WO2025230914A1 - Nucleotides with enzyme-triggered self-immolative linkers for sequencing by synthesis - Google Patents

Abstract

Embodiments of the present disclosure relate to nucleotide molecules having an enzymatically cleavable self-immolative linker group. Also provided herein are methods and kits for sequencing applications using such nucleotides.

Description

IP-2771-PCT PATENT NUCLEOTIDES WITH ENZYME-TRIGGERED SELF-IMMOLATIVE LINKERS FOR SEQUENCING BY SYNTHESIS BACKGROUND Field [0001] The present disclosure generally relates to nucleotides, nucleosides, or oligonucleotides comprising enzymatically cleavable self-immolative linker groups and their uses in polynucleotide sequencing methods. Methods of preparing the nucleotides, nucleosides, or oligonucleotides having enzymatically cleavable self-immolative linker groups 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 (“blocking group” or “protecting group”) is included in each labeled nucleotide that is added to the growing chain to ensure that only one nucleotide is incorporated. After the nucleotide with the protecting group has been added, the protecting 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 3ʹ-hydroxy protecting 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 protecting 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 protecting 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 protecting 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 3ʹ-modified 2-deoxyribonucleoside 5’-triphosphates (3ʹ-modified dNTPs) and testing in two DNA template assays for incorporation activity. WO 2002/029003 describes a sequencing method which may include the use of an allyl protecting group to cap the 3ʹ-OH group on a growing strand of DNA in a polymerase reaction. [0008] In addition, the development of a number of reversible protecting groups and methods of deprotecting them under DNA compatible conditions was previously reported in International Application Publication Nos. WO 2004/018497, WO 2014/139596, and U.S. Pub. Nos.2020/0216891 A1, and 2023/0332197 A1, each of which is hereby incorporated by reference in its entirety. SUMMARY [0009] One aspect of the present disclosure relates to a nucleotide comprising a nucleobase, a ribose or 2´ deoxyribose, and a detectable moiety, wherein the detectable moiety is covalently attached to the nucleotide via a self-immolative linker, and wherein the self-immolative linker comprises an optionally substituted benzyl moiety, a leaving group comprising a carbamate moiety or an acetyl moiety, and an enzymatically cleavable moiety. [0010] Another aspect of the present disclosure relates to an oligonucleotide or polynucleotide comprising a nucleotide described herein incorporated thereof. [0011] Another aspect of the present disclosure relates to a kit comprising a nucleotide described in the present disclosure. [0012] Another aspect of the present disclosure relates to a method of preparing a growing polynucleotide complementary to a target single-stranded polynucleotide, comprising incorporating a nucleotide of the present disclosure into a growing complementary polynucleotide. [0013] Another aspect of the present disclosure relates to a method of determining the sequences of a plurality of target polynucleotides, comprising: (a) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides; (b) contacting the solid support with an aqueous solution comprising DNA polymerase and one or more of four different types of nucleotides (A, G, C, and T or U; dATP, dGTP, dCTP and dTTP or dUTP) under conditions suitable for DNA polymerase- mediated primer extension, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides, wherein at least one type of nucleotide is a nucleotide of the present disclosure carrying a fluorescent label through the self- immolative linker, and wherein each of the one or more of four different type of nucleotides comprises a 3´ blocking group; (c) imaging the solid support and performing one or more fluorescent measurements of the extended copy polynucleotides; and (d) removing the fluorescent label and the 3ʹ blocking group from nucleotides incorporated into the extended copy polynucleotides. [0014] A further aspect of the present disclosure relates to a method of determining the sequences of a plurality of target polynucleotides, comprising: (a’) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides; (b’) contacting the solid support with an aqueous 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, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides, wherein at least one type of nucleotide is an unlabeled nucleotide of the present disclosure having a first functional group attached via the self-immolative linker, and wherein each of the one or more of four different type of nucleotides comprises a 3´ blocking group; (c’) contacting the extended copy polynucleotides with an aqueous labeling mixture comprising a first labeling reagent, wherein the first labeling reagent comprises one or more first fluorescent labels and a first binding moiety that is capable of specific binding to the first functional group of the unlabeled nucleotide; (d’) imaging the solid support and performing one or more fluorescent measurements of the extended copy polynucleotides; and (e’) removing the one or more fluorescent labels and the 3ʹ blocking group from the nucleotides incorporated into the extended copy polynucleotides. BRIEF DESCRIPTION OF THE DRAWINGS [0015] FIGs. 1A and 1B are HPLC analysis plots showing self-immolation of nucleotide containing a carbamate arabinofuranoside (c-ABF) linker moiety. FIG.1A shows the HPLC peak of a negative control without enzymatic cleavage, while FIG. 1B shows the HPLC peak of the nucleotide self-immolation product relative to the starting material. [0016] FIG.2 is a line chart showing percent residual starting material as a functional of time of nucleotides comprising various cleavable linker moieties. [0017] FIG.3A–3C are HPLC analysis plots demonstrating reaction kinetics of self- immolation of a nucleotide containing an acetal arabinofuranoside (a-ABF) linker moiety, taken at reaction times t = 0 seconds (FIG. 3A), t = 30 seconds (FIG. 3B), and t = 30 minutes (FIG. 3C). DETAILED DESCRIPTION [0018] Embodiments of the present disclosure relate to nucleosides and nucleotides with an enzymatically cleavable self-immolative linkers for sequencing applications, for example, sequencing-by-synthesis (SBS). The self-immolative linker allow for enzymatic cleavage. In particular, enzymatic cleavage is likely to be less damaging to DNA and may generate less signal decay during SBS. Additionally, enzymes for cleaving the self-immolative linker may have improved stability during storage and shipping. For example, such enzymes can be freeze dried. Recombinant SBS cleaving enzymes may be produced at scale from relatively low costs. Definitions [0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. The use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting. The use of the term “having” as well as other forms, such as “have,” “has,” and “had,” is not limiting. As used in this specification, whether in a transitional phrase or in the body of the claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the above terms are to be interpreted synonymously with the phrases “having at least” or “including at least.” For example, when used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components. [0020] Where a range of values is provided, it is understood that the upper and lower limit, and each intervening value between the upper and lower limit of the range is encompassed within the embodiments. [0021] As used herein, common organic abbreviations are defined as follows: °C Temperature in degrees Centigrade ABF or Abf Arabinofuranosidyl dATP Deoxyadenosine triphosphate dCTP Deoxycytidine triphosphate dGTP Deoxyguanosine triphosphate dTTP Deoxythymidine triphosphate ddNTP Dideoxynucleotide triphosphate ffN Fully functionalized nucleotide RT Room temperature SBS Sequencing by Synthesis SM Starting material [0022] As used herein, the term “array” refers to a population of different probe molecules that are attached to one or more substrates such that the different probe molecules