Nucleotides having 3'-hydroxy blocking groups

Abstract

Embodiments of the present disclosure relate to nucleotide and nucleoside molecules with 3'-OH blocking groups. Also provided herein are kits including and methods using the fully functionalized nucleotides containing the 3'-OH blocking group for sequencing applications.

Description

IP-2770-PCT PATENTNUCLEOTIDES HAVING 3 -HYDROXY BLOCKING GROUPSBACKGROUNDField

[0001] The present disclosure generally relates to nucleotides, nucleosides, or oligonucleotides comprising 3 '-hydroxy blocking groups and their use in polynucleotide sequencing methods. Methods of preparing the 3 '-hydroxy protected nucleotides, nucleosides, or oligonucleotides are also disclosed.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. Sci. 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] In order to ensure that only a single incorporation occurs, a structural modification (“protecting group” or “blocking group”) is included in each labeled nucleotide that is added to the growing chain to ensure that only one nucleotide is incorporated. After thenucleotide with the blocking group has been added, the blocking group is then removed, under reaction conditions which do not interfere with the integrity of the DNA being sequenced. The sequencing cycle can then continue with the incorporation of the next protected, labeled nucleotide.

[0006] To be useful in DNA sequencing, nucleotides, which are usually nucleotide triphosphates, generally require a 3 '-hydroxy blocking group so as to prevent the polymerase used to incorporate it into a polynucleotide chain from continuing to replicate once the base on the nucleotide is added. There are many limitations on the types of groups that can be added onto a nucleotide and still be suitable. The blocking group should prevent additional nucleotide molecules from being added to the polynucleotide chain whilst simultaneously being easily removable from the sugar moiety without causing damage to the polynucleotide chain. Furthermore, the modified nucleotide needs to be compatible with the polymerase or another appropriate enzyme used to incorporate it into the polynucleotide chain. The ideal blocking group must therefore exhibit long-term stability, be efficiently incorporated by the polymerase enzyme, cause blocking of secondary or further nucleotide incorporation, and have the ability to be removed under mild conditions that do not cause damage to the polynucleotide structure, preferably under aqueous conditions.

[0007] Reversible blocking groups have been described previously. For example, Metzker et al., (Nucleic Acids Research, 22 (20): 4259-4267, 1994) discloses the synthesis and use of eight 3 '-modified 2-deoxyribonucleoside 5 ’-triphosphates (3 '-modified dNTPs) and testing in two DNA template assays for incorporation activity. International Application Publication No. WO 2002 / 029003 describes a sequencing method which may include the use of an allyl blocking group to cap the 3'-OH group on a growing strand of DNA in a polymerase reaction.

[0008] In addition, the development of a number of reversible blocking groups and methods of deprotecting them under DNA compatible conditions was previously reported in International Application Publication Nos. WO 2004 / 018497 and WO 2014 / 139596, and U.S. Publication No. 2020 / 0216891 Al, each of which is hereby incorporated by reference in its entirety.SUMMARY

[0009] Some embodiments of the present disclosure relate to a nucleotide or nucleoside comprising a ribose or a 2' deoxyribose having a removable 3'-OH blocking group,wherein the 3'-OH blocking group has a structurecovalently attached to the 3 '-oxygen atom of the ribose or 2' deoxyribose, wherein each R1and R2is independently H, C1-C4 alkyl, C1-C4 haloalkyl, cyano, or halogen; and each R3, R4a, R4b, R5a, R5b, R6a, and R6bis independently H, C1-C3 alkyl, cyano, or halogen.

[0010] Some embodiments of the present disclosure relate to an oligonucleotide comprising a 3'-OH blocked nucleotide molecule described herein incorporated thereto.

[0011] Some embodiments of the present disclosure relate to a kit comprising one or more nucleotides, wherein one type of nucleotide is a 3'-OH blocked nucleotide molecule described herein.

[0012] Some embodiments of the present disclosure relate to a method of preparing a polynucleotide, comprising incorporating a 3'-OH blocked nucleotide molecule described herein into a growing polynucleotide in the presence of a polymerase, wherein the incorporation of the nucleotide prevents the introduction of any subsequent nucleotide into the growing polynucleotide.

[0013] Some embodiments of the present disclosure relate to a method of determining sequences of a plurality of different target polynucleotides in parallel, the method 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 solution comprising DNA polymerase and one or more of four different types of nucleotides (A, G, C and T or U) under conditions suitable for DNA polymerase-mediated primer extension, wherein:(i) at least a portion of one type of nucleotide in the aqueous incorporation solution is the 3 ’-OH blocked nucleotide described herein having a fluorescent label covalently attached to the nucleobase via a cleavable linker, or an unlabeled 3' blocked nucleotide as described herein that does not contain any detectable moiety; and<ii) each type of nucleotide is a nucleotide triphosphate having a 3'-OH blocking group covalently attached to the 2' deoxyribose of the nucleotide;(c) imaging the solid support and performing one or more fluorescent measurements to determine the identity of incorporated nucleotides;(d) contacting the solid support with an aqueous deblocking solution;(e) contacting said solid support with an aqueous wash solution; and(f) repeating steps (b)-(e) to determine target polynucleotide sequences.

[0014] Some embodiments of the present disclosure relate to a method of determining sequences of a plurality of different target polynucleotides in parallel, the method 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 solution comprising DNA polymerase and one or more of four different types of nucleotides (A, G, C and T or U) under conditions suitable for DNA polymerase-mediated primer extension to produce extended copy polynucleotides, wherein:(i) at least a portion of one type of unlabeled nucleotide in the aqueous incorporation solution is the unlabeled nucleotide having a 3 ’-OH blocking group as described herein having non-fluorescent functional moiety covalently attached to the nucleobase via a cleavable linker, or an unlabeled 3 ' blocked nucleotide as described herein that does not contain any detectable moiety; and(ii) at least two types of nucleotides are unlabeled; and(iii) each type of nucleotide is a nucleotide triphosphate having a 3'-OH blocking group covalently attached to the 1' deoxyribose of the nucleotide;(c) contacting the extended copy polynucleotides with a labeling reagent comprising one or more fluorescent labels and a binding moiety that is capable of specific binding to the unlabeled nucleotide;(d) imaging the solid support and performing one or more fluorescent measurements to determine the identity of incorporated nucleotides;(e) contacting the solid support with an aqueous deblocking solution;(f) contacting said solid support with an aqueous wash solution; and(g) repeating steps (b)-(f) to determine target polynucleotide sequences.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is box plot showing differences in primary sequencing metrics including error rate, Q30 score, phasing, and prephasing for sequencing by synthesis (SBS) runs using a standard fully functionalized nucleotides (ffNs) set as compared to a standard ffNs set spiked with 10% molar percentage of fully functionalized G nucleotide (ffG) having a 3'-OH blocking group according to an embodiment of the present disclosure.

[0016] FIG. 2A is a line chart illustrating the percentage incorporation as a function of time for a standard fully functionalized G nucleotide (ffG) with AOM blocking group, as compared to a modified ffG having a 3 ’-OH blocking group according to an embodiment of the present disclosure. Incorporation was measured at ffG concentrations of 0. 1 pM and 0.5 pM.

[0017] FIG. 2B is a bar chart showing kcat of incorporation for the test condition of FIG. 2A.

[0018] FIG. 3 A is a plot showing blocking group cleavage (i.e., 3'-OH %) as a function of time for fully functionalized A nucleotide (ffA) with AOM blocking group as compared to ffA having a 3'-OH blocking group according to an embodiment of the present disclosure.

[0019] FIG. 3B is a bar chart showing vmaxand kmfor incorporation of ffA with AOM blocking group, and ffA having a 3’ blocking group according to an embodiment of the present disclosure.

[0020] FIG. 4 is a bar chart showing constant kObs for various standard ffNs and corresponding ffNs having a blocking group in accordance with the present disclosure at concentrations of 0.1 ,u M. The ffNs included fully functionalized T nucleotide (ffT), two different fully functionalized C nucleotides (ffCs) labeled with different dyes, and dark ffG.

[0021] FIG. 5 is a plot showing constant kcat as a function of standard ffG and ffG having a blocking group in accordance with the present disclosure.DETAILED DESCRIPTION

[0022] Embodiments of the present disclosure relate to nucleosides and nucleotides with 3'-OH blocking groups for sequencing applications, for example, sequencing-by-synthesis (SBS). These blocking groups may offer better stability in solution compared to those known in the art. In particular, the 3'-OH blocking groups have improved stability during the synthesis of the fully functionalized nucleotides (ffNs) and also great stability in solution during formulation, storage and operation on the sequencing instruments. In addition, the 3'-OH blocking groups described herein may also achieve low pre-phasing, lower signal decay for improved data quality, which enables longer reads from the sequencing applications.Definitions

[0023] 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 theclaim, 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.

[0024] 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 ffN Fully functionalized nucleotide ffA Fully functionalized A nucleotide ffC Fully functionalized C nucleotide ffl Fully functionalized T nucleotide ffG Fully functionalized G nucleotideRT Room temperatureSBS Sequencing by SynthesisSM Starting material

[0025] 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 inventioninclude, 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.

[0026] 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 means in addition to covalent attachment.

[0027] 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-, -CH2CH(CH3)CH2-, and the like. Other radical naming conventions clearly indicate that the radical is a di-radical such as “alkylene” or “alkenylene.”

[0028] 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.

