0064] As used herein, a “nucleoside” is structurally similar to a nucleotide, but is missing the phosphate moieties. An example of a nucleoside analogue would be one in which the label is linked to the base and there is no phosphate group attached to the sugar molecule. The term “nucleoside” is used herein in its ordinary sense as understood by those skilled in the art. Examples include, but are not limited to, a ribonucleoside comprising a ribose moiety and a deoxyribonucleoside comprising a deoxyribose moiety. A modified pentose moiety is a pentose moiety in which an oxygen atom has been replaced with a carbon and / or a carbon has been replaced with a sulfur or an oxygen atom. A “nucleoside” is a monomer that can have a substituted base and / or sugar moiety. Additionally, a nucleoside can be incorporated into larger DNA and / or RNA polymers and oligomers.

[0065] The term “purine base” is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers. Similarly, the term “pyrimidine base” is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers. A non-limiting list of optionally substituted purine-bases includes purine, adenine, guanine, deazapurine, 7-deaza adenine, 7-deaza guanine, hypoxanthine, xanthine, alloxanthine, 7- alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid and isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil and 5-alkylcytosine (e.g., 5 -methylcytosine).

[0066] As used herein, when an oligonucleotide or polynucleotide is described as “comprising” a nucleoside or nucleotide described herein, it means that the nucleoside or nucleotide described herein forms a covalent bond with the oligonucleotide or polynucleotide. Similarly, when a nucleoside or nucleotide is described as part of an oligonucleotide orpolynucleotide, such as “incorporated into” an oligonucleotide or polynucleotide, it means that the nucleoside or nucleotide described herein forms a covalent bond with the oligonucleotide or polynucleotide. In some such embodiments, the covalent bond is formed between a 3' hydroxy group of the oligonucleotide or polynucleotide with the 5' phosphate group of a nucleotide described herein as a phosphodiester bond between the 3' carbon atom of the oligonucleotide or polynucleotide and the 5' carbon atom of the nucleotide.

[0067] As used herein, the term “cleavable linker” is not meant to imply that the whole linker is required to be removed. The cleavage site can be located at a position on the linker that ensures that part of the linker remains attached to the detectable label and / or nucleoside or nucleotide moiety after cleavage.

[0068] As used herein, “derivative” or “analog” means a synthetic nucleotide or nucleoside derivative having modified base moieties and / or modified sugar moieties. Such derivatives and analogs are discussed in, e.g., Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90:543-584, 1990. Nucleotide analogs can also comprise modified phosphodiester linkages, including phosphorothioate, phosphorodithioate, alkyl-phosphonate, phosphoranilidate and phosphoramidate linkages. “Derivative,” “analog,” and “modified” as used herein, may be used interchangeably, and are encompassed by the terms “nucleotide” and “nucleoside” defined herein.

[0069] As used herein, the term “phosphate” is used in its ordinary sense as understoodOHby those skilled in the art, and includes its protonated forms (for example, O' andused herein, the terms “monophosphate,” “diphosphate,” and “triphosphate” are used in their ordinary sense as understood by those skilled in the art, and include protonated forms.

[0070] As used herein, the term “phasing” refers to a phenomenon in SBS that is caused by incomplete removal of the 3' terminators and fluorophores, and / or failure to complete the incorporation of a portion of DNA strands within clusters by polymerases at a given sequencing cycle. Prephasing is caused by the incorporation of nucleotides without effective 3' terminators, wherein the incorporation event goes 1 cycle ahead due to a termination failure. Phasing and prephasing cause the measured signal intensities for a specific cycle to consist of the signal from the current cycle as well as noise from the preceding and following cycles. As the number of cycles increases, the fraction of sequences per cluster affected by phasing andprephasing increases, hampering the identification of the correct base. Prephasing can be caused by the presence of a trace amount of unprotected or unblocked 3'-OH nucleotides during sequencing by synthesis (SBS). The unprotected 3'-OH nucleotides could be generated during the manufacturing processes or possibly during the storage and reagent handling processes. Accordingly, the discovery of nucleotide analogues which decrease the incidence of prephasing is surprising and provides a great advantage in SBS applications over existing nucleotide analogues. For example, the nucleotide analogues provided can result in faster SBS cycle time, lower phasing and prephasing values, and longer sequencing read lengths.Nucleotides with Terminally Modified Triphosphate Groups

[0071] One aspect of the present disclosure relates to a nucleotide comprising a nucleobase, a ribose or 2' deoxyribose having a 3' hydroxy blocking group, and a terminally modified triphosphate group, wherein at least one hydroxy group of the terminal phosphate moiety of the terminally modified triphosphate group is replaced by:(a) a substituent RTPCselected from the group consisting of a halo, unsubstituted or substituted (C1-C6alkyl)amino, unsubstituted or substituted (C6-C10aryl)amino, unsubstituted or substituted (5 to 10 membered heteroaryl)amino, unsubstituted or substituted C1-C6alkyl, unsubstituted or substituted Ci-G, alkoxy, unsubstituted or substituted C6-C10aryloxy, unsubstituted or substituted (C1-C6alkoxy)C1-C6alkyl, unsubstituted or substituted -O-(C1-C6alkoxy)C1-C6alkyl, unsubstituted or substituted -0-(C6-C10aryl)C1-C6alkyl, unsubstituted or substituted -O-(5 to 10 membered heteroaryl)C1-C6alkyl, (unsubstituted or substituted amino)(C1-C6alkylene)-O-, and (unsubstituted or substituted amino)(2 to 20 heteroalkylene)-O-;(b) a monophosphate, diphosphate, or triphosphate group having at least one terminal hydroxy group independently substituted with the substituent RTPC; or(c) a second nucleotide comprising a second nucleobase, a ribose or 2' deoxyribose, and a monophosphate, diphosphate or triphosphate group, wherein the second nucleotide is connected to the nucleotide via the monophosphate, diphosphate or triphosphate group of the second nucleotide.

[0072] In some embodiments of the nucleotide described herein, the nucleotide has the structure of formula (I):salt thereof,wherein B is the nucleobase;L is an optionally present cleavable linker;X is a detectable label or a functional moiety that can attach to a labeling reagent; PG is the hydroxy blocking group attached to the 3 "oxygen of the nucleotide; and n is an integer of 1, 2, 3 or 4.

[0073] In some embodiments of the nucleotide described herein, R1PCis a halo (e.g., fluoro, chloro or bromo). In some other embodiments, RTPCis unsubstituted or substituted -O- (C6-C10aryl)C1-C6alkyl, in which a C6-C10aryl is covalently attached to a C1-C6alkylene-O-, in which the aryl and C1-C6alkylene can be either unsubstituted or substituted. In one such embodiment, RTPCisIn some other embodiments, R™ is unsubstituted or substituted -O-(5 to 6 membered heteroaryl)C1-C6alkyl, in which a 5 or 6 membered heteroaryl (e.g., containing 1 to 3 heteroatoms selected from N, O and S) is covalently attached to a C1-C6, alkylene-O-, in which the heteroaryl and C1-C6alkylene can be either unsubstituted or substituted. In some other embodiments, RT PCis an unsubstituted or substituted C1-C6alkoxy, for example, methoxy, ethoxy, n-propyloxy, isopropyloxy, or t-butoxy. In some other embodiments, RTPCis unsubstituted or substituted (C6-C10aryl)amino, in which an amino group is substituted with a C6-C10aryl and the aryl (e.g., phenyl or naphthyl) can be either unsubstituted or substituted. In some such embodiments, the aryl is substituted with one or more water solubilizing groups such t , asor a quaternary ammonium ion. In one such example, RTPCis. in some other embodiments, RTPCis (unsubstituted or substituted amino)(C1-C6alkylene)-O- In some such embodiment, Rrptis-O(CH2)I-6-NH2. In one embodiment, Rrp(isIn some other embodiments, R™ is (unsubstituted or substituted amino)(2 to 20 heteroalkylene)-O-. In some such embodiment, RTPCis -O(CH2CH2O)M-CH2CH2NH2. In one such embodiment, Rl ptisIn some embodiments, n is 1. In other embodiments, n is 2 or 4.

[0074] In some other embodiments of the nucleotide described herein, the nucleotide has the structure of formula (11):salt thereof, wherein B is the nucleobase;B' is the second nucleobase; each of L and L' is independently an optionally present cleavable linker as described herein;X is a detectable label or a functional moiety that can attach to a labeling reagent;X' is a second detectable label or a second functional moiety that can attach to a second labeling reagent;PG is the 3' hydroxy blocking group attached to the 3 'oxygen of the nucleotide;PG' is a 3' hydroxy blocking group attached to the 3 'oxygen of the second nucleotide; and m is an integer of 2, 3 or 4.

[0075] In some embodiments of the nucleotide of formula (II), B and B ' are the same.In other embodiments, B and B' are different. In further embodiments, PG and PG' are the same.In other embodiments, PG and PG" are different. In further embodiments, L and L' are the same.In other embodiments, L and L' are different. For example, the nucleotide dimer of formula (II) may include two A nucleotide, each contacting a 3 ' blocking group and also each is labeled with a dye via a cleavable linker. A particular homodimer example is illustrated below:and the synthesis of exemplary dimers is described in Mohsen et al., Nucleic Acids Research, 2019, 47, 18, 9495-9501.

[0076] In some embodiments of the nucleotide described herein (e.g., formula (I) or(II)), PG is azidomethyl (-CH2N3), allyl (-CH2CH=CH2), or -CH2OCH2CH=CH2(“AOM”). In some embodiments, L is present and wherein L and the 3' hydroxy blocking group are cleavable under the same reaction condition. In other embodiments, PG is a glycoside blocking group that is subject to enzymatic cleavage. For example, -OPG may have thethe squiggle line indicates the point of 3'-oxygen’s attachment to the 3'- carbon atom.

[0077] In another aspect, the present disclosure provides for an oligonucleotide or polynucleotide comprising the nucleotide as described herein incorporated thereto. In some embodiments, the oligonucleotide or polynucleotide is at least partially complementary and hybridized to a target polynucleotide immobilized on a surface of a solid support. In further embodiments, the solid support comprises an array of different target polynucleotides immobilized thereon.