can be differentiated from each other according to relative location. An array can include different probe molecules that are each located at a different addressable location on a substrate. Alternatively, or additionally, an array can include separate substrates each bearing a different probe molecule, wherein the different probe molecules can be identified according to the locations of the substrates on a surface to which the substrates are attached or according to the locations of the substrates in a liquid. Exemplary arrays in which separate substrates are located on a surface include, without limitation, those including beads in wells as described, for example, in U.S. Patent No.6,355,431 B1, US 2002/0102578 and PCT Publication No. WO 00/63437. Exemplary formats that can be used in the invention to distinguish beads in a liquid array, for example, using a microfluidic device, such as a fluorescent activated cell sorter (FACS), are described, for example, in US Pat. No. 6,524,793. Further examples of arrays that can be used in the invention include, without limitation, those described in U.S. Pat Nos. 5,429,807; 5,436,327; 5,561,071; 5,583,211; 5,658,734; 5,837,858; 5,874,219; 5,919,523; 6,136,269; 6,287,768; 6,287,776; 6,288,220; 6,297,006; 6,291,193; 6,346,413; 6,416,949; 6,482,591; 6,514,751 and 6,610,482; and WO 93/17126; WO 95/11995; WO 95/35505; EP 742287; and EP 799897. [0023] As used herein, the term “covalently attached” or “covalently bonded” refers to the forming of a chemical bonding that is characterized by the sharing of pairs of electrons between atoms. For example, a covalently attached polymer coating refers to a polymer coating that forms chemical bonds with a functionalized surface of a substrate, as compared to attachment to the surface via other means, for example, adhesion or electrostatic interaction. It will be appreciated that polymers that are attached covalently to a surface can also be bonded via means in addition to covalent attachment. [0024] As used herein, any “R” group(s) represent substituents that can be attached to the indicated atom. An R group may be substituted or unsubstituted. If two “R” groups are described as “together with the atoms to which they are attached” forming a ring or ring system, it means that the collective unit of the atoms, intervening bonds and the two R groups are the recited ring. For example, when the following substructure is present: and R¹ and R² are defined as selected consisting of hydrogen and alkyl, or R¹ and R² together with the atoms to which they are attached form an aryl or carbocyclyl, it is meant that R¹ and R² can be selected from hydrogen or alkyl, or alternatively, the substructure has structure: where A is an aryl ring or a the depicted double bond. [0025] 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.” [0026] 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. [0027] As used herein, “Ca to 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 “C1 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 C4 cycloalkyl group refers to all cycloalkyl groups having from 3 to 4 carbon atoms, that is, cyclopropyl and cyclobutyl. Similarly, a “4 to 6 membered heterocyclyl” group refers to all heterocyclyl groups with 4 to 6 total ring atoms, for example, azetidine, oxetane, oxazoline, pyrrolidine, piperidine, piperazine, morpholine, and the like. If no “a” and “b” are designated with regard to an alkyl, alkenyl, alkynyl, cycloalkyl, or aryl group, the broadest range described in these definitions is to be assumed. As used herein, the term “C1-C6” includes C1, C2, C3, C4, C5 and C6, and a range defined by any of the two numbers_. For example, C₁-C₆ alkyl includes C₁, C₂, C₃, C₄, C₅ and C₆ alkyl, C2-C6 alkyl, C1-C3 alkyl, etc. Similarly, C2-C6 alkenyl includes C2, C3, C4, C5 and C6 alkenyl, C₂-C₅ alkenyl, C₃-C₄ alkenyl, etc.; and C₂-C₆ alkynyl includes C₂, C₃, C₄, C₅ and C₆ 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. [0028] 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 “C₁-C₄ alkyl” or similar designations. By way of example only, “C₁-C₆ 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. [0029] As used herein, “alkoxy” refers to the formula –OR wherein R is an alkyl as is defined above, such as “C₁-C₉ alkoxy”, including but not limited to methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy, and the like. [0030] As used herein, “alkenyl” refers to a straight or branched hydrocarbon chain containing one or more double bonds. The alkenyl group may have 2 to 20 carbon atoms, although the present definition also covers the occurrence of the term “alkenyl” where no numerical range is designated. The alkenyl group may also be a medium size alkenyl having 2 to 9 carbon atoms. The alkenyl group could also be a lower alkenyl having 2 to 6 carbon atoms. The alkenyl group may be designated as “C2-C6 alkenyl” or similar designations. By way of example only, “C2-C6 alkenyl” indicates that there are two to six carbon atoms in the alkenyl chain, i.e., the alkenyl chain is selected from the group consisting of ethenyl, propen-1-yl, propen-2-yl, propen-3-yl, buten-1- yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl- ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl, buta-1,2,-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groups include, but are in no way limited to, ethenyl, propenyl, butenyl, pentenyl, and hexenyl, and the like. [0031] As used herein, “alkynyl” refers to a straight or branched hydrocarbon chain containing one or more triple bonds. The alkynyl group may have 2 to 20 carbon atoms, although the present definition also covers the occurrence of the term “alkynyl” where no numerical range is designated. The alkynyl group may also be a medium size alkynyl having 2 to 9 carbon atoms. The alkynyl group could also be a lower alkynyl having 2 to 6 carbon atoms. The alkynyl group may be designated as “C₂-C₆ alkynyl” or similar designations. By way of example only, “C₂-C₆ alkynyl” indicates that there are two to six carbon atoms in the alkynyl chain, i.e., the alkynyl chain is selected from the group consisting of ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and 2-butynyl. Typical alkynyl groups include, but are in no way limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl, and the like. [0032] As used herein, “heteroalkyl” refers to a straight or branched hydrocarbon chain containing one or more heteroatoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur, in the chain backbone. The heteroalkyl group may have 1 to 20 carbon atoms, although the present definition also covers the occurrence of the term “heteroalkyl” where no numerical range is designated. The heteroalkyl group may also be a medium size heteroalkyl having 1 to 9 carbon atoms. The heteroalkyl group could also be a lower heteroalkyl having 1 to 6 carbon atoms. The heteroalkyl group may be designated as “2 to 10 membered heteroalkyl” or similar designations. The heteroalkyl group may contain one or more heteroatoms. By way of example only, “2 to 10 membered heteroalkyl” indicates that the total number of carbon atoms and one or more heteroatoms (excluding hydrogen atoms) in the backbone of the chain is 2 to 10. [0033] As used herein, “heteroalkylene” refers to an alkylene group, as defined herein, containing one or more heteroatoms in the carbon back bone (i.e., an alkylene group in which one or more carbon atoms is replaced with a heteroatom, for example, nitrogen atom, oxygen atom or sulfur atom). For example, a -CH2- may be replaced with -O-, -S-, or -NH-. Heteroalkylene groups include, but are not limited to ether, thioether, amino-alkylene, and alkylene-amino-alkylene moieties. In some embodiments, the heteroalkylene may include one, two, three, four, or five - CH2CH2O- unit(s). Alternatively and/or additionally, one or more carbon atoms can also be substituted with an oxo (=O) to become a carbonyl. For example, a -CH2- may be replaced with - C(=O)-. [0034] The term “aromatic” refers to a ring or ring system having a conjugated pi electron system and includes both carbocyclic aromatic (e.g., phenyl) and heterocyclic aromatic groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of atoms) groups provided that the entire ring system is aromatic. [0035] As used herein, “aryl” refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent carbon atoms) containing only carbon in the ring backbone. When the aryl is a ring system, every ring in the system is aromatic. The aryl group may have 6 to 18 carbon atoms, although the present definition also covers the occurrence of the term “aryl” where no numerical range is designated. In some embodiments, the aryl group has 6 to 10 carbon atoms. The aryl group may be designated as “C6-C10 aryl,” “C6 or C10 aryl,” or similar designations. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl. [0036] An “aralkyl” or “arylalkyl” is an aryl group connected, as a substituent, via an alkylene group, such as “C7-14 aralkyl” and the like, including but not limited to benzyl, 2- phenylethyl, 3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., a C1-C6 alkylene group). [0037] As used herein, “heteroaryl” refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent atoms) that contain(s) one or more heteroatoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur, in the ring backbone. When the heteroaryl is a ring system, every ring in the system is aromatic. The heteroaryl group may have 5-18 ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms), although the present definition also covers the occurrence of the term “heteroaryl” where no numerical range is designated. In some embodiments, the heteroaryl group has 5 to 10 ring members or 5 to 7 ring members. The heteroaryl group may be designated as “5-7 membered heteroaryl,” “5-10 membered heteroaryl,” or similar designations. Examples of heteroaryl rings include, but are not limited to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothienyl. [0038] A “heteroaralkyl” or “heteroarylalkyl” is heteroaryl group connected, as a substituent, via an alkylene group. Examples include but are not limited to 2-thienylmethyl, 3- thienylmethyl, furylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., a C1-C6 alkylene group). [0039] As used herein, “carbocyclyl” means a non-aromatic cyclic ring or ring system containing only carbon atoms in the ring system backbone. When the carbocyclyl is a ring system, two or more rings may be joined together in a fused, bridged or spiro-connected fashion. Carbocyclyls may have any degree of saturation provided that at least one ring in a ring system is not aromatic. Thus, carbocyclyls include cycloalkyls, cycloalkenyls, and cycloalkynyls. The carbocyclyl group may have 3 to 20 carbon atoms, although the present definition also covers the occurrence of the term “carbocyclyl” where no numerical range is designated. The carbocyclyl group may also be a medium size carbocyclyl having 3 to 10 carbon atoms. The carbocyclyl group could also be a carbocyclyl having 3 to 6 carbon atoms. The carbocyclyl group may be designated as “C3-C6 carbocyclyl” or similar designations. Examples of carbocyclyl rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro- indene, bicycle[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl. [0040] As used herein, “cycloalkyl” means a fully saturated carbocyclyl ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. [0041] As used herein, “heterocyclyl” means a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring backbone. Heterocyclyls may be joined together in a fused, bridged or spiro-connected fashion. Heterocyclyls may have any degree of saturation provided that at least one ring in the ring system is not aromatic. The heteroatom(s) may be present in either a non-aromatic or aromatic ring in the ring system. The heterocyclyl group may have 3 to 20 ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms), although the present definition also covers the occurrence of the term “heterocyclyl” where no numerical range is designated. The heterocyclyl group may also be a medium size heterocyclyl having 3 to 10 ring members. The heterocyclyl group could also be a heterocyclyl having 3 to 6 ring members. The heterocyclyl group may be designated as “3-6 membered heterocyclyl” or similar designations. In preferred six membered monocyclic heterocyclyls, the heteroatom(s) are selected from one up to three of O, N or S, and in preferred five membered monocyclic heterocyclyls, the heteroatom(s) are selected from one or two heteroatoms selected from O, N, or S. Examples of heterocyclyl rings include, but are not limited to, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidinyl, pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathianyl, 1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2- oxazinyl, trioxanyl, hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl, 1,3- dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl, thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, and tetrahydroquinoline. [0042] As used herein, “alkoxyalkyl” or “(alkoxy)alkyl” refers to an alkoxy group connected via an alkylene group, such as C2-C8 alkoxyalkyl, or (C1-C6 alkoxy)C1-C6 alkyl, for example, –(CH₂)_1-3-OCH_3. [0043] As used herein, “-O-alkoxyalkyl” or “-O-(alkoxy)alkyl” refers to an alkoxy group connected via an –O-(alkylene) group, such as –O-(C₁-C₆ alkoxy)C₁-C₆ alkyl, for example, –O-(CH2)1-3-OCH3. [0044] As used herein, “(heterocyclyl)alkyl” refer to a heterocyclic or a heterocyclyl group, as defined above, connected, as a substituent, via an alkylene group, as defined above. The alkylene and heterocyclyl groups of a (heterocyclyl)alkyl may be substituted or unsubstituted. Examples include but are not limited to (tetrahydro-2H-pyran-4-yl)methyl, (piperidin-4-yl)ethyl, (piperidin-4-yl)propyl, (tetrahydro-2H-thiopyran-4-yl)methyl, and (1,3-thiazinan-4-yl)methyl. [0045] As used herein, “(cycloalkyl)alkyl” or “(carbocyclyl)alkyl” refers to a cycloalkyl or carbocyclyl group (as defined herein) connected, as a substituent, via an alkylene group. Examples include but are not limited to cyclopropylmethyl, cyclobutylmethyl, cyclopentylethyl, and cyclohexylpropyl. [0046] An “O-carboxy” group refers to a “-OC(=O)R” group in which R is selected from hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl, C₆-C₁₀ aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein. [0047] A “C-carboxy” group refers to a “-C(=O)OR” group in which R is selected from the group consisting of hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclyl, C₆-C₁₀ aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein. A non-limiting example includes carboxyl (i.e., -C(=O)OH). [0048] A “sulfonyl” group refers to an “-SO₂R” group in which R is selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein. [0049] A “sulfino” group refers to a “-S(=O)OH” group. [0050] A “S-sulfonamido” group refers to a “-SO2NRARB” group in which RA and RB are each independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein. [0051] An “N-sulfonamido” group refers to a “-N(RA)SO2RB” group in which RA and Rb are each independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3- C7 carbocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein. [0052] A “C-amido” group refers to a “-C(=O)NRARB” group in which RA and RB are each independently selected from hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein. [0053] An “N-amido” group refers to a “-N(RA)C(=O)RB” group in which RA and RB are each independently selected from hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein. [0054] An “amino” group refers to a “-NRARB” group in which RA and RB are each independently selected from hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein. A non-limiting example includes free amino (i.e., -NH₂). [0055] An “aminoalkyl” group refers to an amino group connected via an alkylene group. [0056] The term “hydroxy” as used herein refers to a –OH group. [0057] The term “cyano” group as used herein refers to a “-CN” group. [0058] The term “azido” as used herein refers to a –N3 group. [0059] When a group is described as “optionally substituted” it may be either unsubstituted or substituted. Likewise, when a group is described as being “substituted,” the substituent may be selected from one or more of the indicated substituents. As used herein, a substituted group is derived from the unsubstituted parent group in which there has been an exchange of one or more hydrogen atoms for