[0029] As used herein, “Cato Cb” 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 C3 to 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 “Ci-Ce” includes Ci, C2, C3, C4, C5 and Ce, and a range defined by any of the two numbers. For example, Ci-G, alkyl includes Ci, C2, C3, C4, C5 and Ce alkyl, C2-C6 alkyl, C1-C3 alkyl, etc. Similarly, C2-C6 alkenyl includes C2, C3, C4, C5 and Ce alkenyl,C2-C5 alkenyl, C3-C4 alkenyl, etc.; and C2-C6 alkynyl includes C2, C3, C4, C5 and Cr, alkynyl, C2- C5 alkynyl, C3-C4 alkynyl, etc. C3-C8 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 C5-C6 cycloalkyl.

[0030] 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. The alkyl group may be designated as “Ci-C4alkyl” or similar designations. By way of example only, “Ci-Ce alkyl” 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.

[0031] The term “allyl” as used herein refers to a CH2=CH-CH2 — group.

[0032] The term “-O-allyl” as used herein refers to a CH2=CH-CH2O — group.

[0033] The term “hydroxy” as used herein refers to a -OH group.

[0034] The term “cyano” group as used herein refers to a “-CN” group.

[0035] The term “azido” as used herein refers to a -N3 group.

[0036] 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 hydroxyl group that is present in ribose. The nitrogen containing heterocyclic base can be purine or pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof, such as deaza adenine and 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.

[0037] 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 adeoxyribonucleoside 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.

[0038] 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, deazapurine, adenine, 7-deaza adenine, guanine, 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).

[0039] 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 or polynucleotide, 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.

[0040] 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.

[0041] As used herein, “derivative” or “analogue” 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.

[0042] 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.

[0043] The term “protecting group”, “protecting groups”, “blocking group” or “blocking groups” as used herein refer to any atom or group of atoms that is added to a molecule in order to prevent existing groups in the molecule from undergoing unwanted chemical reactions. Sometimes, “protecting group” and “blocking group” can be used interchangeably.

[0044] As used herein, the prefixes “photo” or “photo-” mean relating to light or electromagnetic radiation. The term can encompass all or part of the electromagnetic spectrum including, but not limited to, one or more of the ranges commonly known as the radio, microwave, infrared, visible, ultraviolet, X-ray or gamma ray parts of the spectrum. The part of the spectrum can be one that is blocked by a metal region of a surface such as those metals set forth herein. Alternatively, or additionally, the part of the spectrum can be one that passes through an interstitial region of a surface such as a region made of glass, plastic, silica, or other material set forth herein. In particular embodiments, radiation can be used that is capable of passing through a metal. Alternatively, or additionally, radiation can be used that is masked by glass, plastic, silica, or other material set forth herein.

[0045] 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 failure to complete the incorporation of a portion of DNA strands within clusters by polymerases at a given sequencing cycle. Pre-phasing 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 and pre-phasing increases, hampering the identification of the correct base. Pre-phasing 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 ofnucleotide analogues which decrease the incidence of pre-phasing 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 pre-phasing values, and longer sequencing read lengths.3'-QH Blocking Groups

[0046] Some embodiments of the present disclosure relate to a nucleotide or nucleoside molecule comprising a ribose or 2' deoxyribose having a removable 3'-OH blocking group, wherein the 3'-OH blocking group has a structurecovalently attached to the 3'-oxygen atom of the ribose or 2' deoxyribose, wherein each R1and R2is independently H, C1-C4 alkyl, C1-C4 haloalkyl, cyano, or halogen; and each R3, R4a, R4b, R5a, R5b, R6a, and R6bis independently H, C1-C3 alkyl, cyano, or halogen. In some embodiments, each of R4aand R4bis H. In other embodiments, at least one of R4aand R4bis methyl, cyano, or halo (e.g., F). In some embodiments, each of RSaand R5bis H. In other embodiments, at least one of R5aand R5bis methyl, cyano, or halo (e.g., F). In some embodiments, each of R6aand R6bis H. In other embodiments, at least one of R6aand R6bis methyl, cyano, or halo (e.g., F). In some further embodiments, each of R1, R2and R3is independently H, F, Cl, methyl, ethyl, isopropyl or trifluoromethyl. In some such embodiments, two of R1, R2and R3is H, and one of R1, R2and R3is independently H, F, Cl, methyl, ethyl, isopropyl or trifluoromethyl. In some further

[0047] In some embodiments, the nucleoside or nucleotide is covalently attached to a detectable moiety, optionally via a cleavable linker. In some further embodiments, the detectablelabel is covalently attached to a nucleobase of the nucleoside or nucleotide via the cleavable linker. In some further embodiments, the cleavable linker comprising an azido moiety, a -O-allyl moiety, a disulfide moiety, an acetal moiety, or a thiocarbamate moiety. In some further embodiments, the 3'-OH blocking group and the cleavable linker are cleavable under the same chemical reaction conditions. In some further embodiments, the detectable moiety is a fluorescent label. In other further embodiments, the detectable moiety comprises a non-fluorescent functional moiety that is capable of being bound to a labeling reagent via covalent bonding or noncovalent interaction.

[0048] In any embodiments of the present disclosure, nucleoside or nucleotide as provided by the present disclosure comprises a 2' deoxyribose. In some further embodiments, the nucleotide is a nucleotide triphosphate. In further embodiments, the nucleoside or nucleotide is a deoxy guanosine, deoxyguanosine triphosphate, deoxy (7-deaza) guanosine, or deoxy (7- deaza)guanosine triphosphate bearing the nucleobaseoptionally substituted variants thereof.

[0049] Provided herein is an oligonucleotide comprising a nucleotide in accordance with the present disclosure incorporated thereto.Additional d'-OH Blocking Groups

[0050] The nucleoside or nucleotide described herein may include 3'-OH blocking groups in accordance with the present disclosure. Additionally or alternatively, the nucleoside or nucleotide may include an 3'-OH blocking group selected from the group consisting of}r. / xr.z\ Si(Me)3 u U (SEM), wherein the squiggly line refers to the point attachment to the 3' carbon of the nucleotide. Additional 3' OH blocking group are disclosed in W02004 / 018497 and WO2014 / 139596, which are hereby incorporated by references. For example, the blocking group may be azidomethyl (-CH2N3) or substituted azidomethyl (e.g., -CH(CHF2)N3 or CH(CH2F)N3), or allyl connecting to the 3’ oxygen atom of the ribose or deoxyribose moiety.Nucleotides with Detectable Moieties

[0051] According to an aspect of the disclosure, the described 3'-OH blocked nucleotide includes a detectable moiety. In some such embodiments, the detectable moiety is a fluorescent label (e.g., a fluorescent dye). Such nucleotide is also commonly referred to herein as a “labeled nucleotide.” In other embodiments, the detectable moiety is a non-fluorescent functional moiety that is capable of binding to a labeling reagent (e.g., a reagent containing one or more fluorescent dyes) via covalent bonding or noncovalent interaction. Such nucleotide is also commonly referred to as a “unlabeled nucleotide” because the nucleotide itself does not carry any fluorescent label prior to binding with a labeling reagent.

[0052] The detectable moiety (e.g., a fluorescent dye or a non-fluorescent functional moiety) can be conjugated via an optional cleavable linker by a variety of means including covalent attachment, hydrophobic attraction, or ionic attraction. In some embodiments, the detectable moiety may be covalently attached to the nucleotide 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 cleavable linker moiety.

[0053] Nucleosides or nucleotides labeled with the detectable moiety described herein may have the formula:B-L-detectable moietywhere B is a nucleobase, such as, for example uracil, thymine, cytosine, adenine, 7-deaza adenine, guanine, 7-deaza guanine, and the like; L is an optional cleavable linker which may or may not be present; R’ can be H, or -OR’ is monophosphate, diphosphate, triphosphate, thiophosphate, a phosphate ester analog, -O- attached to a reactive phosphorous containing group, or -O- protected by a blocking group; R" is H or OH; and R’" is H, a 3’ blocking group described herein, or -OR"’ forms a phosphoramidite. Where -OR’" is phosphoramidite, R’ is an acid- cleavable hydroxyl protecting group which allows subsequent monomer coupling underderivatives and analogs thereof. In some further embodiments, the nucleobase comprises the

[0054] The use of a blocking group allows polymerization to be controlled, such as by stopping extension when a labeled nucleotide is incorporated. If the blocking effect is reversible, for example, by way of non-limiting example by changing chemical conditions or by removal of a chemical block, extension can be stopped at certain points and then allowed to continue.Cleavable Linkers

[0055] 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.

[0056] Useful linker groups may be found in PCT Publication No. WO 2004 / 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. 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.

[0057] Particular linkers include those disclosed in PCT Publication No. WO 2004 / 018493 (herein incorporated by reference) such as those that include moieties of the formulae:

[0058] (wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a C1-C10 substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a C1-C10 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.

[0059] 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:(wherein * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside). The linker moieties illustrated herein may comprise the whole or partial linker structure between the nucleotides / nucleosides and the labels. The linker moieties illustrated herein may comprise the whole or partial linker structure between the nucleotides / nucleosides and the labels.

[0060] Additional examples of linkers are disclosed in U.S. Publication No. 2020 / 0216891 Al, which is incorporated by reference in its entirety:2, 3, 4, 5; k is 1; Z is -N3 (azido), -O-Ci-Ce alkyl, -O-C2-C6 alkenyl, or -O-C2-C6 alkynyl; and R comprises the detectable moiety described herein, which may contain additional linker and / or spacer structure. One of ordinary skill in the art understands that the detectable moiety described herein is covalently bound to the linker by reacting a functional group of the detectable moiety (e.g., carboxyl) with a functional group of the linker (e.g., amino) to form an amide bond. In one I embodiment, the cleavable 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).