[0078] Additional embodiments of the present disclosure relate to a solid support comprising an array of a plurality of immobilized template or target polynucleotides and at least a portion of such immobilized template or target polynucleotides is hybridized to an oligonucleotide or a polynucleotide comprising the nucleotide described herein.Labeled Nucleotides

[0079] According to an aspect of the disclosure, the described nucleotide also comprises a detectable label, (e.g., X and / or X'). Such a nucleotide is referred to herein as a “labeled nucleotide.” The label (e.g., a fluorescent dye) can be conjugated via an optionally present linker by a variety of means including hydrophobic attraction, ionic attraction, and covalent attachment. In some aspect, the detectable label is conjugated to the substrate by covalent attachment. More particularly, the covalent attachment is by means of a linker group (e.g., L and / or L'). In some instances, such labeled nucleotides are also referred to as “modified nucleotides.”

[0080] Various fluorescent dyes may be used in the present disclosure as detectable labels, in particularly those dyes that may be excitation by a blue light (e.g., about 450 nm to about 460 nm) or a green light (e.g., about 520 nm to about 540 nm). These dyes may also be referred to as “blue dyes” and “green dyes” respectively. Examples of various type of blue dyes, including but not limited to coumarin dyes, chromenoquinoline dyes, bisboron containing heterocycles, and naphthalimide dyes that are disclosed in U.S. Publication Nos. 2018 / 0094140, 2018 / 0201981, 2020 / 0277529, 2020 / 0277670, 2021 / 0188832, 2022 / 0033900, 2022 / 0195196, 2022 / 0195517, 2022 / 0380389, 2023 / 0313292, and 2023 / 0416279, and U.S. Ser. Nos. 63 / 492896 and 63 / 593489, each of which is incorporated by reference in its entirety. Examples of green dyes including cyanine or polymethine dyes disclosed in International Publication Nos. W02013 / 041117,WO2014 / 135221, WO 2016 / 189287, W02017 / 051201 and W02018 / 060482A1, as well as U.S. Ser. No. 63 / 616289, each of which is incorporated by reference in its entirety.

[0081] Labeled nucleosides and nucleotides are useful for labeling polynucleotides formed by enzymatic synthesis, such as, by way of non-limiting example, in PCR amplification, isothermal amplification, solid phase amplification, polynucleotide sequencing (e.g., solid phase sequencing), nick translation reactions and the like.

[0082] In some embodiments, the dye may be covalently attached to oligonucleotides or nucleotides via the nucleotide base. For example, the labeled nucleotide or oligonucleotide may have the label attached to the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base through a linker moiety.Linkers

[0083] In some embodiments described herein, the purine or pyrimidine base of the nucleotide or nucleoside molecules described herein can be linked (e.g., via linkers L or L') to a detectable label (e.g., X or X") as described above. In some such embodiments, the linkers used are cleavable. The use of a cleavable linker ensures that the label can, if required, be removed after detection, avoiding any interfering signal with any labeled nucleotide or nucleoside incorporated subsequently. In some embodiments, the cleavable linker comprises an azido moiety, a -O-CL-G, alkenyl moiety (e.g., -O-allyl), a disulfide moiety, an acetal moiety (same or similar to the 3 '-acetal blocking group AOM described herein), or a thiocarbamate moiety.

[0084] In some other embodiments, the linkers used are non-cleavable. For instance, where a nucleotide of the present disclosure is incorporated, no nucleotides need to be subsequently incorporated and thus the label need not be removed from the nucleotide.

[0085] Cleavable linkers are known in the art, and conventional chemistry can be applied to attach a linker to a nucleotide base and a label. The linker can be cleaved by any suitable method, including exposure to acids, bases, nucleophiles, electrophiles, radicals, metals, reducing or oxidizing agents, light, temperature, enzymes etc. The linker as discussed herein may also (or alternatively) be cleaved using an enzyme. In some examples, the same enzymatic catalyst used to cleave the 3'-O-blocking group bond can be used to cleave the linker. In some further embodiments, the linker group may be enzymatically cleavable by the same enzyme that can cleave the blocking group (e.g., PG or PG") in accordance with the present disclosure. In other embodiments, the linker group may be enzymatically cleavable by a different enzyme than the enzyme capable of cleaving the blocking group. Suitable linkers can be adapted from standard chemical protecting groups, as disclosed in Greene & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons. Further suitable cleavable linkers used in solid-phase synthesis are disclosed in Guillier et al. (Chem. Rev. 100:2092-2157, 2000).

[0086] Where the detectable label is attached to the base, the linker can be attached at any position on the nucleotide base provided that Watson-Crick base pairing can still be carried out. In the context of purine bases, it is preferred if the linker is attached via the 7-position of the purine or the preferred deazapurine analogue, via an 8-modified purine, via an N-6 modified adenosine or an N-2 modified guanine. For pyrimidines, attachment is preferably via the 5- position on cytosine, thymidine or uracil and the N-4 position on cytosine.

[0087] In some embodiments, the linker can comprise a spacer unit. It may be desirable that the length of the linker holds the label at a sufficient distance from the nucleotide so as not to interfere with any interaction between the nucleotide and an enzyme, for example, a polymerase.

[0088] In some embodiments, the linker may consist of the similar functionality as the 3'-OH protecting group. This may make the deprotection and deprotecting process more efficient, as only a single treatment will be required to remove both the label and the protecting group.

[0089] Use of the term “cleavable linker” is not meant to imply that the whole linker is required to be removed. The cleavage site can be located at a position on the linker that ensures that part of the linker remains attached to the dye and / or substrate moiety after cleavage. Cleavable linkers may be, by way of non-limiting example, electrophilically cleavable linkers, nucleophilically cleavable linkers, photocleavable linkers, cleavable under reductive conditions (for example disulfide or azide containing linkers), oxidative conditions, cleavable via use of safety-catch linkers and cleavable by elimination mechanisms. The use of a cleavable linker to attach the dye compound to a substrate moiety ensures that the label can, if required, be removed after detection, avoiding any interfering signal in downstream steps.

[0090] Useful linker groups may be found in PCT Publication No. W02004 / 018493 (herein incorporated by reference), examples of which include linkers that may be cleaved using water-soluble phosphines or water-soluble transition metal catalysts formed from a transition metal and at least partially water-soluble ligands, for example, a Pd(II) complex and THP. In aqueous solution the latter form at least partially water-soluble transition metal complexes. Such cleavable linkers can be used to connect bases of nucleotides to labels such as the dyes set forth herein.

[0091] Particular linkers include those disclosed in PCT Publication No. W02004 / 018493 (herein incorporated by reference) such as those that include moieties of the formulae:(wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a CMO substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a Ci-Cio substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside). In some aspect, the linkers connect the bases of nucleotides to labels such as, for example, the dye compounds described herein.

[0092] Additional examples of linkers include those disclosed in U.S. Publication No.2016 / 0040225 (herein incorporated by reference), such as those include moieties of the formulae:The linker moieties illustrated herein may comprise the whole or partial linker structure between the nucleotides / nucleosides and the labels.

[0093] Additional examples of linkers are disclosed in U.S. Publication No. 2020 / 0216891 Al, which is incorporated by reference in its entirety:is 1, 2, 3, 4, 5; k is 1; Z is -N3(azido), -O-C1-C6alkyl, -O-C2-C6alkenyl, or -O-C2-C6alkynyl; and R comprises a detectable label (e.g., a fluorescent dye described herein) or a binding moiety for post-incorporation labeling, which may contain additional linker and / or spacer structure. One of ordinary skill in the art understands that the detectable label or the binding moiety described herein is covalently bound to the linker by reacting a functional group of the binding moiety containing compound (e.g., carboxyl) with a functional group of the linker (e.g., amino) to form an amide bond. Tn one embodiment, the cleavahle linker comprises (“AOL”linker moiety) where Z is-O-allyl. For the purpose of the present disclosure, the nucleotide may contain multiple cleavable linkers repeating units (e.g., k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). The linker can also comprise a spacer unit, such as one or more PEG unit(s) (-OCTECFE-ln, where n is an integer of 1-20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The spacer distances, for example, the nucleotide base from a cleavage site or label.

[0094] In particular embodiments, the length of the linker between a fluorescent dye (fluorophore) and a guanine base can be altered, for example, by introducing a polyethylene glycol spacer group, thereby increasing the fluorescence intensity compared to the same fluorophore attached to the guanine base through other linkages known in the art. Exemplary linkers and their properties are set forth in PCT Publication No. WO 2007 / 020457 (herein incorporated by reference). The design of linkers, and especially their increased length, can allow improvements in the brightness of fluorophores attached to the guanine bases of guanosine nucleotides when incorporated into polynucleotides such as DNA. Thus, when the dye is for use in any method of analysis which requires detection of a fluorescent dye label attached to a guanine-containingnucleotide, it is advantageous if the linker comprises a spacer group of formula -((CkhhOfn-, wherein n is an integer between 2 and 50, as described in WO 2007 / 020457.

[0095] Nucleotides may be labeled at sites on the sugar or nucleobase. As known in the art, a “nucleotide” consists of a nitrogenous base, a sugar, and one or more phosphate groups. In RNA, the sugar is ribose and in DNA is a deoxyribose, i.e., a sugar lacking a hydroxy group that is present in ribose. The nitrogenous base is a derivative of purine or pyrimidine. The purines are adenine (A) and guanine (G), and the pyrimidines are cytosine (C) and thymine (T) or in the context of RNA, uracil (U). The C-1 atom of deoxyribose is bonded to N-1 of a pyrimidine or N- 9 of a purine. A nucleotide is also a phosphate ester of a nucleoside, with esterification occurring on the hydroxy group attached to the C-3 or C-5 of the sugar. Nucleotides are usually mono-, di- or triphosphates.

[0096] Although the base is usually referred to as a purine or pyrimidine, the skilled person will appreciate that derivatives and analogues are available which do not alter the capability of the nucleotide to undergo Watson-Crick base pairing. “Derivative” or “analogue” means a compound or molecule whose core structure is the same as, or closely resembles that of a parent compound but which has a chemical or physical modification, such as, for example, a different or additional side group, which allows the derivative nucleotide or nucleoside to be linked to another molecule. For example, the base may be a deazapurine. In particular embodiments, the derivatives should be capable of undergoing Watson-Crick pairing. “Derivative” and “analogue” also include, for example, a synthetic nucleotide or nucleoside derivative having modified base moieties and / or modified sugar moieties. Such derivatives and analogues are discussed in, for example, Scheit, Nucleotide analogs (John Wiley & Son, 1980) and Uhlman el al., Chemical Reviews 90:543-584, 1990. Nucleotide analogues can also comprise modified phosphodiester linkages including phosphorothioate, phosphorodithioate, alkyl-phosphonate, phosphoranilidate, phosphoramidate linkages and the like.