another atom or group. Unless otherwise indicated, when a group is deemed to be “substituted,” it is meant that the group is substituted with one or more substituents independently selected from C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl, C₁-C₆ heteroalkyl, C3-C7 carbocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1- C₆ haloalkyl, and C₁-C₆ haloalkoxy), C₃-C₇carbocyclyl-C₁-C₆-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 3-10 membered heterocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 3-10 membered heterocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), aryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), (aryl)C1-C6 alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 5-10 membered heteroaryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), (5-10 membered heteroaryl)C1-C6 alkyl (optionally substituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), halo, -CN, hydroxy, C1-C6 alkoxy, (C1-C6 alkoxy)C1-C6 alkyl, -O(C1-C6 alkoxy)C1- C₆ alkyl; (C₁-C₆ haloalkoxy)C₁-C₆ alkyl; -O(C₁-C₆ haloalkoxy)C₁-C₆ alkyl; aryloxy, sulfhydryl (mercapto), halo(C1-C6)alkyl (e.g., –CF3), halo(C1-C6)alkoxy (e.g., –OCF3), C1-C6 alkylthio, arylthio, amino, amino(C₁-C₆)alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl, -SO₃H, sulfino, -OSO₂C_1- 4alkyl, monophosphate, diphosphate, triphosphate, and oxo (=O). Wherever a group is described as “optionally substituted” that group can be substituted with the above substituents. [0060] Wherever a substituent is depicted as a di-radical (i.e., has two points of attachment to the rest of the molecule), it is to be understood that the substituent can be attached in any directional configuration unless otherwise indicated. Thus, for example, a substituent depicted as –AE– or includes the substituent being oriented such that the A is attached at the leftmost attachment point of the molecule as well as the case in which A is attached at the rightmost attachment point of the molecule. In addition, if a group or substituent is depicted as , and L is defined an optionally present linker moiety; when L is not present (or group or substituent is equivalent to . [0061] As used herein, a nitrogen containing heterocyclic base, a sugar, and one or more phosphate groups. are 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), deaza adenine (e.g., 7-deaza adenine), guanine (G), deaza guanine (e.g., 7-deaza guanine) and modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof. The C-1 atom of deoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine. [0062] As used herein, a “nucleoside” is structurally similar to a nucleotide, but is missing the phosphate moieties. An example of a nucleoside analogue would be one in which the label is linked to the base and there is no phosphate group attached to the sugar molecule. The term “nucleoside” is used herein in its ordinary sense as understood by those skilled in the art. Examples include, but are not limited to, a ribonucleoside comprising a ribose moiety and a deoxyribonucleoside comprising a deoxyribose moiety. A modified pentose moiety is a pentose moiety in which an oxygen atom has been replaced with a carbon and/or a carbon has been replaced with a sulfur or an oxygen atom. A “nucleoside” is a monomer that can have a substituted base and/or sugar moiety. Additionally, a nucleoside can be incorporated into larger DNA and/or RNA polymers and oligomers. [0063] 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). [0064] As used herein, when an oligonucleotide or polynucleotide is described as “comprising” or “labeled with” 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 3ʹ hydroxy group of the oligonucleotide or polynucleotide with the 5ʹ phosphate group of a nucleotide described herein as a phosphodiester bond between the 3ʹ carbon atom of the oligonucleotide or polynucleotide and the 5ʹ carbon atom of the nucleotide. [0065] 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. [0066] As used herein, the term “self-immolative linker” refers to a linker moiety (e.g., a covalent construct) that can degrade spontaneously in response to specific stimuli (e.g., by an enzyme), results in cleavage of two or more chemical bonds. It is to be understood that a self- immolative linker is a type of cleavable linker. [0067] As used herein, “derivative” or “analog” means a synthetic nucleotide or nucleoside derivative having modified base moieties and/or modified sugar moieties. Such derivatives and analogs are discussed in, e.g., Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90:543-584, 1990. Nucleotide analogs can also comprise modified phosphodiester linkages, including phosphorothioate, phosphorodithioate, alkyl-phosphonate, phosphoranilidate and phosphoramidate linkages. “Derivative,” “analog,” and “modified” as used herein, may be used interchangeably, and are encompassed by the terms “nucleotide” and “nucleoside” defined herein. [0068] As used herein, the term “phosphate” is used in its ordinary sense as understood OH O P O by those skilled in the art, and includes its protonated forms (for example, O- and OH used herein, the terms “monophosphate,” “diphosphate,” and “triphosphate” sense as understood by those skilled in the art, and include protonated forms. [0069] The terms “protecting group” and “protecting 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. [0070] As used herein, the term “phasing” refers to a phenomenon in SBS that is caused by incomplete removal of the 3ʹ 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 3ʹ terminators, wherein the incorporation event goes 1 cycle ahead due to a termination failure. Phasing and pre- phasing 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 3ʹ-OH nucleotides during sequencing by synthesis (SBS). The unprotected 3ʹ-OH nucleotides could be generated during the manufacturing processes or possibly during the storage and reagent handling processes. Accordingly, the discovery of nucleotide analogues which decrease the incidence of 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. Nucleotides with Enzymatically Cleavable Self-Immolative Linker [0071] One aspect of the present disclosure relates to a nucleotide comprising a nucleobase, a ribose or 2´ deoxyribose, and a detectable moiety, wherein the detectable moiety is covalently attached to the nucleotide via a self-immolative linker, and wherein the self-immolative linker comprises an optionally substituted benzyl moiety, a leaving group comprising a carbamate moiety or an acetyl moiety, and an enzymatically cleavable moiety. In some embodiments of the nucleotide described herein, the self-immolative linker has the structure of formula (I): (I), wherein each of L¹ and L² is independently an optionally present spacer moiety; X is O or NH; R^EC is the enzymatically cleavable moiety; R^BZ is H, unsubstituted or substituted C₁-C₆ alkyl, unsubstituted or substituted C₆-C₁₀ aryl, or unsubstituted or substituted 5 to 10 membered heteroaryl; the phenylene moiety is optionally substituted with one or more electron withdrawing groups or one or more electron donating groups; the asterisk indicates the point of connection to the nucleobase; and the detectable moiety is covalently attached to the leaving group, the benzyl moiety (if L² is present), each optionally via a spacer L³. In some embodiments of the nucleotide described herein, each of L¹, L² and L³ optionally substituted C2-C10 alkylene; an optionally substituted 2 to 20 membered, 2 to 10 membered, 3 to 8 membered, or 4 to 6 membered heteroalkylene (each independently containing 1 to 6 heteroatoms selected from N, O, =O, S, and =S); an optionally substituted 2 to 20 membered, 2 to 10 membered, 3 to 8 membered, or 4 to 6 membered heteroalkenylene (each independently containing 1 to 6 heteroatoms selected from N, O, =O, S, and =S); or an optionally substituted 2 to 20 membered, 2 to 10 membered, 3 to 8 membered, or 4 to 6 membered heteroalkynylene (each independently containing 1 to 6 heteroatoms selected from N, O, =O, S, and =S). In further embodiments, one or more of the methylene units of the C2-C10 alkylene, the 2 to 20 membered (e.g., 2 to 10 membered, 3 to 8 membered, or 4 to 6 membered) heteroalkylene, the 2 to 20 membered (e.g., 2 to 