[0061] In particular embodiments, the 3 ' blocked nucleotide may be enzymatically incorporate 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 replication enzyme. Thus, the linker can also comprise a spacer unit, such as one or more PEG unit(s) (-OCH2CH2-)n, 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 detectable moiety. Exemplary linkers and their properties are set forth in PCT Publication No. WO 2007 / 020457 (herein incorporated by reference). A PEG linker may be introduced by reacting thecarboxyl group of the detectable moiety with a PEG containing reagent with a terminal amino group to form amide bonding (e.g., H2N-(CH2)20)2-IO-(CH2)2C(0)OH).

[0062] In some embodiments, the cleavable linker (between the detectable moiety and nucleotide) and the 3' 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.

[0063] Non-limiting exemplary nucleotides as described herein include:wherein L represents a linker, including a cleavable linker described herein; Rxrepresents a ribose or deoxyribose moiety as described above, or a ribose or deoxyribose moiety with the 5' position substituted with mono-, di- or tri- phosphates; R represents the detectable moiety described herein.

[0064] In some embodiments, non-limiting exemplary nucleotide containing a detectable moiety covalently attached via a cleavable linker are shown below:wherein R comprises or is a detectable moiety (e.g., a fluorescent dye or a non-fluorescent functional moiety); PG stands for the 3' blocking groups described herein; 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 one embodiment, -O-PG is AOM. In another embodiment, -PG is. In one embodiment, m is 5. In another embodiment, m is 0. In another embodiment, m is 2. In some further embodiments, p is 1, 2, 3, 4-bHNy° or 5. (CH2)mRrefersto the connection point of the detectable moiety with the cleavable linker as a result of a reaction between an amino group of the linker moiety and the carboxyl group of the detectable moiety. In further embodiments, the nucleotide may be attached to the detectable moiety via more than one of the same cleavable linkers (such as LN3-LN3, sPA-sPA, AOL-AOL). In other embodiments, the nucleotide may be attached to the hapten moiety via two or more different cleavable linkers (such sPA-LN3, sPA-sPA-LN3, sPA-LN3-LN3, etc.). In addition, the linker may further include additional PEG spacers as described herein, for example, between R and-(CH2)m-. In any embodiments of the nucleotide described herein, the nucleotide is a nucleotide triphosphate. In further embodiments, the nucleotide has a 2" deoxyribose.Kits

[0065] The present disclosure provides for kits including one or more nucleotides, wherein one type of nucleotide is a 3' blocked nucleotide in accordance with the present disclosure. In some embodiments, the kit comprises an incorporation mixture used for sequencing by synthesis. In some embodiments, the 3' blocked nucleotide according to the present disclosure is about or at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% portion of the one type of nucleotide in the incorporation mixture, or within a range defined by any two of the previous values. In some further embodiments, the 3 ' blocked nucleotide according to the present disclosure is about 5% or 10% of the one type of nucleotide in the aqueous incorporation mixture. The percentage “%”refers to the molar percentage of the 3' blocked nucleotide according to the present disclosure relative to the total amount of the particular type of nucleotide in the incorporation mixture. In further embodiment, the 3 ' blocked nucleotide according to the present disclosure having the 3 ' blocking groupattached to the 3' oxygen atom of the ribose or deoxyribose. In further embodiments, the particular type of nucleotide having the 3' blocking groupis a deoxyguanosine, deoxy guanosine triphosphate, deoxy (7- deaza)guanosine, or deoxy (7-deaza) guanosine triphosphate. In further embodiments, the kit comprises four types of nucleotides, and each type of nucleotides comprises a 3' blocking groupattached to 3" carbon atom of the ribose or 2' deoxyribose. In some such embodiments, at least one type of nucleotide (e.g., dGTP or ffG) contains a mixture of the nucleotide containing either a 3 ' blocking groupattached to 3 ' oxygen atom or a 3 ' blocking groupattached to 3' carbon atom. In some embodiments, the molar ratio of the two different types of blocking groups may be 1:99, 2:98, 3:97, 4:96, 5:95, 6:94, 7:93, 8:92, 9:91, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40,65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, or 99: 1.

[0066] In some embodiments, the kit comprises an enzyme and a buffer appropriate for the action of the enzyme. In further embodiments, the enzyme is a polymerase, a terminal deoxynucleotidyl transferase, or a reverse transcriptase. In yet further embodiments, the polymerase is a DNA polymerase.

[0067] In a particular embodiment, a kit can include at least one labeled 3' blocked nucleotide or nucleoside together with labeled or unlabeled nucleotides or nucleosides (e.g., unlabeled nucleotides or nucleosides in accordance with the present disclosure). For example, nucleotides labeled with dyes may be supplied in combination with unlabeled or native nucleotides, and / or with fluorescently labeled 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).

[0068] Where kits comprise a plurality, particularly two types of, three types of, or four types of 3' blocked nucleotides labeled with a dye compound, the different nucleotides may be labeled with different dye compounds, or one of the different nucleotides 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. Asused 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 laser. 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 450 nm to 460 nm, 480-490 nm (e.g., 488 nm), or 500 nm to 540 nm (e.g., 523 nm).

[0069] In some embodiments, the first and second type labeled nucleotides are excitable using a single excitation source, which may be a first light source having a first excitation wavelength. For example, the excitation bands for the first and the second labels may be at least partially overlapping such that excitation in the overlap region of the spectrum causes both labels to emit fluorescence. In some other embodiments, the second type of labeled nucleotides is excitable using a second excitation source, which may be a second light source having a second excitation wavelength that is different from the first excitation wavelength. In some further embodiments, the kit may include a third type nucleotide, wherein the third nucleotide is labeled with a third compound that is different from the first and the second labels (i.e., a third label). Alternatively, the third type of nucleotide is a mixture of the third type of nucleotide labeled with the first label and the third type of nucleotide labeled with the second label. In some such embodiments, the third type labeled nucleotide is excitable using the first light source having the first excitation wavelength. That is, each of the first type, second type and the third type of nucleotide is excitable using the same light source with a single wavelength. In some other embodiments, the third type labeled nucleotide is excitable using both the first light source having the first excitation wavelength, or the second light source having the second excitation wavelength. In still other embodiments, the third type of nucleotide is excitable using a third light source with a third excitation wavelength. In some further embodiments, the kit may further comprise a fourth type of nucleotide. In some such embodiments, the fourth nucleotide is unlabeled (dark). In other embodiments, the fourth nucleotide is labeled with a different compound than the first, second and the third nucleotide, and each label has a distinct absorbance maximum that is distinguishable from the other labels. In still other embodiments, the fourth nucleotide is unlabeled. In some embodiments, the first light source has an excitation wavelength from about 400 nm to about 490 nm, from about 420nm to about 470 nm, or from 450 nm to about 460 nm(e.g., 450 nm or 488 nm). In some other embodiments, the first excitation light source has a wavelength from about 500 nm to about 550 nm, from about 510 to about 540 nm, or from about 520 to about 530 nm (e.g., 523 nm or 525 nm). In other embodiments, when two excitation light sources are used, the first excitation light source has an excitation wavelength from about 400 nm to about 490 nm, from about 420nm to about 470 nm, or from 450 nm to about 460 nm (e.g., 450 nm or 488 nm), and the second excitation light source a wavelength from about 500 nm to about 550 nm, from about 510 to about 540 nm, or from about 520 to about 530 nm (e.g., 523 nm or 525 nm). In some embodiments, the emissions of the first type of labeled nucleotide, the second type of labeled nucleotide and the third type of labeled nucleotide are detectable in two detection channels with different wavelengths (e.g., at a blue region with a wavelength ranging from about 472 nm to about 520 nm, and at a green region with a wavelength ranging from about 540 nm to about 640nm). In some other embodiments, the emissions of the first type of labeled nucleotide, the second type of labeled nucleotide and the third type of labeled nucleotide are detectable in two detection channels with different wavelengths (e.g., at a green region with a wavelength ranging from about 540 nm to about 585 nm, and a second green region with a wavelength ranging from about 585 nm to about 640nm). In other embodiments, each of the first type, the second type and the third type of nucleotide has an emission spectrum that can be collected in a single emission collection filter or channel.

[0070] In addition to the labeled nucleotides, the kit may comprise together at least one additional component. The further component(s) may be one or more of the components identified in a method set forth herein or in the Examples section below. Some non-limiting examples of components that can be combined into a kit of the present disclosure are set forth below. In some embodiments, the kit further comprises a DNA polymerase (such as a mutant 9°N DNA polymerase) and one or more buffer compositions. Non-limiting examples of DNA polymerase may be used in the present disclosure include those disclosed in WO 2005 / 024010, U.S. Publication Nos. 2020 / 0131484 Al, 2020 / 0181587 Al, and 2024 / 0141427, each of which is incorporated by reference herein in its entirety. Exemplary polymerases include but not limited to Pol 812, Pol 1901, Pol 1558, Pol 963, or Pol A. 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. The amino acid sequence of Pol A is disclosed as SEQ ID NO:5 of U.S. Publication No. 2024 / 0141427. One buffer composition may comprise antioxidants such as ascorbic acid or sodium ascorbate, which can be used to protect the dye compounds from photo damage during detection. Additional buffer composition may comprise a reagent can may be used to cleave the 3' blocking group and / or the cleavable linker. For example, a water-soluble phosphines or water-soluble transition metal catalysts formed from a transitionmetal and at least partially water-soluble ligands, such as a palladium complex. Various components of the kit may be provided in a concentrated form to be diluted prior to use. In such embodiments a suitable dilution buffer may also be included. Again, one or more of the components identified in a method set forth herein can be included in a kit of the present disclosure. In any embodiments of the nucleotide or labeled nucleotide described herein, the nucleotide contains a 3' blocking group.Methods of Sequencing

[0071] Nucleotides according to the present disclosure may be used in any method of analysis such as a method that include detection of a fluorescent label attached to a nucleotide whether on its own or incorporated into or associated with a larger molecular structure or conjugate. In some embodiments, unlabeled nucleotides according to the present disclosure may be used in sequencing methods alongside labeled nucleotides. In this context the term “incorporated into a polynucleotide” can mean that the 5’ phosphate is joined in phosphodiester linkage to the 3’-OH group of a second (modified or unmodified) nucleotide, which may itself form part of a longer polynucleotide chain. The 3’ end of a nucleotide set forth herein may or may not be joined in phosphodiester linkage to the 5’ phosphate of a further (modified or unmodified) nucleotide. Thus, in one non-limiting embodiment, the disclosure provides a method of detecting a nucleotide incorporated into a polynucleotide which comprises: (a) incorporating at least one nucleotide of the disclosure into a polynucleotide and (b) detecting the nucleotide(s) incorporated into the polynucleotide by detecting the fluorescent signal from the dye compound attached to said nucleotide(s).