[0097] A dye may be attached to any position on the nucleotide base, for example, through a linker. In particular embodiments, Watson-Crick base pairing can still be carried out for the resulting analog. Particular nucleobase labeling sites include the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base. As described above a linker group may be used to covalently attach a dye to the nucleotide.

[0098] In particular embodiments the labeled nucleotide may be enzymatically incorporable and enzymatically extendable. Accordingly, a linker moiety may be of sufficient length to connect the nucleotide to the compound such that the compound does not significantly interfere with the overall binding and recognition of the nucleotide by a nucleic acid replicationenzyme. Thus, the linker can also comprise a spacer unit. The spacer distances, for example, the nucleotide base from a cleavage site or label.

[0099] In a particular embodiment, the linker (between dye and nucleotide) and blocking group are both present and are separate moieties. In particular embodiments, the linker and blocking group are both cleavable under substantially similar conditions. Thus, deprotection and deblocking processes may be more efficient because only a single treatment will be required to remove both the dye compound and the blocking group. However, in some embodiments a linker and blocking group need not be cleavable under similar conditions, instead being individually cleavable under distinct conditions.

[0100] The disclosure also encompasses polynucleotides incorporating dye compounds. Such polynucleotides may be DNA or RNA comprised respectively of deoxyribonucleotides or ribonucleotides joined in phosphodiester linkage. Polynucleotides may comprise naturally occurring nucleotides, non-naturally occurring (or modified) nucleotides other than the labeled nucleotides described herein or any combination thereof, in combination with at least one modified nucleotide (e.g., labeled with a dye compound) as set forth herein. Polynucleotides according to the disclosure may also include non-natural backbone linkages and / or non-nucleotide chemical modifications. Chimeric structures comprised of mixtures of ribonucleotides and deoxyribonucleotides comprising at least one labeled nucleotide are also contemplated.

[0101] Non-limiting exemplary labeled nucleotides as described herein include:wherein L represents a linker and R represents a sugar residue as described above, or a sugar residue with the 5’ position substituted with one, two or three phosphates.

[0102] In some embodiments, non-limiting exemplary fully functionalized nucleotide conjugates are shown below:wherein PG stands for a blocking group as described herein; each of n and p is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and m is 0, 1, 2, 3, 4, or 5. In some embodiments, n is 2 or 5. In some embodiments, p is 2 or 5. In some embodiments, m is 0 orrefers to the connection point of the Dye with the cleavable linker as a resultof a reaction between an amino group of the linker moiety and the carboxyl group of the Dye.refers to the connection point of a functional moiety of the nucleotide with the cleavable linker as a result of a reaction between an amino group of the linker moiety and the carboxyl group of the functional moiety. In further embodiments, the nucleotide may be attached to the functional moiety via more than one of the same cleavable linkers (such as AOL-AOL). In addition, the linker may furtherinclude additional PEG spacers as described herein, for example, between RFand -(CH2)m-. To generate the modified nucleotide with terminal phosphate capping, the terminal phosphate moiety of the exemplary fully functionalized nucleotide conjugates can be modified with RTFCas described herein.Kits

[0103] One aspect of the present disclosure relates to a kit comprising one or more nucleotides according to the present disclosure. Another aspect of the present disclosure relates to a kit comprising a first type of nucleotide according to the present disclosure.

[0104] In some embodiments, the kit may contain four types of labeled nucleotides (A, C, G and T or U; dATP, dCTP, dGTP and dTTP or dUTP), where one or more of the four types of nucleotides is labeled. In some embodiments, the first type of nucleotide carries a first detectable label. In further embodiments, the kit comprises four types of nucleotides, wherein a second type of nucleotide carries a second detectable label, a third type of nucleotide carries a third detectable label, and a fourth type of nucleotide is unlabeled (dark), and wherein each of the detectable labels has a distinct emission maximum that is distinguishable from the other detectable labels. In other embodiments, a second type of nucleotide carries a second detectable label, a third type of nucleotide comprises a mixture of the third type of nucleotide carrying the first detectable label and the third type of nucleotide carrying the second detectable label, and a fourth type of nucleotide is unlabeled (dark). Thus, one or more of the label nucleotides can have a distinct absorbance maximum and / or emission maximum such that the compound(s) is(are) distinguishable from other compounds. For example, each compound can have a distinct absorbance maximum and / or emission maximum such that each of the compounds is spectrally distinguishable from the other three compounds (or two compounds if the fourth nucleotide is unlabeled). It will be understood that parts of the absorbance spectrum and / or emission spectrum other than the maxima can differ and these differences can be exploited to distinguish the compounds. The kit may be such that two or more of the compounds have a distinct absorbance maximum.

[0105] The compounds, nucleotides, or kits that are set forth herein may be used to detect, measure, or identify a biological system (including, for example, processes or components thereof). Exemplary techniques that can employ the compounds, nucleotides or kits include sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, cellular assay (e.g., cell binding or cell function analysis), or protein assay (e.g., protein binding assay or protein activity assay). The use may be on an automated instrument for carrying out a particulartechnique, such as an automated sequencing instrument. The sequencing instrument may contain two light sources operating at different wavelengths.

[0106] In a particular embodiment, the labeled nucleotide(s) described herein may be supplied in combination with unlabeled or native nucleotides, or any combination thereof. Combinations of nucleotides may be provided as separate individual components (e.g., one nucleotide type per vessel or tube) or as nucleotide mixtures (e.g., two or more nucleotides mixed in the same vessel or tube).

[0107] Where kits comprise a plurality, particularly two, or three, or more particularly four, nucleotides, the different nucleotides may be labeled with different dye compounds, or one may be dark, with no dye compounds. Where the different nucleotides are labeled with different dye compounds, it is a feature of the kits that the dye compounds are spectrally distinguishable fluorescent dyes. As used herein, the term "spectrally distinguishable fluorescent dyes" refers to fluorescent dyes that emit fluorescent energy at wavelengths that can be distinguished by fluorescent detection equipment (for example, a commercial capillary-based DNA sequencing platform) when two or more such dyes are present in one sample. When two nucleotides labeled with fluorescent dye compounds are supplied in kit form, it is a feature of some embodiments that the spectrally distinguishable fluorescent dyes can be excited at the same wavelength, such as, for example by the same light source. When four nucleotides labeled with fluorescent dye compounds are supplied in kit form, it is a feature of some embodiments that two of the spectrally distinguishable fluorescent dyes can both be excited at one wavelength and the other two spectrally distinguishable dyes can both be excited at another wavelength. Particular excitation wavelengths for the dyes are between 450-460 nm, 490-500 nm, or 520 nm or above (e.g., 532 nm).

[0108] In some embodiments, the kit further comprises a DNA polymerase and one or more buffer compositions.Post-incorporation labeling kits

[0109] In other embodiments, one or more types of nucleotides (A, G, C, and T or U; dATP, dCTP, dGTP and dTTP or dUTP) are unlabeled. In some embodiments, the first type of nucleotide is unlabeled and has a first functional moiety that can attach to a labeling reagent. In further embodiments, the kit further comprises a first labeling reagent, wherein the first labeling reagent comprises one or more first detectable labels and a first binding moiety that is capable of specific binding to the first functional moiety of the first type of unlabeled nucleotide. In some such embodiments, the first functional moiety of the first type of unlabeled nucleotide is bound to the first labeling reagent by either covalent bonding or noncovalent interaction via a cleavablelinker described herein. In some embodiments, each of the four types of nucleotides is unlabeled, and wherein the second type of unlabeled nucleotides comprises a second functional moiety, and the kit further comprises a second labeling reagent, wherein the second labeling reagent comprises one or more second detectable labels and a second binding moiety that is capable of specific binding to the second functional moiety of the second type of unlabeled nucleotide. In some such embodiments, the second functional moiety of the second type of unlabeled nucleotide is bound to the second labeling reagent by either covalent bonding or noncovalent interaction via a cleavable linker described herein. In some further embodiments, the third type of unlabeled nucleotides comprises a third functional moiety, and the kit further comprises a third labeling reagent, wherein the third labeling reagent comprises one or more third detectable labels and a third binding moiety that is capable of specific binding to the third functional moiety of the third type of unlabeled nucleotide. In some such embodiments, the third functional moiety of the third type of unlabeled nucleotide is bound to the third labeling reagent by either covalent bonding or noncovalent interaction via a cleavable linker described herein. In other embodiments, the third type of unlabeled nucleotides comprises a mixture of the third type of unlabeled nucleotides comprising the first functional moiety and the third type of unlabeled nucleotides comprising the second functional moiety, and wherein both the first labeling reagent and the second labeling reagent are capable of specific binding to the third type of unlabeled nucleotides. The fourth type of unlabeled nucleotides is not capable of specific binding with any of the first, second, or third labeling reagent.

[0110] Post-incorporation labeling kits and methods have been described in U.S. Publication No. 2023 / 0383342 Al, which is incorporated by reference in its entirety. Non-limiting examples of noncovalent interaction between a functional moiety (X or X') of the nucleotide and a binding moiety of the labeling reagent include but are not limited to avidin (e.g., streptavidin or neutravidin) and biotin; dinitrophenyl (DNP) moiety and anti-DNP antibody; digoxigenin (DIG) and anti-DIG antibody; P-N-acetyl glucosamine (O-GlcNAc) and WGA (lectin); alkyl guanine moiety and SNAP-Tag®, alkyl chloride moiety and HaloTag®; 3 -nitrotyrosine and anti- nitrotyrosine antibody; nickel or cobalt complex such as Ni-nitrilotriacetic acid (NT A) and His- Tag; zinc complex and oligo- aspartate protein. Non-limiting examples of covalent interaction between a functional moiety and a labeling reagent include but are not limited to a biorthogonal reaction selected from the group consisting of a [3+21 dipolar cycloaddition, a Diels-Alder cycloaddition, a [4+1] cycloaddition, a phosphine ligation, or condensation with 2-acylphenyl boronic acid. For example, one of the functional moiety and the binding moiety comprises or is norbornene, transcyclooctene (TCO), dibenzocyclooctyne (DBCO), or bicyclo[6.1.0]nonyne (BCN), and the other one of the functional moiety and the binding moiety comprises or is azido.In some other embodiments, one of the functional moiety and the binding moiety comprises or is TCO, and the other one of the functional moiety and the binding moiety comprises or is an optionally substituted 1,2,4,5-tetrazine moiety.