10 membered, 3 to 8 membered, or 4 to 6 membered) heteroalkenylene, or the 2 to 20 membered (e.g., 2 to 10 membered, 3 to 8 membered, or 4 to 6 membered) heteroalkynylene described herein may be replaced by a ring or ring system selected from unsubstituted or substituted C6-C10 arylene, or unsubstituted or substituted 5 to 10 membered heteroaryl containing 1, 2, 3, 4, 5 or 6 heteroatoms selected from N, O and S. In some embodiments, L² is not present. In some such embodiments, the leaving group comprises a carbamate moiety. In some further embodiments, the detectable moiety is covalently attached to the phenylene moiety, optionally via a spacer L³. In some further embodiments, the self-immolative linker of the nucleotide has the structure of formula (Ia-1) or (Ia-2): (Ia-2), and R³ is H, an electron donating group, or an electron withdrawing group. [0073] In some other embodiments, the leaving group comprises an acetal moiety. In some further embodiments, the detectable moiety is covalently attached to the leaving group moiety, optionally via a spacer L³. In some further embodiments, the self-immolative linker of the nucleotide has the structure of formula (Ib-1), (Ib-2) or (Ib-3): or R² withdrawing group. [0074] In some embodiments of the nucleotide with linker moiety of formula (I), (Ia- 1), (Ia-2), (Ib-1), (Ib-2) or (Ib-3), the electron donating group is an optionally substituted amino, hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkyl or C₂-C₆ alkenyl. In one embodiment, the electron donating group is methoxy. In some embodiments, the electron withdrawing group is halo (e.g., fluoro, chloro, bromo or iodo), C₁-C₆ haloalkyl (e.g., trifluoromethyl), cyano, nitro, sulfonyl, trifluoromethylsulfonyl, substituted ammonium group, –CH(=O), acyl, carboxy, or C-amido. In some such embodiments, both of R¹ and R² are H. In some other embodiments, R¹ is H and R² is an electron withdrawing group as described herein. In some other embodiments, R² is H and R¹ is an electron donating group as described herein. In still other embodiments, R¹ is an electron donating group as described herein and R² is an electron withdrawing group as described herein. In some embodiments, R³ is H. In some other embodiments, R³ is an electron donating group as described herein. In some other embodiments, R³ is an electron withdrawing group as described herein. In some embodiments, R^BZ is H. In some other embodiments, R^BZ is C₁-C₆ alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl). In some other embodiments, R^BZ is optionally substituted phenyl. In some embodiments, X is O. In some other embodiment, X is NH. In further embodiments, –X–R^EC is –O–glycoside. In such embodiments, the –O–glycoside forms an –O–glycosidic bond with the carbon atom of the phenylene to which it is attached. In yet further embodiments, R^EC is an arabinofuranosidyl (ABF) group. In some further embodiments, some further embodiment, the self-immolative linker has the structure: . In some further group such that the dye is covalently attached to the self-immolative linker via a reaction of the carboxy group with a terminal amino group to form an amido moiety (i.e., -NH-C(=O)-, in which the carbonyl portion comes from the carboxy group of the dye). In other embodiment, the detectable moiety comprises a functional group that form covalent or noncovalent bonding with a labeling reagent. [0075] In some embodiments of the nucleotide with linker moiety of formula (I), (Ia- 1), (Ia-2), (Ib-1), (Ib-2) or (Ib-3), L¹ is a 2 to 10 membered heteroalkylene spacer, 2 to 10 membered heteroalkenylene spacer, or 2 to 10 membered heteroalkynylene spacer, each containing one to three heteroatoms selected from N, O and S. In some further embodiments, L¹ comprises In some embodiments, L³ is a 2 to 10 selected from N, O, and S, wherein one methylene unit is optionally replaced by an optionally substituted phenylene, or an optionally substituted 5 or 6 membered heteroarylene moiety (e.g., a triazole). In one example, L³ . of the nucleotide with linker moiety of formula (I), (Ia- 1), (Ia-2), (Ib-1), (Ib-2) or (Ib-3), the nucleotide comprises a 3´ blocking group. In further embodiments, the 3´ blocking group is enzymatically cleavable. In some embodiments, the 3´ blocking group and the enzymatic cleavable moiety of the self-immolative linker are removable by a single enzymatic reaction, and the enzymatic reaction results in the self-immolation of the linker and the removal of the detectable moiety of the nucleotide. In some embodiments, the 3´ blocking group is –O–glycoside, forming an –O–glycosidic bond with the 3ʹ carbon atom of the nucleotide. In further embodiments, the 3ʹ blocking group has a structure: (alpha-L-arabinofuranoside), where the squiggle line carbon atom of the ribose or 2´ deoxyribose. In addition to the or 3ʹ blocking group described herein, beta- L-arabinofuranoside 3ʹ blocking group may also be used. In some other embodiments, the 3ʹ blocking group has a structure (alpha-D-glucoside), (alpha-D-galactoside), beta-D- other embodiments, the removable 3ʹ blocking group has a (alpha-D-xyloside) or beta-D- xyloside. In some other group has a structure (alpha-N-Acetyl-D-glucosamine), or beta-N-Acetyl-D- the removable 3ʹ blocking group has a structure (alpha-D-glucuronide), or beta-D-glucuronide. In addition, of the self-immolative linker moiety (e.g., R^EC in formula (I), (Ia-1), (Ia-2), (Ib-1), (Ib-2) or (Ib-3)) may also include the same moiety(in which X is O in formula (I), (Ia-1), (Ia-2), (Ib-1), (Ib-2) or (Ib-3)) as the 3ʹ blocking group described herein such that a single enzymatic reaction results in the self-immolation of the linker and the removal of the detectable moiety of the nucleotide. [0077] In some embodiments of the nucleotide described herein, the detectable moiety is a fluorescent dye. In other embodiments, the detectable moiety is a functional group that is capable of attaching to a labeling reagent. In such embodiments, the functional group can attach to the labeling reagent via covalent bonding (e.g., a chemical reaction between the functional group of the nucleotide and a reactive group of the labeling reagent to form covalent bonding). In some other embodiments, the functional group can attach to the labeling reagent via noncovalent interaction (e.g., the functional group is a biotin which can bind to a labeled avidin such as streptavidin. [0078] In any embodiments described herein, the nucleotide may be a nucleotide triphosphate comprising 2´ deoxyribose. [0079] Other aspects of the present disclosure relate to an oligonucleotide or polynucleotide comprising a nucleotide in accordance with the present disclosure incorporated therein. In such embodiments, the nucleotide may be a nucleotide triphosphate comprising 2´ deoxyribose. In some embodiments, the oligonucleotide or polynucleotide is at least partially complementary and hybridized to a target polynucleotide immobilized on a surface of a solid support. In further embodiments, the solid support comprises an array of a plurality of target polynucleotides immobilized thereon. Enzymes Capable of Cleaving Self-Immolative Linkers [0080] Certain enzymes may be capable of cleaving self-immolative linkers described herein. For example, glycoside hydrolases or glycosyl hydrolases may be suitable for cleaving linker groups in accordance with the present disclosure. Table 1 lists particular example glycoside hydrolases that can cleave, for example, the glycoside 3´ blocking group or the enzymatic cleavable moiety of the self-immolative linker. Enzymes in accordance with the present disclosure may be suitable for inclusion in a kit for sequencing by synthesis. Enzymes in accordance with the present disclosure may be suitable in methods of growing a polynucleotide strand. Enzymes in accordance with the present disclosure may be suitable for use in methods of sequencing. Table 1. Exemplary enzymes and corresponding E.C. Number and CAZy family Enzyme Name E.C. Number CAZy Family (as of July 2023) α-L-arabinofuranosidase E.C.3.2.1.55 GH 2, 3, 10, 43, 51, 54, 62 and 159 1, 1, 7, [0081] The enzymatically triggered self-immolation of nucleotide containing a carbamate/ABF self-immolative linker is illustrated in the scheme A below.