[0072] This method can include: a synthetic step (a) in which one or more nucleotides according to the disclosure are incorporated into a polynucleotide and a detection step (b) in which one or more nucleotide(s) incorporated into the polynucleotide are detected by detecting or quantitatively measuring their fluorescence or lack of fluorescence, where unlabeled nucleotides are used.

[0073] The present disclosure also provides for a method of determining a sequence of a single-stranded target polynucleotide, comprising:(a) contacting a copy polynucleotide strand with an aqueous incorporation mixture comprising DNA polymerase and one or more of four different types of nucleotides (A, C, G and T or U), and incorporating one type of nucleotide into the copy polynucleotide strand to produce an extended copy polynucleotide strand, wherein at least a portion of one type of nucleotide in the aqueous incorporation mixture is a 3 ' blocked nucleotide according to the present disclosure carrying a fluorescent label, or an unlabeled 3 ' blockednucleotide according to the present disclosure that does not contain any detectable moiety, each type of nucleotide is a nucleotide triphosphate having a 3'-OH blocking group covalently attached to the 2' deoxyribose of the nucleotide, and wherein the copy polynucleotide strand is complementary to at least a portion of the target polynucleotide strand;(b) imaging and performing one or more fluorescent measurements to determine the identity of the nucleotide incorporated into the copy polynucleotide strand;(c) removing the 3'-OH blocking group from the nucleotide incorporated into the copy polynucleotide strand;(d) washing the 3'-OH blocking group away from the extended copy polynucleotide strand; and repeating steps (a) to (d) until a sequence of the portion of the target polynucleotide strand is determined. In some embodiments, the 3' blocked nucleotide according to the present disclosure is about or at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% portion of the one type of nucleotide in the aqueous incorporation mixture, or within a range defined by any two of the previous values. In some further embodiments, the 3 ' blocked nucleotide according to the present disclosure is about 5% or 10% of the one type of nucleotide in the aqueous incorporation mixture. In some further embodiments, the portion is at least 5%. In one embodiment, the portion is at least 10%. The percentage “%” refers to the molar percentage of the 3' blocked nucleotide according to the present disclosure relative to the total amount of the particular type of nucleotide in the incorporation mixture.

[0074] The present disclosure also provides for a method of determining a sequence of a single-stranded target polynucleotide, comprising:(a) contacting a copy polynucleotide strand with an aqueous incorporation mixture comprising DNA polymerase and one or more of four different types of nucleotides (A, C, G and T or U), and incorporating one type of nucleotide into the copy polynucleotide strand to produce an extended copy polynucleotide strand, wherein at least a portion of one type of nucleotide in the aqueous incorporation mixture is an unlabeled 3' blocked according to the present disclosure having a functional moiety, or an unlabeled 3 ' blocked nucleotide according to the present disclosure that does not contain any detectable moiety; each type of nucleotide is a nucleotide triphosphate having a 3'-OH blocking group covalently attached to the 2' deoxyribose of the nucleotide, and wherein the copy polynucleotide strand is complementary to at least a portion of the target polynucleotide strand;(b) contacting the extended copy polynucleotide strand with a labeling reagent, wherein the labeling reagent comprises one or more fluorescent labels and a binding moiety that is capable of specific binding to the functional moiety of the unlabeled nucleotide;(c) imaging and performing one or more fluorescent measurements to determine the identity of the nucleotide incorporated into the copy polynucleotide strand;(d) removing the 3'-OH blocking group from the nucleotide incorporated into the copy polynucleotide strand;(e) washing the 3'-OH blocking group away from the extended copy polynucleotide strand; and repeating steps (a) to (e) until a sequence of the portion of the target polynucleotide strand is determined. In some embodiments, the 3' blocked unlabeled nucleotide in accordance with the present disclosure is about or at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% portion of the one type of nucleotide in the aqueous incorporation mixture, or within a range defined by any two of the previous values. In some further embodiments, the portion is at least 5%. In one embodiment, the portion is about 10%. The percentage “%” refers to the molar percentage of the 3 ' blocked nucleotide according to the present disclosure relative to the total amount of the particular type of nucleotide in the incorporation mixture.

[0075] In some embodiments of the methods described herein, at least a portion of one type of nucleotides in the aqueous incorporation mixture each has the 3'-OH blocking group of the structureattached to the 3’ oxygen atom of the nucleotide. In further embodiments, the other nucleotides in the aqueous incorporation mixture each has the 3'-OH blocking group of the structureattached to the 3' oxygen atom. In some embodiments, the steps (a) to (d) or steps (a) to (e) is repeated at least 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 times. In some embodiments, the fluorescent label and the removable 3'-OH blocking group from the nucleotide incorporated into the copy polynucleotide strand are removed in a single chemical reaction. In some embodiments, removing the 3'-OH blocking group comprises contacting the incorporated nucleotide with a cleavage solution comprising a palladium catalyst. In some further embodiments, the cleavage solution comprising the palladium catalyst further comprises one or more buffer reagents selected from the group consisting of a primary amine, a secondary amine, a tertiary amine, a carbonate salt, a phosphate salt, and a borate salt, and combinations thereof.

[0076] The present disclosure also provides for a method of determining sequences of a plurality of different target polynucleotides in parallel, the method 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 solution comprising DNA polymerase and one or more of four different types of nucleotides (A, G, C and T or U) under conditions suitable for DNA polymerase-mediated primer extension, wherein:(i) at least a portion of one type of nucleotide in the aqueous incorporation solution is a 3' blocked nucleotide according to the present disclosure having a fluorescent label covalently attached to the nucleobase via a cleavable linker, or an unlabeled 3 ' blocked nucleotide according to the present disclosure that does not contain any detectable moiety; and(ii) each type of nucleotide is a nucleotide triphosphate having a 3'-OH blocking group covalently attached to the 1' deoxyribose of the nucleotide;(c) imaging the solid support and performing one or more fluorescent measurements to determine the identity of incorporated nucleotides;(d) contacting the solid support with an aqueous deblocking solution;(e) contacting said solid support with an aqueous wash solution; and(f) repeating steps (b)-(e) to determine target polynucleotide sequences.

[0077] In some embodiments, at least a portion of one type of nucleotides in the aqueous incorporation solution each has the 3'-OH blocking group of the structureattached to the 3’ oxygen atom of the nucleotide. In some embodiments, the 3' blocked nucleotide having 3 '-OH blocking group of the structureis about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% portion of the one type of nucleotide in the aqueous incorporation mixture, or within a range defined by any two of the previous values. In some further embodiments, the portion is at least 5%. In one embodiment, the portion is about 10%. The percentage “%” refers to the molar percentage of the 3' blocked nucleotide according to the present disclosure relative to the total amount of the particular type of nucleotide in the incorporation mixture. In some further embodiments, the other nucleotides in the aqueous incorporation solution each has the 3'-OH blocking group of the structureattached to the 3’ oxygen atom of the nucleotide (i.e., AOM blocking group). 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 cleavable linker comprises a moiety selected from the group consisting of:, wherein X is O, Y is O, and * indicates where the moiety is connected to the remainder of the nucleotide. In some further embodiments, the cleavable linkercomprises . In some further embodiments, the cleavable linker is selected from the group consisting of: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 nucleobase; and ** indicates the attachment point of the cleavable linker to the fluorescent label.

[0078] Additional embodiments of the present disclosure also provides for a method of determining sequences of a plurality of different target polynucleotides in parallel, the method 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 solution comprising DNA polymerase and one or more of four different types of nucleotides (A, G, C and T or U) under conditions suitable for DNA polymerase-mediated primer extension to produce extended copy polynucleotides, wherein:(i) at least a portion of one type of unlabeled nucleotide in the aqueous incorporation solution is the unlabeled 3' blocked nucleotide in accordance according to the present disclosure, having a non-fluorescent functional moiety covalently attached to the nucleobase via a cleavable linker, or an unlabeled 3'blocked nucleotide according to the present disclosure that does not contain any detectable moiety, and(ii) at least two types of nucleotides are unlabeled; and(hi) each type of nucleotide is a nucleotide triphosphate having a 3'-OH blocking group covalently attached to the 2' deoxyribose of the nucleotide;(c) contacting the extended copy polynucleotides with a labeling reagent comprising one or more fluorescent labels and a binding moiety that is capable of specific binding to the unlabeled nucleotide;(d) imaging the solid support and performing one or more fluorescent measurements to determine the identity of incorporated nucleotides;(e) contacting the solid support with an aqueous deblocking solution;(f) contacting said solid support with an aqueous wash solution; and(g) repeating steps (b)-(f) to determine target polynucleotide sequences.