[0111] Some additional embodiments of the present disclosure relate to a sequencing kit comprising:(a) an incorporation mixture comprising DNA polymerase and nucleotides A, G, C, and T or U (e.g. dATP, dCTP, dGTP and dTTP or dUTP) as described herein (e.g., the DNA polymerase is an altered archaeal DNA polymerase);(b) an aqueous deblocking solution comprising a palladium catalyst, tris(hydroxyalkyl)phosphine, and one or more buffer reagents that is suitable to chemically remove (i) 3' blocking groups from incorporated nucleotides to expose a 3 ’-OH group for further nucleotide incorporation on the solid support, and (ii) any detectable labels attached via cleavable linkers; and(c) an aqueous wash solution comprising a Pd(II) scavenger; wherein said kit is configured for performing at least about 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 cycles of sequencing-by-synthesis.

[0112] In some further embodiment, the kit may comprise four types of labeled nucleotides of fully functionalized nucleotides described herein (A, C, T and G), where each type of nucleotide comprises the 3' blocking group (such as the AOM blocking group) and the AOL linker moiety described herein. In further embodiments, G is unlabeled and does not comprise any cleavable linker. In still further embodiments, one or more the remaining three nucleotides (i.e., A, C and T) comprises an allylamine or allylamide linker moiety described herein. In one embodiment, the kit comprises unlabeled ffG, labeled ffA(s), labeled ffC, and labeled ffT (wherein ffT contains an allylamine or allylamide linker moiety between the nucleobase and the detectable label; also referred to as double bond or DB linker). In further embodiment, the kit comprises unlabeled ffG, labeled ffA(s), labeled ffC-DB, and labeled ftT-DB described herein. In some further embodiments, at least one type of the nucleotides comprises a base that is attached to a detectable label via a cleavable linker, and the cleavable linker is, wherein Z is -O-CH2-CH=CH2; n is an integer of 1, 2, 3, 4 or 5; * indicates the attachment point of the cleavable linker to the base; and ** indicates the attachment point of the cleavable linker to the detectable label. In further embodiments, the nucleobase of T nucleotide is attached to the detectable label via the cleavable linker. In some further embodiments, wherein each of at least three of the nucleotidesindependently comprises a base that is attached to a detectable label (or a functional moiety) via a cleavable linker, and the cleavable linker is selected from the group consisting of:, herein Z is -O-CH2-CH=CH2; n is an integer of 1, 2, 3, 4 or 5; * indicates the attachment point of the cleavable linker to the base; and ** indicates the attachment point of the cleavable linker to the detectable label or the functional moiety that is capable of attaching to a labeling reagent as described herein.

[0113] The modified nucleotide triphosphate with fluoro substitution on the terminal phosphate moiety is also referred to as y-F modified nucleotide. The fluoro substitution can be removed by subject to the y-F modified nucleotide to a basic environment ( such as 10 eq. methylamine) to generate a hydroxy group, thus transform the F-capped triphosphate to the normal triphosphate. In some embodiments, the kit may include y-F modified nucleotide and a chemical reagent for removal of the fluoro group.Methods of Sequencing

[0114] An aspect of the present disclosure relates to a method of determining the sequences of a plurality of different target polynucleotides, comprising:(a) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides;(b) contacting the solid support with an aqueous incorporation mixture comprising DNA polymerase and one or more of four different types of nucleotides (A, G, C, and T or U; dATP, dGTP, dCTP and dTTP or dUTP) under conditions suitable for DNA polymerase-mediated primer extension, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides, wherein at least one type of nucleotide is a nucleotide having a terminally modified triphosphate group as described herein carrying a detectable label through a cleavable linker, and wherein each of the one or more of four different type of nucleotides comprises a 3' blocking group covalently attached to the 2' deoxyribose of the nucleotide;(c) imaging and performing one or more fluorescent measurements of the extended copy polynucleotides to determine the identity of incorporated nucleotides;(d) removing the 3' blocking group from the nucleotides incorporated into the extended copy polynucleotides;(e) washing the solid support after the removal of the 3' blocking group from the incorporated nucleotides; and(f) repeating steps (b) to (e) until the sequences of at least a portion of the target polynucleotides are determined.

[0115] In some embodiments, steps (b) to (e) are repeated at least 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 cycles. In some embodiments, the removal of the 3' blocking group also removes the detectable label of the incorporated nucleotides.Post-Incorporation Labeling SBS Workflow

[0116] Another aspect of the present disclosure relates to a method of determining the sequences of a plurality of different target polynucleotides, comprising:(a) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides;(b) contacting the solid support with an aqueous incorporation mixture comprising DNA polymerase and one or more of four different types of nucleotides (A, G, C, and T or U; dATP, dGTP, dCTP and dTTP or dUTP) under conditions suitable for DNA polymerase-mediated primer extension, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides, wherein at least two types of nucleotides are unlabeled and at least one type of unlabeled nucleotide is a unlabeled nucleotide as described herein having a first functional moiety, and wherein each of the one or more of four different type of nucleotides comprises a 3' blocking group covalently attached to the 2' deoxyribose of the nucleotide;(c) contacting the extended copy polynucleotides with an aqueous labeling mixture comprising a first labeling reagent, wherein the first labeling reagent comprises one or more first detectable labels and a first binding moiety that is capable of specific binding to the first functional moiety of the unlabeled nucleotide;(d) imaging the solid support and performing one or more fluorescent measurements to determine the identity of incorporated nucleotides;(e) removing the 3' blocking group from the nucleotides incorporated into the extended copy polynucleotides;(f) washing the solid support after the removal of the 3' blocking group from the incorporated nucleotides; and(g) repeating steps (b) to (f) until the sequences of at least a portion of the target polynucleotides are determined.

[0117] In some embodiments, steps (b) to (f) are repeated at least 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 cycles. In some embodiments, the first functional moiety of the first type of unlabeled nucleotide is bound to the first labeling reagent by either covalent bonding or noncovalent interaction via a cleavable linker. In some embodiments, the removal of the 3' blocking group also removes the first labeling reagent. In some embodiments, the method is performed on an automated sequencing instrument comprising a single light source. In further embodiments, the single light source operates at a wavelength from about 500 nm to about 540 nm, or about 520 nm to about 525 nm. In some embodiments, the method is performed on an automated sequencing instrument comprising two light sources operating at two different wavelengths. In further embodiments, one light source operates at a wavelength from about 450 nm to about 460 nm, and the other light source operates at a wavelength from about 520 nm to about 525 nm.

[0118] In some embodiments, step (c) is performed under conditions where the first labeling reagent binds specifically to the incorporated unlabeled first type of nucleotides to provide labeled extended copy polynucleotides. In some embodiments, the first functional moiety of the first type of unlabeled nucleotide is bound to the first labeling reagent by covalent bonding, optionally via the cleavable linker as described herein. In some other embodiments, the first functional moiety of the first type of unlabeled nucleotide is bound to the first labeling reagent by noncovalent interaction, optionally via the cleavable linker as described herein.

[0119] In some embodiments, each of the four types of nucleotides in the aqueous incorporation mixture is unlabeled, the second type of unlabeled nucleotides comprises a second functional moiety, wherein the aqueous labeling mixture comprises a second labeling reagent, and the second labeling reagent comprises one or more second detectable labels and a second binding moiety that is capable of specific binding to the second functional moiety of the second type of unlabeled nucleotides. In some such embodiments, step (c) is performed under conditions where the first labeling reagent binds specifically to the incorporated unlabeled first type of nucleotides, and the second labeling reagent binds specifically to the incorporated unlabeled second type of nucleotides to provide labeled extended copy polynucleotides. In some such embodiments, the first functional moiety of the first type of unlabeled nucleotide is bound to the first labeling reagent by covalent bonding, optionally via a cleavable linker as described herein. In some other embodiments, the second functional moiety of the first type of unlabeled nucleotide is bound tothe second labeling reagent by noncovalent interaction, optionally via the cleavable linker as described herein. In some such embodiments, the third type of unlabeled nucleotides comprises a third functional moiety, wherein the aqueous labeling mixture comprises a third labeling reagent, and the third labeling reagent comprises one or more third detectable labels and a third binding moiety that is capable of specific binding to the third functional moiety of the third type of unlabeled nucleotides. In some such embodiments, step (c) is performed under conditions where the first labeling reagent binds specifically to the incorporated unlabeled first type of nucleotides, the second labeling reagent binds specifically to the incorporated unlabeled second type of nucleotides, and the third labeling reagent binds specifically to the incorporated unlabeled third type of nucleotides to provide labeled extended copy polynucleotides. In some other embodiments, the third type of unlabeled nucleotide comprises a mixture of the third type of unlabeled nucleotides comprising the first functional moiety and the third type of unlabeled nucleotides comprising the second functional moiety, and wherein both the first labeling reagent and the second labeling reagent are capable of specific binding to the third type of unlabeled nucleotides. In further embodiments, the fourth type of unlabeled nucleotides is not capable of specific binding with any of the first, second, or third labeling reagent. In some embodiments of the any of the sequencing methods described herein, each type of nucleotides has a terminally modified triphosphate group as described herein.