H N _O Nucleotide Scheme A: , wherein the dashed line refers to optionally present [0082] The enzymatically triggered self-immolation of nucleotide containing an acetal/ABF self-immolative linker is illustrated in the scheme B below:

hed line ref Labeled Nucleotides [0083] According to an aspect of the disclosure, the described nucleotide also comprises a detectable fluorescent label. Such a nucleotide is referred to herein as a “labeled nucleotide.” In some instances, such labeled nucleotides are also referred to as “modified nucleotides.” The label (e.g., a fluorescent dye) can be conjugated via the self-immolative linker in accordance with the present disclosure by a variety of means including hydrophobic attraction, ionic attraction, and covalent attachment. In some aspect, the detectable label is conjugated to the substrate by covalent attachment. More particularly, the covalent attachment is by means of a self-immolative linker. [0084] Various fluorescent dyes may be used in the present disclosure as detectable fluorescent labels, in particularly those dyes that may be excitation by a blue light (e.g., about 450 nm to about 460 nm) or a green light (e.g., about 520 nm to about 540 nm). These dyes may also be referred to as “blue dyes” and “green dyes” respectively. Examples of various type of blue dyes, including but not limited to coumarin dyes, chromenoquinoline dyes, and bisboron containing heterocycles are disclosed in U.S. Publication Nos. 2018/0094140, 2018/0201981, 2020/0277529, 2020/0277670, 2021/0188832, 2022/0033900, 2022/0195517, 2022/0380389, 2023/0313292, and 2023/0416279, each of which is incorporated by reference in its entirety. Examples of green dyes including cyanine or polymethine dyes disclosed in International Publication Nos. WO2013/041117, WO2014/135221, WO 2016/189287, WO2017/051201 and WO2018/060482A1, each of which is incorporated by reference in its entirety. [0085] Labeled nucleosides and nucleotides are useful for labeling polynucleotides formed by enzymatic synthesis, such as, by way of non-limiting example, in PCR amplification, isothermal amplification, solid phase amplification, polynucleotide sequencing (e.g., solid phase sequencing), nick translation reactions and the like. [0086] In some embodiments, the dye may be covalently attached to oligonucleotides or nucleotides via the nucleotide base. For example, the labeled nucleotide or oligonucleotide may have the label attached to the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base through a self-immolative linker moiety. [0087] Unless indicated otherwise, the reference to nucleotides is also intended to be applicable to nucleosides. The present application will also be further described with reference to DNA, although the description will also be applicable to RNA, PNA, and other nucleic acids, unless otherwise indicated. Methods of Sequencing [0088] One aspect of the present disclosure relates to a method of preparing a growing polynucleotide complementary to a target single-stranded polynucleotide, comprising incorporating a nucleotide as described herein into a growing complementary polynucleotide. In some embodiments, the incorporation of the nucleotide prevents the introduction of any subsequent nucleotide into the growing complementary polynucleotide. In some embodiments, the incorporation of the nucleotide is accomplished by a polymerase, a terminal deoxynucleotidyl transferase, or a reverse transcriptase. In some embodiments, the method may be used to synthesize oligonucleotide or polynucleotides. In other embodiments, the method is used in the context of SBS. [0089] Another aspect of the present disclosure relates to a method of determining the sequences of a plurality of target polynucleotides, comprising: (a) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides; (b) contacting the solid support with an aqueous 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, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides, wherein at least one type of nucleotide is a nucleotide described herein carrying a fluorescent label through the self-immolative linker, and wherein each of the one or more of four different type of nucleotides comprises a 3´ blocking group and a 2´ deoxyribose; (c) imaging the solid support and performing one or more fluorescent measurements of the extended copy polynucleotides; and (d) removing the fluorescent label and the 3ʹ blocking group from nucleotides incorporated into the extended copy polynucleotides. [0090] In some embodiments, step (d) comprises enzymatically removing the fluorescent label from the incorporated nucleotides. In some embodiments, the fluorescent label and the 3´ blocking group are removed in a single reaction. In some embodiments, the method further comprises: (e) washing the solid support after the removal of the 3ʹ blocking group and the fluorescent label from the incorporated nucleotides. In further embodiments, the method further comprises repeating steps (b) to (e) until the sequences of at least a portion of the target polynucleotides are determined. In yet further embodiments, steps (b) to (e) are repeated at least 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 cycles. [0091] Another aspect of the present disclosure relates to an alternative sequencing by synthesis method in which at least one labeling reagent is introduced after the incorporation of unlabeled nucleotides. In particular, the present disclosure relates to a method of determining the sequences of a plurality of target polynucleotides, comprising: (a’) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides; (b’) contacting the solid support with an aqueous 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, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides, wherein at least one type of nucleotide is an unlabeled nucleotide described herein having a first functional group attached to the self-immolative linker, and wherein each of the one or more of four different type of nucleotides comprises a 3´ blocking group and a 2´ deoxyribose; (c’) contacting the extended copy polynucleotides with an aqueous labeling mixture comprising a first labeling reagent, wherein the first labeling reagent comprises one or more first fluorescent labels and a first binding moiety that is capable of specific binding to the first functional group of the unlabeled nucleotide; (d’) imaging the solid support and performing one or more fluorescent measurements of the extended copy polynucleotides; and (e’) removing the one or more fluorescent labels and the 3ʹ blocking group from the nucleotides incorporated into the extended copy polynucleotides. [0092] In some embodiments, step (e’) comprises enzymatically removing the one or more fluorescent labels from the incorporated nucleotides. In some embodiments, the one or more fluorescent labels and the 3´ blocking group are removed in a single reaction. In some embodiments, the method further comprises: (f’) washing the solid support after the removal of the 3ʹ blocking group and the one or more fluorescent labels from the incorporated nucleotides. In further embodiments, the method comprises repeating steps (b’) to (e’) until the sequences of at least a portion of the target polynucleotides are determined. In yet further 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 fluorescent label is removed by contacting the solid support with an aqueous cleavage solution comprising a glycoside hydrolase or glycosidase that is capable of catalyzing the degradation of the self-immolative linker. In further embodiments, the glycoside hydrolase or glycosidase is an arabinofuranosidase, a glucosidase, a mannosidase, a xylosidase, a galactosidase, an N-acetyl-glucosaminidase, or a glucuronidase. In some embodiments, the concentration of the glycoside hydrolase or glycosidase in the aqueous