[0079] In some embodiments, at least a portion of one type of unlabeled nucleotides in the aqueous incorporation solution each has the 3'-OH blocking group of the structureattached to the 3’ oxygen atom of the nucleotide. In some embodiments, the 3' blocked nucleotide having 3'-OH blocking group of the structureabout 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% portion of the one type of nucleotide in the aqueous incorporation mixture, or within a range defined by any two of the previous values. The percentage “%” refers to the molar percentage of the 3 ' blocked nucleotide according to the present disclosure relative to the total amount of the particular type of nucleotide in the incorporation mixture. In some further embodiments, the portion is at least 5%. In one embodiment, the portion is about 10%. In further embodiments, the other nucleotides in the aqueous incorporation solution each has the 3'-OH blocking group of the structure(i.e.;AQM blocking group) attached to the 3' oxygen atom. 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 cleavable linker comprises a moiety selected from the group consisting of:wherein X is O, Y is O, and * indicates where the moiety is connected to the remainder of the nucleotide. In further embodiments, thecleavable linker comprises . In yet further embodiments, the cleavable linker is selected from the group consisting of: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 non-fluorescent functional moiety.

[0080] In some embodiments of the methods of sequencing described herein, the DNA polymerase is an altered family B archaeal DNA polymerase. In further embodiments, the DNA polymerase comprises a 3-amino acid region that is functionally equivalent or homologous to amino acids 408-410 in 9° N DNA polymerase, wherein the first amino acid of the 3-amino acid region is an amino acid selected from the group consisting of isoleucine (I), alanine (A), valine (V), and serine (S); the second amino acid of the 3-amino acid region is an amino acid selected from the group consisting of alanine (A) and glycine (G); and the third amino acid of the 3-amino acid region is an amino acid selected from the group consisting of alanine (A), isoleucine (I), valine (V), leucine (L), threonine (T), and proline (P).

[0081] In some embodiments of the methods of sequencing described herein, at least one type of nucleotide is a 3' blocked G nucleotide that does not contain any detectable moiety. Such 3' blocked G nucleotide has a structure selected from the group consisting of:is a G nucleotide has a structure, wherein Linker refers to a cleavable linker according to the present disclosure. In other embodiments, the 3 ' blocked nucleotide having the blocking group as described herein may be A, T, C or U nucleotide.Deprotection of the 3'-OH Blocking Groups

[0082] The 3'-OH blocking groups described herein may be removed or cleaved under various chemical conditions. Non-limiting cleaving condition includes a Pd(II) complex, such as Pd(OAc)2or allylPd(II) chloride dimer, in the presence of a phosphine ligand (e.g., a water solublephosphine), for example tris(hydroxymethyl)phosphine (THMP), or tris(hydroxylpropyl)phosphine (THP or THPP). For those blocking groups containing an alkynyl group (e.g., an ethynyl), they may also be removed by a Pd(II) complex (e.g., Pd(OAc)2 or allyl Pd(II) chloride dimer) in the presence of a phosphine ligand (e.g., THP or THMP). In some embodiments of the methods described herein, the aqueous deblocking solution comprises a palladium catalyst and tris(hydroxypropyl)phosphine. In some embodiments, the aqueous wash solution comprises a palladium scavenger. In some embodiments, the deblocking solution further comprises one or more buffer reagents selected from the group consisting of a primary amine, a secondary amine, a tertiary amine, a carbonate salt, a phosphate salt, and a borate salt, and combinations thereof.Palladium Cleavage Reagents

[0083] In some other embodiments, the 3’ blocking group described herein such asAOM ormay be cleaved by a palladium catalyst. In some such embodiments, the Pd catalyst is water soluble. In some such embodiments, is a Pd(0) complex (e.g., Tris(3,3',3"-phosphinidynetris(benzenesulfonato)palladium(0) nonasodium salt nonahydrate). In some instances, the Pd(0) complex may be generated in situ from reduction of a Pd(II) complex by reagents such as alkenes, alcohols, amines, phosphines, or metal hydrides. Suitable palladium sources include Na2PdC14, LizPdCU, PdfCHjCN Ch, (PdCl(C3Hs))2, [Pd(C3H5)(THP)]Cl, [Pd(C3H5)(THP)2]Cl, Pd(OAc)2, Pd(Ph3)4, Pd(dba)2, Pd(Acac)2, PdCh(COD), Pd(TFA)2, Na2PdBr4, K2PdBr4, PdCl2, PdBr2, and Pd(NO3)2. In one such embodiment, the Pd(0) complex is generated in situ from Na2PdCl4or K2PdCl4. In another embodiment, the palladium source is allyl palladium(II) chloride dimer [(PdClfCsHs))!]. In some embodiments, the Pd(0) complex is generated in an aqueous solution by mixing a Pd(ll) complex with a phosphine. Suitable phosphines include water soluble phosphines, such as THP, THMP, PTA, TCEP, bis(p-sulfonatophenyl)phenylphosphine dihydrate potassium salt, or triphenylphosphine-3,3’,3”-trisulfonic acid trisodium salt.

[0084] In some embodiments, the palladium catalyst is prepared by mixing [(Allyl)PdCl]2 with THP in situ. The molar ratio of [( Allyl)PdCl]2 and the THP may be about 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5 or 1:10. In one embodiment, the molar ratio of [(Allyl)PdCl]2 to THP is 1:10. In some other embodiment, the palladium catalyst is prepared by mixing a water soluble Pd reagent such as Na2PdCl4or KzPdCU with THP in situ. The molar ratio of Na2PdCl4or K2PdCl4and THP may be about 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1 :5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1 : 9.5 or 1 : 10. In one embodiment, the molar ratio of N a2PdCl4or K2 PdC 14to THP is about 1 : 3. Inanother embodiment, the molar ratio of Na2PdC14 or K^PdCh to THP is about 1 : 3.5. In yet another embodiment, the molar ratio of Na PdCU or K^PdCU to THP is about 1:2.5. In some further embodiments, one or more reducing agents may be added, such as ascorbic acid or a salt thereof (e.g., sodium ascorbate). In some embodiments, the cleavage mixture may contain additional buffer reagents, such as a primary amine, a secondary amine, a tertiary amine, a carbonate salt, a phosphate salt, or a borate salt, or combinations thereof. In some further embodiments, the buffer reagent comprises ethanolamine (EA), tris (hydroxymethyl) aminomethane (Tris), glycine, sodium carbonate, sodium phosphate, sodium borate, 2-dimethylethanolamine (DMEA), 2- diethylethanolamine (DEEA), N,N,N',N'-tetramethylethylenediamine (TEMED), N,N,N',N'- tetraethylethylenediamine (TEEDA), or 2-piperidine ethanol (also known as (2- hydroxyethyl)piperidine, having the structureor combinations thereof. In one embodiment, the buffer reagent comprises or is DEEA. In another embodiment, the buffer reagent comprises or is (2-hydroxyethyl)piperidine. In another embodiment, the buffer reagent contains one or more inorganic salts such as a carbonate salt, a phosphate salt, or a borate salt, or combinations thereof. In one embodiment, the inorganic salt is a sodium salt.Palladium (Pd) Scavengers

[0085] Pd has the capacity to stick on DNA, mostly in its inactive Pd(II) form, which may interfere with the binding between DNA and polymerase, causing increased phasing. A postcleavage wash composition that includes a Pd scavenger compound may be used following the deblocking step. For example, PCT Publication No. WO 2020 / 126593 discloses Pd scavengers such as 3, 3 ’-dithiodipropionic acid (DDPA) and lipoic acid (LA) may be included in the scan composition and / or the post-cleavage wash composition. The use of these scavengers in the postcleave washing solution has the purpose of scavenging Pd(0), converting Pd(0) to the inactive Pd(II) form, thereby improving the prephasing value and sequencing metrics, reducing signal degrade, and extend sequencing read length.Pd( 0) Scavengers

[0086] Certain aspects of the present disclosure relate to employing alternative palladium scavengers in several steps of sequencing by synthesis, where at least one palladium scavenger comprises one or more allyl moieties. For example, -O-allyl, — S-allyl , -NR-allyl, or - N+RR'-allyl, or combinations thereof, wherein R is H, unsubstituted or substituted Ci-G> alkyl, unsubstituted or substituted C2-C6 alkenyl, unsubstituted or substituted C2-C6 alkynyl, unsubstituted or substituted Ce-Cio aryl, unsubstituted or substituted 5 to 10 membered heteroaryl, unsubstituted or substituted C3-C10 carbocyclyl, or unsubstituted or substituted 5 to 10 membered heterocyclyl; and R' is H, unsubstituted Ci-Ce alkyl or substituted Ci-Ce alkyl. The allyl containingPd scavenger acts as a competitive substrate to consume any residual Pd(0) sticking on the nucleic acid (i.e., a Pd(0) scavenger). These palladium scavengers are described in WO 2022 / 243480, which is incorporated by reference in its entirety.

[0087] In some embodiments of any of the methods described herein, the Pd(0) scavenger comprises one or more allyl moieties is the incorporation mix (1MX). In some such embodiments, such palladium scavenger is compatible with the other sequencing reagents in the incorporation mix, which may also include a polymerase (such as DNA polymerase), in addition to the one or more different types of nucleotides. 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). In some embodiments, the Pd(0) scavenger is premixed with the DNA polymerase and / or the one or more of four types of nucleotides (e.g., dATP, dCTP, dGTP, and dTTP or dUTP). In other embodiments, the Pd(0) scavenger is stored separately form the DNA polymerase and / or the one or more of four types of nucleotides and is mixed with these components shortly before sequencing run starts.