[0120] In any embodiments of the sequencing methods described herein, the solid support comprises at least 500,000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, or 5,000,000 spatially distinguishable sites / cm2that comprise multiple copies of target polynucleotides.Incorporation Mix

[0121] In some embodiments of the method described herein, step (b) is also referred to as the incorporation step, includes contacting a mixture containing one or more nucleotides (e.g., dATP, dCTP, dGTP, and dTTP or dUTP) with a copy polynucleotide / target polynucleotide complex in an incorporation solution comprising a polymerase and one or more buffering agents. In some such embodiments, the polymerase is a DNA polymerase, such as a mutant of 9°N polymerase (e.g., those disclosed in WO 2005 / 024010, which is incorporated by reference), for example, Pol 812, Pol 1901, Pol 1558 or Pol 963. The amino acid sequences of Pol 812, Pol 1901, Pol 1558 or Pol 963 DNA polymerases are described, for example, in U.S. Patent Publication Nos. 2020 / 0131484 Al and 2020 / 0181587 Al, both of which are incorporated by reference herein. Additional polymerases that may be used in the method include those disclosed in U.S. Ser. Nos. 63 / 412,241 and 63 / 433,971, both of which are incorporated by reference. In some embodiments, the one or more buffering agents comprise a primary amine, a secondary amine, a tertiary amine, a natural amino acid, or a non-natural amino acid, or combinations thereof. In furtherembodiments, the buffering agents comprise ethanolamine or glycine, or a combination thereof. In one embodiment, the buffer agent comprises or is glycine. In further embodiments, the mutant of 9°N polymerase may be engineered for high efficient incorporation of the nucleotide in accordance with the present disclosure.Cleavage Mix

[0122] In some embodiments of the method described herein, step (d) of the standard SBS or step (e) of the post-incorporation labeling SBS, also referred to as the cleaving step, includes contacting the incorporated nucleotide and the copy polynucleotide strand with a cleavage solution comprising a catalyst (e.g., a Pd(II) catalyst or a glycoside hydrolase or glycosidase such as an arabinofuranosidase, a glucosidase, a mannosidase, a xylosidase, a galactosidase, an N-acetyl-glucosaminidase, or a glucuronidase). In some embodiments, the cleavage solution comprises a catalyst capable of cleaving the linker group in accordance with the present disclosure. In further embodiments, the 3'-OH blocking group and the detectable label are removed in a single step of reaction.Embodiments and Alternatives of Sequencing-By-Synthesis

[0123] Alternatively, the sequencing methods described herein may also be carried out using unlabeled nucleotides and affinity reagents containing a fluorescent dye described herein. For example, one, two, three or each of the four different types of nucleotides (e.g., dATP, dCTP, dGTP and dTTP or dUTP) in the incorporation mixture of step (1) may be unlabeled. Each of the four types of nucleotides (e.g., dNTPs) has a 3' blocking group to ensure that only a single base can be added by a polymerase to the 3' end of the primer polynucleotide. After incorporation of an unlabeled nucleotide, the remaining unincorporated nucleotides are washed away. An affinity reagent is then introduced that specifically recognizes and binds to the incorporated dNTP to provide a labeled extension product comprising the incorporated dNTP. Uses of unlabeled nucleotides and affinity reagents in sequencing by synthesis have been disclosed in WO 2018 / 129214 and WO 2020 / 097607. A modified sequencing method of the present disclosure using unlabeled nucleotides may include the following steps:(1) contacting a solid support with sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides;(2) contacting the solid support with an aqueous solution comprising DNA polymerase and one or more of four different types of unlabeled nucleotides (A, G, C and T or U) under conditions suitable for DNA polymerase-mediated primer extension,wherein each of the nucleotides comprises a 3' blocking group and at least one type of nucleotide comprising a blocking group as described herein;(3) contacting the extended copy polynucleotides with a set of affinity reagents under conditions wherein one affinity reagent binds specifically to the incorporated unlabeled nucleotides to provide labeled extended copy polynucleotides;(4) imaging the solid support to determine the identity of the incorporated nucleotides (e.g., by performing one or more fluorescent measurements of the extended copy polynucleotides); and(5) contacting the solid support with an aqueous deblocking solution comprising a palladium catalyst and tris(hydroxyalkyl)phosphine under conditions suitable to chemically remove 3' allyl blocking groups from incorporated nucleotides to expose a 3’- OH group for further nucleotide incorporation on the solid support; and(6) repeating steps (2)-(5) to determine target polynucleotide sequences.

[0124] In some embodiments of the modified sequencing method described herein, the method further comprises removing the affinity reagents from the incorporated nucleotides. In still further embodiments, the 3' blocking group and the affinity reagent are removed in the same reaction. In some embodiments, the method further comprises a step (6) washing the solid support with a third aqueous wash solution. In further embodiments, steps (2) through (6) are repeated at least 50, 100, 150, 200, 250, or 300 cycles to determine the target polynucleotide sequences. In some embodiments, the set of affinity reagents may comprise a first affinity reagent that binds specifically to the first type of nucleotide, a second affinity reagent that binds specifically to the second type of nucleotide, and a third affinity reagent that binds specifically to the third type of nucleotide. In some further embodiments, each of the first, second and the third affinity reagents comprises a detectable labeled that is spectrally distinguishable. In some embodiments, the affinity reagents may include protein tags, antibodies (including but not limited to binding fragments of antibodies, single chain antibodies, bispecific antibodies, and the like), aptamers, knottins, affimers, or any other known agent that binds an incorporated nucleotide with a suitable specificity and affinity. In one embodiment, at least one affinity reagent is an antibody or a protein tag. In another embodiment, at least one of the first type, the second type, and the third type of affinity reagents is an antibody or a protein tag comprising one or more detectable labels (e.g., multiple copies of the same detectable label), wherein the detectable label is or comprises a bis-boron dye moiety described herein.

[0125] Some embodiments include pyrosequencing techniques. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the nascent strand (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P.(1996) “Real-time DNA sequencing using detection of pyrophosphate release.” Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) “Pyrosequencing sheds light on DNA sequencing.” Genome Res. 11(1), 3-11; Ronaghi, M., Uhlen, M. and Nyren, P. (1998) “A sequencing method based on real-time pyrophosphate.” Science 281(5375), 363; U.S. Pat. Nos. 6,210,891; 6,258,568 and 6,274,320, the disclosures of which are incorporated herein by reference in their entireties). In pyrosequencing, released PPi can be detected by being immediately converted to adenosine triphosphate (ATP) by ATP sulfurase, and the level of ATP generated is detected via luciferase-produced photons. The nucleic acids to be sequenced can be attached to features in an array and the array can be imaged to capture the chemiluminescent signals that are produced due to incorporation of a nucleotides at the features of the array. An image can be obtained after the array is treated with a particular nucleotide type (e.g., A, T, C or G). Images obtained after addition of each nucleotide type will differ with regard to which features in the array are detected. These differences in the image reflect the different sequence content of the features on the array. However, the relative locations of each feature will remain unchanged in the images. The images can be stored, processed and analyzed using the methods set forth herein. For example, images obtained after treatment of the array with each different nucleotide type can be handled in the same way as exemplified herein for images obtained from different detection channels for reversible terminator- based sequencing methods.

[0126] In another exemplary type of SBS, cycle sequencing is accomplished by stepwise addition of reversible terminator nucleotides containing, for example, a cleavable or photobleachable dye label as described, for example, in WO 04 / 018497 and U.S. Pat. No. 7,057,026, the disclosures of which are incorporated herein by reference. This approach is being commercialized by Solexa (now Illumina, Inc.), and is also described in WO 91 / 06678 and WO 07 / 123,744, each of which is incorporated herein by reference. The availability of fluorescently - labeled terminators in which both the termination can be reversed, and the fluorescent label cleaved facilitates efficient cyclic reversible termination (CRT) sequencing. Polymerases can also be co-engineered to efficiently incorporate and extend from these modified nucleotides.

[0127] Preferably in reversible terminator-based sequencing embodiments, the labels do not substantially inhibit extension under SBS reaction conditions. However, the detection labels can be removable, for example, by cleavage or degradation. Images can be captured following incorporation of labels into arrayed nucleic acid features. In particular embodiments, each cycle involves simultaneous delivery of four different nucleotide types to the array and each nucleotide type has a spectrally distinct label. Four images can then be obtained, each using a detection channel that is selective for one of the four different labels. Alternatively, different nucleotide types can be added sequentially, and an image of the array can be obtained betweeneach addition step. In such embodiments each image will show nucleic acid features that have incorporated nucleotides of a particular type. Different features will be present or absent in the different images due the different sequence content of each feature. However, the relative position of the features will remain unchanged in the images. Images obtained from such reversible terminator-SBS methods can be stored, processed and analyzed as set forth herein. Following the image capture step, labels can be removed, and reversible terminator moieties can be removed for subsequent cycles of nucleotide addition and detection. Removal of the labels after they have been detected in a particular cycle and prior to a subsequent cycle can provide the advantage of reducing background signal and crosstalk between cycles. Examples of useful labels and removal methods are set forth below.

[0128] Some embodiments can utilize detection of four different nucleotides using fewer than four different labels. For example, SBS can be performed utilizing methods and systems described in the incorporated materials of U.S. Pub. No. 2013 / 0079232. As a first example, a pair of nucleotide types can be detected at the same wavelength, but distinguished based on a difference in intensity for one member of the pair compared to the other, or based on a change to one member of the pair (e.g., via chemical modification, photochemical modification or physical modification) that causes apparent signal to appear or disappear compared to the signal detected for the other member of the pair. As a second example, three of four different nucleotide types can be detected under particular conditions while a fourth nucleotide type lacks a label that is detectable under those conditions, or is minimally detected under those conditions (e.g. , minimal detection due to background fluorescence, etc.). Incorporation of the first three nucleotide types into a nucleic acid can be determined based on presence of their respective signals and incorporation of the fourth nucleotide type into the nucleic acid can be determined based on absence or minimal detection of any signal. As a third example, one nucleotide type can include label(s) that are detected in two different channels, whereas other nucleotide types are detected in no more than one of the channels. The aforementioned three exemplary configurations are not considered mutually exclusive and can be used in various combinations. An exemplary embodiment that combines all three examples, is a fluorescent-based SBS method that uses a first nucleotide type that is detected in a first channel (e.g., dATP having a label that is detected in the first channel when excited by a first excitation wavelength), a second nucleotide type that is detected in a second channel (e.g., dCTP having a label that is detected in the second channel when excited by a second excitation wavelength), a third nucleotide type that is detected in both the first and the second channel (e.g., dTTP having at least one label that is detected in both channels when excited by the first and / or second excitation wavelength) and a fourth nucleotidetype that lacks a label that is not, or minimally, detected in either channel (e.g., dGTP having no label).

[0129] Further, as described in the incorporated materials of U.S. Pub. No. 2013 / 0079232, sequencing data can be obtained using a single channel. In such so-called one-dye sequencing approaches, the first nucleotide type is labeled but the label is removed after the first image is generated, and the second nucleotide type is labeled only after a first image is generated. The third nucleotide type retains its label in both the first and second images, and the fourth nucleotide type remains unlabeled in both images.

[0130] Some embodiments can utilize sequencing by ligation techniques. Such techniques utilize DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides. The oligonucleotides typically have different labels that are correlated with the identity of a particular nucleotide in a sequence to which the oligonucleotides hybridize. As with other SBS methods, images can be obtained following treatment of an array of nucleic acid features with the labeled sequencing reagents. Each image will show nucleic acid features that have incorporated labels of a particular type. Different features will be present or absent in the different images due the different sequence content of each feature, but the relative position of the features will remain unchanged in the images. Images obtained from ligation-based sequencing methods can be stored, processed and analyzed as set forth herein. Exemplary SBS systems and methods which can be utilized with the methods and systems described herein are described in U.S. Pat. Nos. 6,969,488, 6,172,218, and 6,306,597, the disclosures of which are incorporated herein by reference in their entireties.