cleavage solution is at least about 0.1 µM. In some embodiments, the removal of the fluorescent label is conducted at a temperature of at least about 30°C. In some embodiments, the removal of the fluorescent label is conducted at a pH between about 5 and about 10. [0093] In some embodiments of the sequencing methods described herein, at least one type of nucleotide with a 3´-O-glycoside blocking group. In some embodiments of the sequencing methods described herein, each type of nucleotides has a 3´-O-glycoside blocking group as described herein. In some embodiments, the 3´-O-glycoside group is removed by contacting the solid support with an aqueous cleavage solution comprising a glycoside hydrolase or glycosidase that is capable of catalyzing the hydrolysis of the –O–glycosidic bond of the nucleotide. In some embodiments, the glycoside hydrolase or glycosidase is an arabinofuranosidase, a glucosidase, a mannosidase, a xylosidase, a galactosidase, an n-acetyl-glucosaminidase, or a glucuronidase. In further embodiments, the concentration of the glycoside hydrolase or glycosidase in the aqueous cleavage solution is at about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 µM or within a range defined by any two of the preceding values. In some embodiments, the concentration of the glycoside hydrolase or glycosidase in the aqueous cleavage solution is at least about 0.1 µM. In some embodiments, the concentration of the glycoside hydrolase or glycosidase in the aqueous cleavage solution is at least about 0.5 µM. In some embodiments, the concentration of the glycoside hydrolase or glycosidase in the aqueous cleavage solution is at least about 1 µM. In some embodiments, the concentration of the glycoside hydrolase or glycosidase in the aqueous cleavage solution is at least about 1.5 µM. In some embodiments, the concentration of the glycoside hydrolase or glycosidase in the aqueous cleavage solution is at least about 2 µM. [0094] In some embodiments, step (d) or (e’) comprises enzymatically removing the 3ʹ blocking group from the nucleotides incorporated into the extended copy polynucleotides. In some embodiments, step (d) or (e’) is conducted at a temperature of 30°C to 100°C. In some embodiments, step (d) or (e’) is conducted at a temperature of at least about 35°C. In some embodiments, step (d) or (e’) is conducted at a temperature of at least about 37°C. In some embodiments, step (d) or (e’) is conducted at a temperature of at least about 60°C. In some embodiments, step (d) or (e’) is conducted at a temperature of at least about 65°C. In some embodiments, step (d) or (e’) is conducted at a temperature of at least about 70°C. In some embodiments, step (d) or (e’) is conducted at a temperature of at least about 80°C. In some embodiments, step (d) or (e’) is conducted at a pH of about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0, in in a range defined by any two of the preceding values. In some embodiments, step (d) or (e’) is conducted at a pH between about 5.5 and about 6.5. In some embodiments, step (d) or (e’) is conducted at a pH between about 5.9 and about 6.1. In some embodiments, step (d) or (e’) is conducted at a pH of about 6.0. [0095] In any embodiments of the sequencing methods described herein, the fourth type of nucleotide is unlabeled. In some further embodiments, the G nucleotide is unlabeled. In some further embodiments, G nucleotide has a structure selected from the group consisting of: , wh [0096] In any embodiments of the sequencing methods described herein, the solid support comprises at least 500,000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, or 5,000,000 spatially distinguishable sites/cm² that comprise multiple copies of target polynucleotides. Incorporation Mix [0097] In some embodiments of the method described herein, step (b) or (b’), also referred to as the incorporation step, includes contacting a mixture containing one or more nucleotides (e.g., dATP, dCTP, dGTP, and dTTP or dUTP) with a copy polynucleotide/target polynucleotide complex in an incorporation solution comprising a polymerase and one or more buffering agents. In some such embodiments, the polymerase is a DNA polymerase, such as a mutant of 9°N polymerase (e.g., those disclosed in WO 2005/024010, which is incorporated by reference), for example, Pol 812, Pol 1901, Pol 1558 or Pol 963. The amino acid sequences of Pol 812, Pol 1901, Pol 1558 or Pol 963 DNA polymerases are described, for example, in U.S. Patent Publication Nos. 2020/0131484 A1 and 2020/0181587 A1, both of which are incorporated by reference herein. Additional polymerases that may be used in the method include those disclosed in U.S. Ser. Nos. 63/412,241 and 63/433,971, both of which are incorporated by reference. In some embodiments, the one or more buffering agents comprise a primary amine, a secondary amine, a tertiary amine, a natural amino acid, or a non-natural amino acid, or combinations thereof. In further embodiments, the buffering agents comprise ethanolamine or glycine, or a combination thereof. In one embodiment, the buffer agent comprises or is glycine. In further embodiments, the mutant of 9°N polymerase may be engineered for high efficient incorporation of the nucleotide with the 3´-O-glycoside blocking group. Cleavage Mix [0098] In some embodiments of the method described herein, steps of removing labels and/or blocking groups (i.e., steps (d) or (e’)), also referred to as the cleaving step, includes contacting the incorporated nucleotide and the copy polynucleotide strand with a cleavage solution comprising an enzyme described herein. In some embodiments, the cleavage solution comprises a catalyst or enzyme capable of cleaving the self-immolative linker group in accordance with the present disclosure. In some embodiments, the cleavage solution comprises an enzyme capable of cleaving the self-immolative linker group described herein. In such embodiments, the enzyme (e.g., those described in Table 1) is capable of triggering the self-immolative linker to self- immolate, resulting in the removal of the detectable moiety. In some embodiments, the cleavage solution comprises a catalyst or enzyme capable of cleaving the blocking group in accordance with the present disclosure. In some embodiments, the cleavage solution comprises an enzyme capable of cleaving the blocking group described herein. In some embodiments, the cleavable self- immolative linker and the blocking group are removed by the same enzyme. In some embodiments, the 3ʹ-OH blocking group and the detectable label are removed in a single step of reaction. Chemical Deprotection of the 3ʹ Blocking Groups [0099] In other embodiments, the self-immolative linker and the 3´ blocking group are removed in two separate steps. In some such embodiments, the , the 3´ blocking group may be removed in a chemical reaction that is separate from the enzymatic reaction resulting in the cleavable of the self-immolative linker. Such 3´ blocking group may include azidomethyl (- CH₂N₃) or substituted azidomethyl (e.g., -CH(CHF₂)N₃ or CH(CH₂F)N₃), or allyl, each connecting to the 3’ oxygen atom of the ribose or deoxyribose moiety of the nucleotide. Additional 3ʹ blocking groups are disclosed in U.S. Publication No.2020/0216891 A1, which is incorporated by reference in its entirety. Non-limiting examples of the 3´ blocking group include: (AOM), , , _{each covalently attached to the 3ʹ carbon of the ribose or} [0100] In some embodiments, the 3´ blocking group may be removed or deprotected by a chemical reagent to generate a free hydroxy group, for example, in the presence of a water- soluble phosphine reagent. Non-limiting examples include tris(hydroxymethyl)phosphine (THMP), tris(hydroxyethyl)phosphine (THEP) or tris(hydroxylpropyl)phosphine (THP or THPP). 