[0088] In some other embodiments of the methods and kits described herein, the Pd(0) scavenger comprises one or more allyl moieties is in a solution when performing one or more fluorescent measurements. In such embodiment, such palladium scavenger is compatible with the sequencing reagents of the scanning solution (also known as the scan mix). In further embodiments, the one or more palladium scavengers does not require a separate washing step prior to the next incorporation cycle. In further embodiments, the palladium scavenger in the scan solution is a Pd(0) scavenger described herein. In other embodiments of the methods described herein, the Pd(0) scavenger comprises one or more allyl moieties is in the post cleavage wash solution of step (f). In further embodiments, the palladium scavenger in the post cleavage wash solution is a Pd(0) scavenger described herein. In some such embodiment, the post cleavage wash solution does not comprise lipoic acid or 3,3’ -dithiodipropionic acid (DDPA). In still other embodiments, the Pd(0) scavenger comprises one or more allyl moieties may be present both in the incorporation mixture and the post cleavage wash solution, or present in both the incorporation mix and the scan mix. In some such embodiment, the post cleavage wash solution comprises lipoic acid or DDPA. In other embodiments, the post cleavage wash solution does not comprise lipoic acid or DDPA.

[0089] Non-limiting examples of the Pd(0) scavenger comprising one or more -O-allyl or allyl moieties include the following:, (Compound B, N-Boc tyrosine(allyl)-OH),(Compound C, allyl-0-D-gluocopyranoside),(Compound I), O (Compound J), OH (Compound K), 0(Compound L), 0 (Compound M), and o (Compound N).

[0090] Non-limiting examples of the Pd(0) scavenger comprising one or more -S -allyl moieties include the following:

[0091] Non-limiting examples of the Pd(0) scavenger comprising one or more -NR- allyl or -N+RR'-allyl moieties include the following:where Z is an anion (e.g., a halide anion such as F or Cl ). In one embodiment, the palladiumscavenger isCl (Compound O, diallyldimethylammonium chloride, also known as DADMAC).Pd(Il) Scavengers

[0092] In some embodiments of the methods and kits described herein, the method may further use additional palladium scavenger(s), such as Pd(II) scavenger(s). In some such embodiments, the use of additional Pd(II) scavenger(s) may improve the phasing value of the sequencing metrics. For example, the Pd(II) scavenger(s) may comprise an isocyanoacetate (ICNA) salt, ethyl isocyanoacetate, methyl isocyanoacetate, cysteine (e.g., L-cysteine) or a salt thereof (e.g., A-acetyl-L-cysteine), potassium ethylxanthogenate, potassium isopropyl xanthate, glutathione, ethylenediaminetetraacetic acid (EDTA), iminodiacetic acid, nitrilodiacetic acid, trimercapto-S-triazine, dimethyldithiocarbamate, dithiothreitol, mercaptoethanol, allyl alcohol, propargyl alcohol, thiol, thiosulfate salt (e.g., sodium thiosulfate or potassium thiosulfate), tertiary amine and / or tertiary phosphine, or combinations thereof. In one embodiment, the method also includes the use of L-cysteine or a salt thereof. In another embodiment, the method also includes the use of a thiosulfate salt such as sodium thiosulfate (NazSzOp. In some such embodiments, the Pd(II) scavenger (e.g., L-cysteine or sodium thiosulfate) is in the aqueous solution containing the DNA polymerase and the nucleotides (i.e., incorporation mix). In other embodiments, the Pd(II) scavenger (e.g., L-cysteine or sodium thiosulfate) is in the post cleavage wash solution. In other embodiments, the Pd(II) scavenger (e.g., L-cysteine or sodium thiosulfate) may be both present in the incorporation mixture. In other embodiments, the Pd(II) scavenger (e.g., L-cysteine or sodium thiosulfate) may be present in the scan mixture (i.e., the solution in which one or more fluorescent measurements of the incorporated nucleotide are performed). In other embodiments, the Pd(II) scavenger may be present in one or more of incorporation mixture, the scan mixture, or the post-cleavage wash solution.Compatibility with Linearization

[0093] In order to maximize the throughput of nucleic acid sequencing reactions it is advantageous to be able to sequence multiple template molecules in parallel. Parallel processing of multiple templates can be achieved with the use of nucleic acid array technology. These arrays typically consist of a high-density matrix of polynucleotides immobilized onto a solid support material.

[0094] WO 98 / 44151 and WO 00 / 18957 both describe methods of nucleic acid amplification which allow amplification products to be immobilized on a solid support in order toform arrays comprised of clusters or “colonies” formed from a plurality of identical immobilized polynucleotide strands and a plurality of identical immobilized complementary strands. Arrays of this type are referred to herein as “clustered arrays.” The nucleic acid molecules present in DNA colonies on the clustered arrays prepared according to these methods can provide templates for sequencing reactions, for example as described in WO 98 / 44152. The products of solid-phase amplification reactions such as those described in WO 98 / 44151 and WO 00 / 18957 are so-called “bridged” structures formed by annealing of pairs of immobilized polynucleotide strands and immobilized complementary strands, both strands being attached to the solid support at the 5' end. In order to provide more suitable templates for nucleic acid sequencing, it is preferred to remove substantially all or at least a portion of one of the immobilized strands in the “bridged” structure in order to generate a template which is at least partially single-stranded. The portion of the template which is single-stranded will thus be available for hybridization to a sequencing primer. The process of removing all or a portion of one immobilized strand in a “bridged” double-stranded nucleic acid structure is referred to as “linearization.” There are various ways for linearization, including but not limited to enzymatic cleavage, photo-chemical cleavage, or chemical cleavage. Non-limiting examples of linearization methods are disclosed in PCT Publication No. WO 2007 / 010251, U.S. Patent Publication No. 2009 / 0088327, U.S. Patent Publication Nos. 2009 / 0118128 and 2019 / 0352327, which are incorporated by reference in their entireties.

[0095] In some embodiments, the condition for deprotecting or removal of the 3'-OH blocking groups is also compatible with the linearization processes. In some further embodiments, the deprotection condition is compatible with a chemical linearization process which comprises the use of a Pd complex and a phosphine as described herein.

[0096] In some embodiments, at least one nucleotide is incorporated into a polynucleotide in the synthetic step by the action of a polymerase enzyme. In some such embodiments, the polymerase may be DNA polymerase Pol 812 or Pol 1901. However, other methods of joining nucleotides to polynucleotides, such as, for example, chemical oligonucleotide synthesis or ligation of labeled oligonucleotides to unlabeled oligonucleotides, can be used. Therefore, the term “incorporating,” when used in reference to a nucleotide and polynucleotide, can encompass polynucleotide synthesis by chemical methods as well as enzymatic methods.

[0097] In a specific embodiment, a synthetic step is carried out and may optionally comprise incubating a template polynucleotide strand with a reaction mixture comprising 3' blocked nucleotides of the disclosure. A polymerase can also be provided under conditions which permit formation of a phosphodiester linkage between a free 3’-OH group on a polynucleotide strand annealed to the template polynucleotide strand and a 5’ phosphate group on the nucleotide.Thus, a synthetic step can include formation of a polynucleotide strand as directed by complementary base-pairing of nucleotides to a template strand.

[0098] In all embodiments of the methods, the detection step may be carried out while the polynucleotide strand into which the 3 ' blocked nucleotides are incorporated is annealed to a template strand, or after a denaturation step in which the two strands are separated. Further steps, for example chemical or enzymatic reaction steps or purification steps, may be included between the synthetic step and the detection step. In particular, the target strand incorporating the labeled nucleotide(s) may be isolated or purified and then processed further or used in a subsequent analysis. By way of example, target polynucleotides labeled with nucleotide(s) as described herein in a synthetic step may be subsequently used as labeled probes or primers. In other embodiments, the product of the synthetic step set forth herein may be subject to further reaction steps and, if desired, the product of these subsequent steps purified or isolated.

[0099] Suitable conditions for the synthetic step will be well known to those familiar with standard molecular biology techniques. In one embodiment, a synthetic step may be analogous to a standard primer extension reaction using nucleotide precursors, including nucleotides as described herein, to form an extended target strand complementary to the template strand in the presence of a suitable polymerase enzyme. In other embodiments, the synthetic step may itself form part of an amplification reaction producing a labeled double stranded amplification product comprised of annealed complementary strands derived from copying of the target and template polynucleotide strands. Other exemplary synthetic steps include nick translation, strand displacement polymerization, random primed DNA labeling, etc. A particularly useful polymerase enzyme for a synthetic step is one that is capable of catalyzing the incorporation of nucleotides as set forth herein. A variety of naturally occurring or modified polymerases can be used. By way of example, a thermostable polymerase can be used for a synthetic reaction that is carried out using thermocycling conditions, whereas a thermostable polymerase may not be desired for isothermal primer extension reactions. Suitable thermostable polymerases which are capable of incorporating the nucleotides according to the disclosure include those described in WO 2005 / 024010 or WO 06 / 120433, each of which is incorporated herein by reference. In synthetic reactions which are carried out at lower temperatures such as 37 °C, polymerase enzymes need not necessarily be thermostable polymerases, therefore the choice of polymerase will depend on a number of factors such as reaction temperature, pH, strand-displacing activity and the like.