[0131] Some embodiments can utilize nanopore sequencing (Deamer, D. W. & Akeson, M. “Nanopores and nucleic acids: prospects for ultrarapid sequencing.” Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D. Branton, “Characterization of nucleic acids by nanopore analysis”, Ace. Chem. Res. 35:817-825 (2002); Li, I., M. Gershow, D. Stein, E. Brandin, and J. A. Golovchenko, “DNA molecules and configurations in a solid-state nanopore microscope” Nat. Mater. 2:611-615 (2003), the disclosures of which are incorporated herein by reference in their entireties). In such embodiments, the target nucleic acid passes through a nanopore. The nanopore can be a synthetic pore or biological membrane protein, such as a- hemolysin. As the target nucleic acid passes through the nanopore, each base-pair can be identified by measuring fluctuations in the electrical conductance of the pore. (U.S. Pat. No. 7,001,792; Soni, G. V. & Meller, A. “Progress toward ultrafast DNA sequencing using solid-state nanopores.” Clin. Chem. 53, 1996-2001 (2007); Healy, K. “Nanopore-based single-molecule DNA analysis.” Nanomed. 2, 459-481 (2007); Cockroft, S. L., Chu, J., Amorin, M. & Ghadiri, M. R. “A singlemolecule nanopore device detects DNA polymerase activity with single-nucleotide resolution.” J.Am. Chem. Soc. 130, 818-820 (2008), the disclosures of which are incorporated herein by reference in their entireties). Data obtained from nanopore sequencing can be stored, processed and analyzed as set forth herein. In particular, the data can be treated as an image in accordance with the exemplary treatment of optical images and other images that is set forth herein.

[0132] Some other embodiments of sequencing methods involve the use the 3' blocked nucleotide described herein in nanoball sequencing technique, such as those described in U.S. Patent No. 9,222,132, the disclosure of which is incorporated by reference. Through the process of rolling circle amplification (RCA), a large number of discrete DNA nanoballs may be generated. The nanoball mixture is then distributed onto a patterned slide surface containing features that allow a single nanoball to associate with each location. In DNA nanoball generation, DNA is fragmented and ligated to the first of four adapter sequences. The template is amplified, circularized and cleaved with a type II endonuclease. A second set of adapters is added, followed by amplification, circularization and cleavage. This process is repeated for the remaining two adapters. The final product is a circular template with four adapters, each separated by a template sequence. Library molecules undergo a rolling circle amplification step, generating a large mass of concatemers called DNA nanoballs, which are then deposited on a flow cell. Goodwin et al., “Coming of age: ten years of next-generation sequencing technologies,” Nat Rev Genet. 2016;17(6):333-51.

[0133] Some embodiments can utilize methods involving the real-time monitoring of DNA polymerase activity. Nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and y- phosphate-labeled nucleotides as described, for example, in U.S. Pat. Nos. 7,329,492 and 7,211,414, both of which are incorporated herein by reference, or nucleotide incorporations can be detected with zero-mode waveguides as described, for example, in U.S. Pat. No. 7,315,019, which is incorporated herein by reference, and using fluorescent nucleotide analogs and engineered polymerases as described, for example, in U.S. Pat. No. 7,405,281 and U.S. Pub. No. 2008 / 0108082, both of which are incorporated herein by reference. The illumination can be restricted to a zeptoliter-scale volume around a surface-tethered polymerase such that incorporation of fluorescently labeled nucleotides can be observed with low background (Levene, M. J. et al. “Zero-mode waveguides for single-molecule analysis at high concentrations.” Science 299, 682-686 (2003); Lundquist, P. M. et al. “Parallel confocal detection of single molecules in real time.” Opt. Lett. 33, 1026-1028 (2008); Korlach, J. et al. “Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nano structures.” Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which areincorporated herein by reference in their entireties). Images obtained from such methods can be stored, processed and analyzed as set forth herein.

[0134] Some SBS embodiments include detection of a proton released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons can use an electrical detector and associated techniques that are commercially available from Ion Torrent (Guilford, CT, a Life Technologies subsidiary) or sequencing methods and systems described in U.S. Pub. Nos. 2009 / 0026082; 2009 / 0127589; 2010 / 0137143; and 2010 / 0282617, all of which are incorporated herein by reference. Methods set forth herein for amplifying target nucleic acids using kinetic exclusion can be readily applied to substrates used for detecting protons. More specifically, methods set forth herein can be used to produce clonal populations of amplicons that are used to detect protons.

[0135] The above SBS methods can be advantageously carried out in multiplex formats such that multiple different target nucleic acids are manipulated simultaneously. In particular embodiments, different target nucleic acids can be treated in a common reaction vessel or on a surface of a particular substrate. This allows convenient delivery of sequencing reagents, removal of unreacted reagents and detection of incorporation events in a multiplex manner. In embodiments using surface-bound target nucleic acids, the target nucleic acids can be in an array format. In an array format, the target nucleic acids can be typically bound to a surface in a spatially distinguishable manner. The target nucleic acids can be bound by direct covalent attachment, attachment to a bead or other particle or binding to a polymerase or other molecule that is attached to the surface. The array can include a single copy of a target nucleic acid at each site (also referred to as a feature) or multiple copies having the same sequence can be present at each site or feature. Multiple copies can be produced by amplification methods such as, bridge amplification or emulsion PCR as described in further detail below.

[0136] The methods set forth herein can use arrays having features at any of a variety of densities including, for example, at least about 10 features / cm2, 100 features / cm2, 500 features / cm2, 1,000 features / cm2, 5,000 features / cm2, 10,000 features / cm2, 50,000 features / cm2, 100,000 features / cm2, 1,000,000 features / cm2, 5,000,000 features / cm2, or higher.

[0137] An advantage of the methods set forth herein is that they provide for rapid and efficient detection of a plurality of target nucleic acid in parallel. Accordingly, the present disclosure provides integrated systems capable of preparing and detecting nucleic acids using techniques known in the art such as those exemplified above. Thus, an integrated system of the present disclosure can include fluidic components capable of delivering amplification reagents and / or sequencing reagents to one or more immobilized DNA fragments, the system comprising components such as pumps, valves, reservoirs, fluidic lines and the like. A flow cell can beconfigured and / or used in an integrated system for detection of target nucleic acids. Exemplary flow cells are described, for example, in U.S. Pub. No. 2010 / 0111768 and US Ser. No. 13 / 273,666, each of which is incorporated herein by reference. As exemplified for flow cells, one or more of the fluidic components of an integrated system can be used for an amplification method and for a detection method. Taking a nucleic acid sequencing embodiment as an example, one or more of the fluidic components of an integrated system can be used for an amplification method set forth herein and for the delivery of sequencing reagents in a sequencing method such as those exemplified above. Alternatively, an integrated system can include separate fluidic systems to carry out amplification methods and to carry out detection methods. Examples of integrated sequencing systems that are capable of creating amplified nucleic acids and also determining the sequence of the nucleic acids include, without limitation, the MiSeq™ platform (Illumina, Inc., San Diego, CA) and devices described in US Ser. No. 13 / 273,666, which is incorporated herein by reference.

[0138] Arrays in which polynucleotides have been directly attached to silica-based supports are those for example disclosed in WO 00 / 06770 (incorporated herein by reference), wherein polynucleotides are immobilized on a glass support by reaction between a pendant epoxide group on the glass with an internal amino group on the polynucleotide. In addition, polynucleotides can be attached to a solid support by reaction of a sulfur-based nucleophile with the solid support, for example, as described in WO 2005 / 047301 (incorporated herein by reference). A still further example of solid-supported template polynucleotides is where the template polynucleotides are attached to hydrogel supported upon silica-based or other solid supports, for example, as described in WO 00 / 31148, WO 01 / 01143, WO 02 / 12566, WO 03 / 014392, U.S. Pat. No. 6,465,178 and WO 00 / 53812, each of which is incorporated herein by reference.

[0139] A particular surface to which template polynucleotides may be immobilized is a polyacrylamide hydrogel. Polyacrylamide hydrogels are described in the references cited above and in WO 2005 / 065814, which is incorporated herein by reference. Specific hydrogels that may be used include those described in WO 2005 / 065814 and U.S. Pub. No. 2014 / 0079923. In one embodiment, the hydrogel is PAZAM (poly(N-(5-azidoacetamidylpentyl) acrylamide-co- acrylamide)).

[0140] DNA template molecules can be attached to beads or microparticles, for example, as described in U.S. Pat. No. 6,172,218 (which is incorporated herein by reference). Attachment to beads or microparticles can be useful for sequencing applications. Bead libraries can be prepared where each bead contains different DNA sequences. Exemplary libraries and methods for their creation are described in Nature, 437, 376-380 (2005); Science, 309, 5741, 1728-1732 (2005), each of which is incorporated herein by reference. Sequencing of arrays of such beads using nucleotides set forth herein is within the scope of the disclosure.

[0141] Templates that are to be sequenced may form part of an “array” on a solid support, in which case the array may take any convenient form. Thus, the method of the disclosure is applicable to all types of high-density arrays, including single-molecule arrays, clustered arrays, and bead arrays. Labeled nucleotides of the present disclosure may be used for sequencing templates on essentially any type of array, including but not limited to those formed by immobilization of nucleic acid molecules on a solid support.

[0142] However, labeled nucleotides of the disclosure are particularly advantageous in the context of sequencing of clustered arrays. In clustered arrays, distinct regions on the array (often referred to as sites, or features) comprise multiple polynucleotide template molecules. Generally, the multiple polynucleotide molecules are not individually resolvable by optical means and are instead detected as an ensemble. Depending on how the array is formed, each site on the array may comprise multiple copies of one individual polynucleotide molecule (e.g., the site is homogenous for a particular single- or double-stranded nucleic acid species) or even multiple copies of a small number of different polynucleotide molecules (e.g., multiple copies of two different nucleic acid species). Clustered arrays of nucleic acid molecules may be produced using techniques generally known in the art. By way of example, WO 98 / 44151 and WO 00 / 18957, each of which is incorporated herein, describe methods of amplification of nucleic acids wherein both the template and amplification products remain immobilized on a solid support in order to form arrays comprised of clusters or “colonies” of immobilized nucleic acid molecules. The nucleic acid molecules present on the clustered arrays prepared according to these methods are suitable templates for sequencing using the nucleotides labeled with dye compounds of the disclosure.