[0100] In specific non-limiting embodiments, the disclosure encompasses methods of nucleic acid sequencing, re-sequencing, whole genome sequencing, single nucleotide polymorphism scoring, any other application involving the detection of the labeled nucleotide or nucleoside set forth herein when incorporated into a polynucleotide. Any of a variety of otherapplications benefitting the use of polynucleotides labeled with the nucleotides comprising fluorescent dyes can use labeled nucleotides or nucleosides with dyes set forth herein.

[0101] In a particular embodiment, the disclosure provides use of labeled nucleotides according to the disclosure in a polynucleotide sequencing-by-synthesis (SBS) reaction. Sequencing-by-synthesis generally involves sequential addition of one or more nucleotides or oligonucleotides to a growing polynucleotide chain in the 5' to 3' direction using a polymerase or ligase in order to form an extended polynucleotide chain complementary to the template nucleic acid to be sequenced. The identity of the base present in one or more of the added nucleotide(s) can be determined in a detection or “imaging” step. The identity of the added base may be determined after each nucleotide incorporation step. The sequence of the template may then be inferred using conventional Watson-Crick base-pairing rules. The use of the labeled nucleotides set forth herein for determination of the identity of a single base may be useful, for example, in the scoring of single nucleotide polymorphisms, and such single base extension reactions are within the scope of this disclosure.

[0102] In an embodiment of the present disclosure, the sequence of a template polynucleotide is determined by detecting the incorporation of one or more 3' blocked nucleotides described herein into a nascent strand complementary to the template polynucleotide to be sequenced through the detection of fluorescent label(s) (or lack of fluorescent label) attached to the incorporated nucleotide(s). Sequencing of the template polynucleotide can be primed with a suitable primer (or prepared as a hairpin construct which will contain the primer as part of the hairpin), and the nascent chain is extended in a stepwise manner by addition of nucleotides to the 3’ end of the primer in a polymerase-catalyzed reaction.

[0103] The method, as exemplified above, utilizes the incorporation of fluorescently labeled, 3 '-blocked nucleotides A, G, C, and T into a growing strand complementary to the immobilized polynucleotide, in the presence of DNA polymerase. The polymerase incorporates a base complementary to the target polynucleotide but is prevented from further addition by the 3’-blocking group. The label of the incorporated nucleotide can then be determined, and the blocking group removed by chemical cleavage to allow further polymerization to occur. The nucleic acid template to be sequenced in a sequencing-by-synthesis reaction may be any polynucleotide that it is desired to sequence. The nucleic acid template for a sequencing reaction will typically comprise a double stranded region having a free 3 ’-OH group that serves as a primer or initiation point for the addition of further nucleotides in the sequencing reaction. The region of the template to be sequenced will overhang this free 3'-OH group on the complementary strand. The overhanging region of the template to be sequenced may be single stranded but can be doublestranded, provided that a “nick” is present on the strand complementary to the template strand tobe sequenced to provide a free 3'-OH group for initiation of the sequencing reaction. In such embodiments, sequencing may proceed by strand displacement. In certain embodiments, a primer bearing the free 3'-OH group may be added as a separate component (e.g., a short oligonucleotide) that hybridizes to a single- stranded region of the template to be sequenced. Alternatively, the primer and the template strand to be sequenced may each form part of a partially self- complementary nucleic acid strand capable of forming an intra-molecular duplex, such as for example a hairpin loop structure. Hairpin polynucleotides and methods by which they may be attached to solid supports are disclosed in PCT Publication Nos. WO 01 / 57248 and WO 2005 / 047301, each of which is incorporated herein by reference. Nucleotides can be added successively to a growing primer, resulting in synthesis of a polynucleotide chain in the 5' to 3' direction. The nature of the base which has been added may be determined, particularly but not necessarily after each nucleotide addition, thus providing sequence information for the nucleic acid template. Thus, a nucleotide is incorporated into a nucleic acid strand (or polynucleotide) by joining of the nucleotide to the free 3'-OH group of the nucleic acid strand via formation of a phosphodiester linkage with the 5' phosphate group of the nucleotide.

[0104] The nucleic acid template to be sequenced may be DNA or RNA, or even a hybrid molecule comprised of deoxynucleotides and ribonucleotides. The nucleic acid template may comprise naturally occurring and / or non-naturally occurring nucleotides and natural or nonnatural backbone linkages, provided that these do not prevent copying of the template in the sequencing reaction.

[0105] In certain embodiments, the nucleic acid template to be sequenced may be attached to a solid support via any suitable linkage method known in the art, for example via covalent attachment. In certain embodiments template polynucleotides may be attached directly to a solid support (e.g., a silica-based support). However, in other embodiments of the disclosure the surface of the solid support may be modified in some way so as to allow either direct covalent attachment of template polynucleotides, or to immobilize the template polynucleotides through a hydrogel or polyelectrolyte multilayer, which may itself be non-covalently attached to the solid support.

[0106] In any embodiments of the methods described herein, the nucleotide used in the sequencing application is a 3' blocked nucleotide described herein. In any embodiments, the 3' blocked nucleotide is a nucleotide triphosphate.EXAMPLES

[0107] 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. Sequencing-by-Synthesis Experiment with Modified ffG

[0108] A modified ffG in accordance with the present disclosurealso referred to as “ffG+30” because of the molecular weight difference between the instant blocking group and the standard AOM blocking group) was synthesized.

[0109] Allyloxymethyl chloride (A0M-C1) was added dropwise into a mixture of TBDPS-PAC-dG, AgOTf and MeTHF at -25°C. After 1 h, the reaction was quenched with NEts, filtered through celite and washed with NaHCCh aq. solution. The organic layer was concentrated under reduce pressure to give an orange oil. The crude was subsequently treated with TBAF to give a mixture of G and G+30, which was triturated in EtOAc.

[0110] The mixture of G and G+30 was then converted to the corresponding triphosphate ffG and ffG+30. POCI3 was added dropwise to a mixture of G, G+30, proton sponge and triethylphosphate at -15°C. Once analysis by HPLC had indicated >95% consumption of G and G+30, the reaction mixture was transferred to a second reaction vessel containing TBAPP solution (bis-tri-n-butylammonium pyrophosphate) and tributylamine. The temperature for the reaction mixture was then increased to 0 °C. Once the reaction was complete, TEAB (triethylammonium bicarbonate) buffer (2.0 M) was slowly added to the reaction mixture. The temperature was then increased to 25 °C and the reaction mixture was stirred overnight. The mixture was washed with EtOAc and aqueous layer was concentrated under reduce pressure. Aqueous methylamine (40%) was added to the residue and the reaction mixture was stirred at room temperature overnight. The mixture was concentrated and ffG+30 was isolated by anion- exchange reverse-phase chromatography.

[0111] The ffG+30 was spiked into NextSeq 1000 / 2000 XLEAP reagents (a ffN set having ffG with a structurecreate test reagents. Of the total ffG present in the test reagents, ffG+30 made up 10% molar percentage of the total amount of ffG in the incorporation mixture. The test reagents were compared to P2 2x151 sequencing on an Illumina NextSeq 2000 sequencing instrument using XLEAP SBS reagents and a BP550 library.

[0112] FIG. 1 is box plot showing differences in error rate, %Q30 score, phasing, and prephasing for standard reagents and test reagents. The test reagents showed significantly lower % error rate, phasing, and prephasing and a significantly higher %Q30. These metrics indicate that the ffG+30 is suitable for inclusion in SBS reagents.Example 2. Incorporation Rate of Modified ffG

[0113] Incorporation rate of the ffG+30 was measured and compared to that of the standard ffG discussed with reference to Example 1. The assay was carried out with glycine buffer, a SBS polymerase (polymerase A) at a concentration of 30mg / ml, 20nM template primer, with 4 mM MgSO4 at 30 °C. Standard ffG and ffG+30 were tested at concentrations of 0.1 pM and 0.5 pM.

[0114] FIG. 2A is a line chart illustrating the percentage incorporation as a function of time for standard ffG and ffG+30. For other ffNs, percent incorporation over time of modified ffNs is comparable to that of standard ffNs using various standard SBS polymerases (including, for example, Pol 1901, Polymerase A, among others).

[0115] FIG. 2B is a bar chart showing kcat of incorporation of the same. The results indicate that ffG+30 can be quickly incorporated using existing SBS polymerase.Example 3. Cleavage and Incorporation of Modified ffA

[0116] A modified ffA in accordance with the present disclosure“ffA+30”) was synthesized. The ffA+30 was subjected to an incorporation assay, compared to standard SBS reagents having ffA with a structurecoumarin dye A). The modified ffA and standard ffA were subjected to incorporation assays on an icBot (Illumina) using a standard SBS polymerase (Pol 1901). Coumarin dye A has strong fluorescence. This dye is disclosed in U.S. Publication No. 2020 / 0277670 Al, having the structure moietyconjugated with the ffA.

[0117] FIG.3A is a plot showing blocking group cleavage (i.e,, 3'-OH %) as a function of time for standard ffA against ffA+30. Cleavage speed of ffA+30 was comparable to that of standard ffA in two different incorporation assays.

[0118] The standard ffA and ffA+30 were subjected to incorporation assays using a SBS polymerase on an Illumina cBot instrument. FIG. 3B is a bar chart showing vmfKand kmfor incorporation of standard ffA and ffA+30.Example 4. Kinetics of Modified ffNs

[0119] Modified ffNs were synthesized in accordance with the present disclosure (i.e.,was added as a 3'-0 -blocking group, referred to as “+30”). The modified ffNs included ffT labeled with a green polymethine dye AF550POPOsO, ffC including a green dye (NR550S0), ffC labeled with the blue coumarin dye A, and dark dGTP. Incorporation kinetics of the modified ffNs and corresponding standard ffNs were measured on an icBot (Illumina) and a standard SBS polymerase (polymerase A). The kinetics were measured at a ffN concentration of 0.1 M. Trends were identical to those measured at 0.05 ,u M.

[0120] FIG. 4 is a plot showing the observed incorporation rate (kObs) of the modified and standard ffNs. The incorporation rate k„bs for ffC (green) +30 was higher than that of the standard ffC with green dye. Additionally, the incorporation rate of dGTP+30 was about 5 times higher than that of the standard dGTP.Example 5. Kinetics of Modified dGTP

[0121] Modified dGTP (“dGTP+30”) was synthesized in accordance with Example 1. The modified ffG was subject to an incorporation assay using an icBot (Illumina) and a standard SBS polymerase at 40 °C. Standard dGTP was also run in a parallel assay for comparison.

[0122] FIG. 5 is a plot showing the reaction constant (kcat) as a function of ffN concentration for both dGPT+30 and standard dGTP. At the concentrations tested, dGTP+30 was about 2 times as efficient for incorporation in comparison to the standard dGTP.

Claims

WHAT IS CLAIMED IS:

1. A nucleoside or nucleotide comprising a ribose or 2' deoxyribose having a removable 3'-OH blocking group, wherein the 3'-OH blocking group has a structure ofcovalently attached to the 3 '-oxygen atom of the ribose or 2' deoxyribose, wherein each R1and R2is independently H, C1-C4 alkyl, C1-C4 haloalkyl, cyano, or halogen; and each R3, R4a, R4b, R5a, R5b, R6a, and R6bis independently H, C1-C3 alkyl, cyano, or halogen.

2. The nucleoside or nucleotide of claim 1, wherein each of R4aand R4bis H.

3. The nucleoside or nucleotide of claim 1 or 2, wherein each of R5aand R5bis H.

4. The nucleoside or nucleotide of any one of claims 1 to 3, wherein each of R6aand R6bis H.

5. The nucleoside or nucleotide of any one of claims 1 to 4, wherein each of R1, R2and R3is independently H, F, Cl, methyl, ethyl, isopropyl or trifluoromethyl.

6. The nucleoside or nucleotide of claim 5, wherein7. The nucleoside or nucleotide of claim 6, wherein the 3'-OH blocking group has a8. The nucleoside or nucleotide of any one of claims 1 to 7, wherein the nucleoside or nucleotide is covalently attached to a detectable moiety, optionally via a cleavable linker.

9. The nucleoside or nucleotide of claim 8, wherein the detectable moiety is covalently attached to a nucleobase of the nucleoside or nucleotide via the cleavable linker.

10. The nucleoside or nucleotide of claim 8 or 9, wherein the cleavable linker comprising an azido moiety, a -O-allyl moiety, a disulfide moiety, an acetal moiety, or a thiocarbamate moiety.

11. The nucleoside or nucleotide of any one of claims 8 to 10, wherein the 3'-OH blocking group and the cleavable linker are cleav ble under the same chemical reaction conditions.

12. The nucleoside or nucleotide of any one of claims 8 to 11, wherein the detectable moiety is a fluorescent label.

13. The nucleoside or nucleotide of any one of claims 8 to 11, wherein the detectable moiety comprises a non-fluorescent functional moiety that is capable of binding to a labeling reagent via covalent bonding or noncovalent interaction.

14. The nucleoside or nucleotide of any one of claims 1 to 7, wherein the nucleoside or nucleotide is unlabeled and does not contain any detectable moiety.

15. The nucleoside or nucleotide of any one of claims 1 to 14, comprising a 2' deoxyribose.

16. The nucleoside or nucleotide of claim 15, wherein the nucleotide is a nucleotide triphosphate.

17. An oligonucleotide comprising the nucleotide of any one of claims 1 to 16 incorporated thereto.

18. A kit comprising one or more nucleotides, wherein one type of nucleotide is the nucleotide according to any one of claims 1 to 16.

19. The kit of claim 18, further comprising an enzyme and a buffer appropriate for the action of the enzyme.

20. The kit of claim 19, wherein the enzyme is a polymerase, a terminal deoxynucleotidyl transferase, or a reverse transcriptase.

21. The kit of claim 20, wherein the polymerase is a DNA polymerase.

22. A method of preparing a polynucleotide, comprising incorporating a nucleotide of any one of claims 1 to 16 into a growing polynucleotide in the presence of a polymerase, wherein the incorporation of the nucleotide prevents the introduction of any subsequent nucleotide into the growing polynucleotide.

23. The method of claim 22, wherein the incorporation of the nucleotide is accomplished by a polymerase, a terminal deoxynucleotidyl transferase, or a reverse transcriptase.

24. The method of claim 22 or 23, further comprising: removing the 3" blocking group of the incorporated nucleotide to generate a 3’ hydroxy group on the incorporated nucleotide, and incorporating a second nucleotide.

25. A method of determining sequences of a plurality of different target polynucleotides in parallel, the method comprising:(a) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different targetpolynucleotides 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 solution comprising DNA polymerase and one or more of four different types of nucleotides (A, G, C and T or U) under conditions suitable for DNA polymerase-mediated primer extension, wherein:(i) at least a portion of one type of nucleotide in the aqueous incorporation solution is the nucleotide of claim 12 having a fluorescent label covalently attached to the nucleobase via a cleavable linker, or an unlabeled nucleotide of claim 14; and(ii) each type of nucleotide is a nucleotide triphosphate having a 3'-OH blocking group covalently attached to the 2' deoxyribose of the nucleotide;(c) imaging the solid support and performing one or more fluorescent measurements to determine the identity of incorporated nucleotides;(d) contacting the solid support with an aqueous deblocking solution;(e) contacting said solid support with an aqueous wash solution; and(f) repeating steps (b)-(e) to determine target polynucleotide sequences.

26. The method of claim 25, wherein at least a portion of one type of nucleotides in the aqueous incorporation solution each has the 3'-OH blocking group of the structureattached to the 3’ oxygen atom.

27. The method of claim 26, wherein the portion of one type of unlabeled nucleotideshaving the 3'-OH blocking group of the structure X. is about 5% or more of the total amount of such type of unlabeled nucleotides.

28. The method of claim 26 or 27, wherein the other nucleotides in the aqueous incorporation solution each has the 3'-OH blocking group of the structureattached to the 3’ oxygen atom.

29. The method of any one of claims 25 to 28, wherein steps (b) to (e) are repeated at least50 cycles.

30. The method of any one of claims 25 to 29, wherein the cleavable linker comprises a moiety selected from the group consisting of:, wherein X is O, Y is O, and * indicates where the moiety is connected to the remainder of the nucleotide.Xjt31. The method of claim 30, wherein the cleavable linker comprises32. The method of claim 31, wherein the cleavable linker is selected from the group consisting of: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 nucleobase; and ** indicates the attachment point of the cleavable linker to the fluorescent label.

33. A method of determining sequences of a plurality of different target polynucleotides in parallel, the method 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 solution comprising DNA polymerase and one or more of four different types of nucleotides (A, G, C and T or U) under conditions suitable for DNA polymerase-mediated primer extension to produce extended copy polynucleotides, wherein:(i) at least a portion of one type of unlabeled nucleotide in the aqueous incorporation solution is the unlabeled nucleotide of claim 13 having a non- fluorescent functional moiety covalently attached to the nucleobase via a cleavable linker, or an unlabeled nucleotide of claim 14; and(ii) at least two types of nucleotides are unlabeled; and(iii) each type of nucleotide is a nucleotide triphosphate having a 3'-OH blocking group covalently attached to the 1' deoxyribose of the nucleotide;(c) contacting the extended copy polynucleotides with a labeling reagent comprising one or more fluorescent labels and a binding moiety that is capable of specific binding to the unlabeled nucleotide;(d) imaging the solid support and performing one or more fluorescent measurements to determine the identity of incorporated nucleotides;(e) contacting the solid support with an aqueous deblocking solution;(f) contacting said solid support with an aqueous wash solution; and(g) repeating steps (b)-(f) to determine target polynucleotide sequences.

34. The method of claim 33, wherein at least a portion of one type of unlabeled nucleotides in the aqueous incorporation solution each has the 3'-OH blocking group of the structureattached to the 3’ oxygen atom.

35. The method of claim 34, wherein the portion of one type of unlabeled nucleotideshaving the 3'-OH blocking group of the structure ° is about 5% or more of the total amount of such type of unlabeled nucleotides.

36. The method of claim 34 or 35, wherein the other nucleotides in the aqueous incorporation solution each has the 3'-OH blocking group of the structureattached to the 3’ oxygen atom.

37. The method of any one of claims 33 to 36, wherein steps (b) to (f) are repeated at least 50 cycles.

38. The method of any one of claims 33 to 37, wherein the cleavable linker comprises a moiety selected from the group consisting of:wherein X is O, Y is O, and * indicates where the moiety is connected to the remainder of the nucleotide.

39. The method of claim 38, wherein the cleavable linker comprises40. The method of claim 39, wherein the cleavable linker is selected from the group consisting of: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 non-fluorescent functional moiety.

41. The method of any one of claims 25 to 40, wherein the DNA polymerase is an altered family B archaeal DNA polymerase.

42. The method of any one of claims 25 to 41, wherein at least one type of nucleotide has a structure selected from the group consisting of:

43. The method of any one of claims 25 to 42, wherein the aqueous deblocking solution comprises a palladium catalyst and tris(hydroxypropyl)phosphine.-SO-

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