[0143] The labeled nucleotides of the present disclosure are also useful in sequencing of templates on single molecule arrays. The term “single molecule array” or “SMA” as used herein refers to a population of polynucleotide molecules, distributed (or arrayed) over a solid support, wherein the spacing of any individual polynucleotide from all others of the population is such that it is possible to individually resolve the individual polynucleotide molecules. The target nucleic acid molecules immobilized onto the surface of the solid support can thus be capable of being resolved by optical means in some embodiments. This means that one or more distinct signals, each representing one polynucleotide, will occur within the resolvable area of the particular imaging device used.

[0144] Single molecule detection may be achieved wherein the spacing between adjacent polynucleotide molecules on an array is at least 100 nm, more particularly at least 250nm, still more particularly at least 300 nm, even more particularly at least 350 nm. Thus, each molecule is individually resolvable and detectable as a single molecule fluorescent point, and fluorescence from said single molecule fluorescent point also exhibits single step photobleaching.

[0145] The terms “individually resolved” and “individual resolution” are used herein to specify that, when visualized, it is possible to distinguish one molecule on the array from its neighboring molecules. Separation between individual molecules on the array will be determined, in part, by the particular technique used to resolve the individual molecules. The general features of single molecule arrays will be understood by reference to published applications WO 00 / 06770 and WO 01 / 57248, each of which is incorporated herein by reference. Although one use of the nucleotides of the disclosure is in sequencing-by-synthesis reactions, the utility of the nucleotides is not limited to such methods. In fact, the nucleotides may be used advantageously in any sequencing methodology which requires detection of fluorescent labels attached to nucleotides incorporated into a polynucleotide.

[0146] In particular, the labeled nucleotides of the disclosure may be used in automated fluorescent sequencing protocols, particularly fluorescent dye-terminator cycle sequencing based on the chain termination sequencing method of Sanger and co-workers. Such methods generally use enzymes and cycle sequencing to incorporate fluorescently labeled dideoxynucleotides in a primer extension sequencing reaction. So-called Sanger sequencing methods, and related protocols (Sanger-type), utilize randomized chain termination with labeled dideoxynucleo tides .

[0147] Thus, the present disclosure also encompasses labeled nucleotides which are dideoxynucleotides lacking hydroxyl groups at both of the 3' and 2' positions, such dideoxynucleotides being suitable for use in Sanger type sequencing methods and the like.

[0148] Labeled nucleotides of the present disclosure incorporating 3' blocking groups, it will be recognized, may also be of utility in Sanger methods and related protocols since the same effect achieved by using dideoxy nucleotides may be achieved by using nucleotides having 3'-OH blocking groups: both prevent incorporation of subsequent nucleotides. Where nucleotides according to the present disclosure, and having a 3' blocking group are to be used in Sanger-type sequencing methods it will be appreciated that the dye compounds or detectable labels attached to the nucleotides need not be connected via cleavable linkers, since in each instance where a labeled nucleotide of the disclosure is incorporated; no nucleotides need to be subsequently incorporated and thus the label need not be removed from the nucleotide.EXAMPLES

[0149] Additional embodiments are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the claims.Example 1. Stability Testing of Modified Nucleotides

[0150] Several different modified dTTPs were obtained having the structure. An unmodified dTTP (where RTPCis OH) was used as a control. The modified dTTPs included dTTP-yANS (where RTPCisOH OH OH ). The unmodified and modified dTTPs (2 pM) were subjected to 60 °C for 14 days in 50 mM bis-tris-propane buffer at pH 8.

[0151] FIG. 1 plots the proportion of the degradation products for each of the dTTPs (monophosphate and / or diphosphate), the proportion of the degradation products of deoxynucleotide tetraphosphates (dT4Ps) (e.g. monophosphate, diphosphate, and / or triphosphate), and the degradation products of deoxynucleotide hexaphosphates (dT6Ps) (monophosphate, diphosphate, triphosphate, and / or tetraphosphate). Many of the modified dTTPs (e.g., dTTP-yANS, dTTP-yBz, dTTP-yF, dTTP-yOCH3, dT4P-5F, dT6P-C6-NH2, and dT6P- PEG3-NH2) were very stable under the heat stress condition and have very little degradation products, indicating that these modifications greatly improve the phosphate group stability.Synthesis of y-F modified FFN

[0152] The general synthetic scheme for preparing a y-F modified labeled ffN is illustrated below.

[0153] The synthesis of a modified y-F-FFA with AOM blocking group and AOL cleavable linker and a coumarin blue dye label is described herein. The fluorophosphorylation of AOM-DB-dA was carried out by the addition of POC1 at low temperature followed by TBAPP (tri-n-butylammonium pyrophosphate). Fluoride installation was achieved by the addition of CsF. Removal of the nucleobase protecting groups was achieved using an excess of methylamine. AOM-DB-dA(y-F) was purified using ion exchange and reverse phase chromatography and quantified using UV-vis spectroscopy. Structural analysis was performed using31P and19F NMR to demonstrate that fluoride modification had occurred at the y-P atom. The NMR spectra obtained matched literature examples of y-fluorophosphorylated compounds {J. Am. Chem. Soc. 2019, 141, 38, 15013-15017). Mass spectrometry identified the molecular ion and fragment analysis further supported fluorination at the γ-P atom. Installation of the linker and subsequently the dye moieties were performed using established Illumina chemistry as described in U.S. Publication No. 2021 / 0403500 A1, which is incorporated by reference.Chemical Formula:yF-Dark G

[0154] FIG. 2 is a comparison of proton coupled and decoupled31P NMR spectra of γF-AOM-DB-dATP with proton coupled31P NMR of unmodified AOM-DB-dATP, which demonstrates the presence of only one fluorine atom and also demonstrates that fluorine atom is on the gamma position of the triphosphate.Example 2. Incorporation of Modified dTTPs

[0155] The modified dTTPs discussed with reference to Example 1 above were also subjected to a polymerase-mediated strand displacement assay to determine ability to incorporate such dTTPs. In the assay, a SBS polymerase Pol 1901 was used to incorporate available dTTPs (50 μM). For strands experiencing successful incorporation of the dTTPs, an annealed primer with an attached quencher was eventually displaced, thereby allowing a fluorophore on the strand opposite the quencher to delectably fluoresce.

[0156] FIG. 3 plots the incorporation activity of the modified dTTPs relative to that of unmodified dTTP. The -yF capping group was selected for further testing.Example 3. Stability of yF-FFN in Response to Heat Stress

[0157] FFC was modified to include a fluorine terminal phosphate cap in accordance with the present disclosure (referred to herein as “yF-FFC-1”) as described in Example 1. The yF- FFC-1 and unmodified FFC were aliquoted at a concentration of 2 mM and stored in 15 mM MOPS buffer at pH 7 at 24°C, 40°C, 50°C, or 60°C for up to eleven days. Samples were taken from each aliquot at 0, 1, 2, 4, 7, and 11 days and the amount of remaining triphosphate FFC was measured.

[0158] FIGs. 4A and 4B illustrate the percentage of remaining triphosphate FFC, normalized to the amount measured on day 0. FIG. 4A plots the percentage of remaining unmodified FFC, whereas FIG. 4B plots the percentage of remaining modified FFC-yF. For each temperature condition, yF-FFC-1 had a significantly lower degradation rate as compared to the unmodified FFC. At 24 °C, the rate of degradation was 0.15% per day for the FFC, whereas the rate of degradation for FFC-yF was 0.02% per day. At 40°C, the rate of degradation was 0.86% per day for the FFC, whereas the rate of degradation for FFC-yF was 0.02% per day. At 50°C, the rate of degradation was 3.19% per day for the FFC, whereas the rate of degradation for yF-FFC-1 was 0.09% per day. At 60°C, the rate of degradation was 24.1% per day for the FFC, whereas the rate of degradation for FFC-yF was 0.38% per day. Similar trends were observed in the stability testing between FFT and yF-FFT, between FFA and yF-FFA-1, and between dark G and yF-dark G.Example 4. Stability of yF-FFA-2 in various pH conditions

[0159] FFA was modified to include a fluorine terminal phosphate cap in accordance with the present disclosure (referred to herein as “yF-FFA-2”) as described in Example 1. yF- FFA-2 and unmodified FFA were aliquoted at a concentration of 200 pM and stored in 15 mM MOPS buffer at room temperature for 8 days at three pH conditions - 6.5, 7 and 8. Samples were taken from each aliquot at day 0 and day 8 and the amount of remaining triphosphate FFA or yF- FFA-2 was measured. As illustrated in the table below, yF-FFA-2 showed significant stability at pH 6.5 as compared to the standard unmodified FFA. After 8 days, the standard FFA has completely degraded to 100% diphosphate but there were still about 80% remaining yF-FFA-2.Example 5. Incorporation of yF-dATP and yF-FFA-2

[0160] dATP and FFA were both modified to include a fluorine terminal phosphate cap as described herein by replacing a hydroxy group of the termina phosphate with a fluoro group (referred to herein as “dATP-yF” and “FFA-yF”). yF-dATP, yF-FFA-2, and unmodified FFA were subject to an incorporation assay using an SBS polymerase Pol 1901 at concentrations of 1 pM and 0.5 pM in an ethanolamine buffer. The SBS polymerase was used at a concentration of 330 nM at 40 °C, with 20 nM of primer template and 9 mM of MgSO4.

[0161] FIG. 5 plots the percentage of incorporated nucleotide over time for the three types of nucleotides. Both yF-dATP and yF-FFA-2 were capable of being incorporated using the SBS polymerase Pol 1901.Example 6. Sequencing by synthesis using γF modified FFNs

[0162] The FFA-yF and dark G-yF described herein were tested on an Illumina NextSeq 2000™ instrument using Blue / Green 2ex-2ch chemistry. The instrument was set up to enable multi-read rehybridization runs to allow screening of multiple incorporation mix formulations of the recipe: Dark G at 2 pM, green FFT at 1 pM, red FFA at 1.05 pM, blue FFA at 0.95 pM, green FFC at 1.1 pM and blue FFC at 0.9 pM with 4 mM MgSO4, 0.2% CHAPS, 50 mM NaCl, 1 mM EDTA in 50 mM glycine buffer pH 9.6. In the test example either the standard blue FFA was substituted for yF-FFA-2 at a concentration of 0.95 pM or the standard dark G was substituted for yF-dark G at a concentration of 2 pM. The other FFNs in the incorporation mix were kept as standard. The incorporation time was extended to 30 minutes to account for the unoptimized incorporation rate of the yF modified FFNs. 26 cycles of sequencing-by-synthesis were carried out. It was observed that both yF-FFA-2 and yF-dark G were incorporated by the DNA polymerase and subsequent cycles of SBS were able to be carried out.

Claims

WHAT IS CLAIMED IS:

1. A nucleotide comprising a nucleobase, a ribose or 2 ' deoxyribose having a 3" hydroxy blocking group, and a terminally modified triphosphate group, wherein at least one hydroxy group of the terminal phosphate moiety of the terminally modified triphosphate group is replaced by:(a) a substituent RT PCselected from the group consisting of a halo, unsubstituted or substituted (C1-C6alkyl)amino, unsubstituted or substituted (C6-C10aryl)amino, unsubstituted or substituted (5 to 10 membered heteroaryl)amino, unsubstituted or substituted C1-C6alkyl, unsubstituted or substituted C1-C6alkoxy, unsubstituted or substituted C6-C10aryloxy, unsubstituted or substituted (C1-C6alkoxy)C1-C6alkyl, unsubstituted or substituted -O-(C1-C6alkoxy)C1-C6alkyl, unsubstituted or substituted -0-(C6-C10aryl)C1-C6alkyl, unsubstituted or substituted -O-(5 to 10 membered heteroaryl)C1-C6alkyl, (unsubstituted or substituted amino)(C1-C6alkylene)-O-, and (unsubstituted or substituted amino)(2 to 20 heteroalkylene)- O-;(b) a monophosphate, diphosphate, or triphosphate group having at least one terminal hydroxy group independently substituted with the substituent RT PC; or(c) a second nucleotide comprising a second nucleobase, a ribose or 2' deoxyribose, and a monophosphate, diphosphate or triphosphate group, wherein the second nucleotide is connected to the nucleotide via the monophosphate, diphosphate or triphosphate group of the second nucleotide.

2. The nucleotide of claim 1, having the structure of formula (I):salt thereof, wherein B is the nucleobase;L is an optionally present cleavable linker;X is a detectable label or a functional moiety that can attach to a labeling reagent; PG is the hydroxy blocking group attached to the 3' oxygen of the nucleotide; and n is an integer of 1, 2, 3 or 4.

3. The nucleotide of claim 1 or 2, wherein RTPCis fluoro.

4. The nucleotide of claim 1 or 2, wherein RTPCismethoxy.

5. The nucleotide of claim 1 or 2, wherein RTPCis6. The nucleotide of claim 1 or 2, wherein RT PCis -O(CH2)1-6-NH2or -O(CH2CH2O)1-4- CH2CH2NH2.

7. The nucleotide of any one of claims 2 to 6, wherein n is 1.

8. The nucleotide of any one of claims 2 to 6, wherein n is 2 or 4.

9. The nucleotide of claim 1, having the structure of formula (II):(II), or a salt thereof, wherein B is the nucleobase;B' is the second nucleobase; each of L and L' is independently an optionally present cleavable linker;X is a detectable label or a functional moiety that can attach to a labeling reagent;X' is a second detectable label or a second functional moiety that can attach to a second labeling reagent;PG is the 3 ' hydroxy blocking group attached to the 3 'oxygen of the nucleotide;PG' is a 3' hydroxy blocking group attached to the 3 'oxygen of the second nucleotide; and m is an integer of 2, 3 or 4.

10. The nucleotide of claim 9, wherein B and B' are the same.

11. The nucleotide of claim 9 or 10, wherein PG and PG' are the same.

12. The nucleotide of any one of claims 2 to 11, wherein PG is azidomethyl (-CH2N3), allyl(-CH2CH=CH2), or -CH2OCH2CH=CH2.

13. The nucleotide of any one of claims 2 to 12, wherein L is present and wherein L and the 3' hydroxy blocking group are cleavable under the same reaction condition.

14. An oligonucleotide or polynucleotide comprising the nucleotide of any one of claims 1 to 13 incorporated thereto.

15. The oligonucleotide or polynucleotide of claim 14, wherein the oligonucleotide or polynucleotide is at least partially complementary and hybridized to a target polynucleotide immobilized on a surface of a solid support.

16. The oligonucleotide or polynucleotide of claim 15, wherein the solid support comprises an array of different target polynucleotides immobilized thereon.

17. A kit comprising a first type of nucleotide according to any one of claims 1 to 13.

18. The kit of claim 17, wherein the first type of nucleotide carries a first detectable label.

19. The kit of claim 18, comprising four types of nucleotides, wherein a second type of nucleotide carries a second detectable label, a third type of nucleotide carries a third detectable label, and a fourth type of nucleotide is unlabeled (dark), and wherein each of the detectable labels has a distinct emission maximum that is distinguishable from the other detectable labels.

20. The kit of claim 18, comprising four types of nucleotides, wherein a second type of nucleotide carries a second detectable label, a third type of nucleotide comprises a mixture of the third type of nucleotide carrying the first detectable label and the third type of nucleotide carrying the second detectable label, and a fourth type of nucleotide is unlabeled (dark).

21. The kit of claim 17, wherein the first type of nucleotide is unlabeled and has a first functional moiety that can attach to a labeling reagent.

22. The kit of claim 21 , wherein the kit further comprises a first labeling reagent, wherein the first labeling reagent comprises one or more first detectable labels and a first binding moiety that is capable of specific binding to the first functional moiety of the first type of unlabeled nucleotide.

23. The kit of claim 21 or 22, wherein each of the four types of nucleotides is unlabeled, and wherein the second type of unlabeled nucleotides comprises a second functional moiety, and the kit further comprises a second labeling reagent, wherein the second labeling reagent comprises one or more second detectable labels and a second binding moiety that is capable of specific binding to the second functional moiety of the second type of unlabeled nucleotide.

24. The kit of claim 23, wherein the third type of unlabeled nucleotides comprises a third functional moiety, and the kit further comprises a third labeling reagent, wherein the third labeling reagent comprises one or more third detectable labels and a third binding moiety that is capable of specific binding to the third functional moiety of the third type of unlabeled nucleotide.

25. The kit of claim 23, wherein the third type of unlabeled nucleotides comprises a mixture of the third type of unlabeled nucleotides comprising the first functional moiety and the third type of unlabeled nucleotides comprising the second functional moiety, and wherein both the first labeling reagent and the second labeling reagent are capable of specific binding to the third type of unlabeled nucleotides.

26. The kit of claim 24 or 25, wherein the fourth type of unlabeled nucleotides is not capable of specific binding with any of the first, second, or third labeling reagent.

27. The kit according to any one of claims 17 to 26, further comprising a DNA polymerase and one or more buffer compositions.

28. A method of determining the sequences of a plurality of different target polynucleotides, comprising:(a) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides;(b) contacting the solid support with an aqueous incorporation mixture comprising DNA polymerase and one or more of four different types of nucleotides under conditions suitable for DNA polymerase-mediated primer extension, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides, wherein at least one type of nucleotide is a nucleotide of any one of claims 1 to 13 carrying a detectable label through a cleavable linker, and wherein each of the one or more of four different type of nucleotides comprises a 3' blocking group covalently attached to the 2" deoxyribose of the nucleotide;(c) imaging and performing one or more fluorescent measurements of the extended copy polynucleotides;(d) removing the 3' blocking group from the nucleotides incorporated into the extended copy polynucleotides;(e) washing the solid support after the removal of the 3' blocking group from the incorporated nucleotides; and(f) repeating steps (b) to (e) until the sequences of at least a portion of the target polynucleotides are determined.

29. The method of claim 28, wherein steps (b) to (e) are repeated at least 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 cycles.

30. The method of claim 28 or 29, wherein the removal of the 3' blocking group also removes the detectable label.

31. A method of determining the sequences of a plurality of different target polynucleotides, comprising:(a) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides;(b) contacting the solid support with an aqueous incorporation mixture comprising DNA polymerase and one or more of four different types of nucleotides under conditions suitable for DNA polymerase-mediated primer extension, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides, wherein at least two types of nucleotides are unlabeled and at least one type of unlabeled nucleotide is a unlabeled nucleotide of any one of claims 1 to 13 having a first functional moiety, and wherein each of the one or more of four different type of nucleotides comprises a 3' blocking group covalently attached to the 2' deoxyribose of the nucleotide;(c) contacting the extended copy polynucleotides with an aqueous labeling mixture comprising a first labeling reagent, wherein the first labeling reagent comprises one or more first detectable labels and a first binding moiety that is capable of specific binding to the first functional moiety of the unlabeled nucleotide;(d) imaging the solid support and performing one or more fluorescent measurements;(e) removing the 3' blocking group from the nucleotides incorporated into the extended copy polynucleotides;(f) washing the solid support after the removal of the 3' blocking group from the incorporated nucleotides; and(g) repeating steps (b) to (f) until the sequences of at least a portion of the target polynucleotides are determined.

32. The method of claim 31, wherein steps (b) to (f) are repeated at least 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 cycles.

33. The method of claim 31 or 32, wherein the first functional moiety of the first type of unlabeled nucleotide is bound to the first labeling reagent by either covalent bonding or noncovalent interaction via a cleavable linker.

34. The method of any one of claim 31 to 33, wherein the removal of the 3' blocking group also removes the first labeling reagent.

35. The method of any one of claims 28 to 33, wherein the method is performed on an automated sequencing instrument comprising a single light source.

36. The method of claim 35, wherein the single light source operates at a wavelength from about 500 nm to about 540 nm, or about 520 nm to about 525 nm.

37. The method of any one of claims 28 to 33, wherein the method is performed on an automated sequencing instrument comprising two light sources operating at two different wavelengths.

38. The method of claim 37, wherein one light source operates at a wavelength from about 450 nm to about 460 nm, and the other light source operates at a wavelength from about 520 nm to about 525 nm.

39. A method of preparing a growing polynucleotide complementary to a target singlestranded polynucleotide, comprising: incorporating a nucleotide of any one of claims 1 to 13 into the growing complementary polynucleotide, wherein the incorporation of the nucleotide prevents the introduction of any subsequent nucleotide into the growing complementary polynucleotide.

40. The method of claim 39, wherein the incorporation of the nucleotide is accomplished by a polymerase, a terminal deoxynucleotidyl transferase (TdT), or a reverse transcriptase.

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