3ʹ-acetal blocking groups described herein may be removed or cleaved under various chemical conditions. For 3´ acetal blocking groups such , non-limiting cleaving conditions include a Pd(II) complex, such as Pd chloride dimer, in the presence of a phosphine ligand, 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). Palladium Cleavage Reagents [0101] In some other embodiments, the 3’ blocking group described herein such as allyl or AOM may 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 Na2PdCl4, Li2PdCl4, Pd(CH3CN)2Cl2, (PdCl(C3H5))2, [Pd(C3H5)(THP)]Cl, [Pd(C₃H₅)(THP)₂]Cl, Pd(OAc)₂, Pd(Ph₃)₄, Pd(dba)₂, Pd(Acac)₂, PdCl₂(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 Na₂PdCl₄ or K₂PdCl₄. In another embodiment, the palladium source is allyl palladium(II) chloride dimer [(PdCl(C3H5))2]. In some embodiments, the Pd(0) complex is generated in an aqueous solution by mixing a Pd(II) 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. [0102] 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 Na2PdCl4 or K2PdCl4 with THP in situ. The molar ratio of Na2PdCl4 or K2PdCl4 and 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 Na2PdCl4 or K2PdCl4 to THP is about 1:3. In another embodiment, the molar ratio of Na₂PdCl₄ or K₂PdCl₄ to THP is about 1:3.5. In yet another embodiment, the molar ratio of Na2PdCl4 or K2PdCl4 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 structure ), or combinations thereof. In one embodiment, the buffer reagent comprises or embodiment, the buffer reagent comprises or is (2-hydroxyethyl)piperidine. 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. Kits [0103] One aspect of the present disclosure relates to a kit comprising one or more nucleotides according to the present disclosure. [0104] In some embodiments, the kit further comprises a first enzyme, wherein the first enzyme is a polymerase, a terminal deoxynucleotidyl transferase, or a reverse transcriptase. In further embodiments, the first enzyme and the one or more nucleotides are in a first compartment of the kit. In some embodiments, the polymerase is a DNA polymerase as described herein. [0105] In some embodiments, the kit comprises a second enzyme for removing the detectable moiety of the nucleotide (i.e., through enzymatically triggered cleavage of the self- immolative linker). In further embodiments, the second enzyme can also remove the 3´ blocking group of the nucleotide. In such embodiments, the second enzyme is a glycoside hydrolase or glycosidase. In further embodiments, the second enzyme is an arabinofuranosidase, a glucosidase, a mannosidase, a xylosidase, a galactosidase, an N-acetyl-glucosaminidase, or a glucuronidase. In yet further embodiments, the second enzyme is an L-arabinofuranosidase, a D-glucosidase, a D- mannosidase, a D-xylosidase, a D-galactosidase, an N-acetyl-D-glucosaminidase, or a D- glucuronidase. In yet further embodiments, the second enzyme is an α-L-arabinofuranosidase, a β-L-arabinofuranosidase, an α-D-glucosidase, a β-D-glucosidase, an α-D-mannosidase, a β-D- mannosidase, an α-D-xylosidase, a β-D-xylosidase, an α-D-galactosidase, a β-D-galactosidase, an α-N-acetyl-D-glucosaminidase, a β-N-acetyl-D-glucosaminidase, an α-D-glucuronidase, or a β- D-glucuronidase. None limiting example of these enzymes are illustrated in Table 1. [0106] In other embodiments, the kit further comprises a chemical reagent or a third enzyme for removing the 3´ blocking group of the nucleotide to generate a free hydroxy group. In some embodiments, the kit comprises a third enzyme for removing the blocking group of the nucleotide, wherein the third enzyme is in a separate compartment from the first enzyme. In further embodiments, the second enzyme is a glycoside hydrolase or glycosidase that is capable of catalyzing the hydrolysis of a –O–glycosidic bond of the nucleotide, for example a –O–glycosidic bond of the blocking group. In further embodiments, the third enzyme is an arabinofuranosidase, a glucosidase, a mannosidase, a xylosidase, a galactosidase, an N-acetyl-glucosaminidase, or a glucuronidase. In yet further embodiments, the third enzyme is an L-arabinofuranosidase, a D- glucosidase, a D-mannosidase, a D-xylosidase, a D-galactosidase, an N-acetyl-D- glucosaminidase, or a D-glucuronidase. In yet further embodiments, the third enzyme is an α-L- arabinofuranosidase, a β-L-arabinofuranosidase, an α-D-glucosidase, a β-D-glucosidase, an α-D- mannosidase, a β-D-mannosidase, an α-D-xylosidase, a β-D-xylosidase, an α-D-galactosidase, a β-D-galactosidase, an α-N-acetyl-D-glucosaminidase, a β-N-acetyl-D-glucosaminidase, an α-D- glucuronidase, or a β-D-glucuronidase. Alternatively, the kit may comprise a chemical reagent for cleaving the 3´ blocking group, including those described herein with respect to the cleavage mix, such as a water soluble phosphine reagent and/or a palladium catalyst. [0107] In some embodiments, the kit may contain four types of labeled nucleotides (A, C, G and T or U; dATP, dCTP, dGTP and dTTP or dUTP), where one or more of the four types of nucleotides is labeled. In such a kit, each of the four types of nucleotides can be labeled with a compound that is the same or different from the label on the other three nucleotides. Alternatively, a first type of the four types of nucleotides carries a first label, a second type of nucleotides carries a second label, a third type of nucleotide carries a third label, and a fourth nucleotide is unlabeled (dark). As another example, a first type of nucleotide carries a first label, a second type of nucleotide carries a second label, a third nucleotide is a mixture of the third type of nucleotide carrying the first label and the third type of nucleotide carrying the second label, and a fourth nucleotide is unlabeled (dark). Thus, one or more of the label nucleotides can have a distinct absorbance maximum and/or emission maximum such that the compound(s) is(are) distinguishable from other compounds. For example, each compound can have a distinct absorbance maximum and/or emission maximum such that each of the compounds is spectrally distinguishable from the other three compounds (or two compounds if the fourth nucleotide is unlabeled). It will be understood that parts of the absorbance spectrum and/or emission spectrum other than the maxima can differ and these differences can be exploited to distinguish the compounds. The kit may be such that two or more of the compounds have a distinct absorbance maximum. [0108] The compounds, nucleotides, or kits that are set forth herein may be used to detect, measure, or identify a biological system (including, for example, processes or components thereof). Exemplary techniques that can employ the compounds, nucleotides or kits include sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, cellular assay (e.g., cell binding or cell function analysis), or protein assay (e.g., protein binding assay or protein activity assay). The use may be on an automated instrument for carrying out a particular technique, such as an automated sequencing instrument. The sequencing instrument may contain two light sources operating at different wavelengths (e.g., a blue light source at about 450 nm to about 460 nm and a green light source at about 520 nm to about 560 nm). Alternatively, the sequencing instrument may contain a single light source (e.g., a blue light source at about 450 nm to about 460 nm, or a green light source at about 520 nm to about 560 nm). [0109] In a particular embodiment, the labeled nucleotide(s) described herein may be supplied in combination with unlabeled or native nucleotides, or any combination thereof. Combinations of nucleotides may be provided as separate individual components (e.g., one nucleotide type per vessel or tube) or as nucleotide mixtures (e.g., two or more nucleotides mixed in the same vessel or tube). [0110] Where kits comprise a plurality, particularly two, or three, or more particularly four, nucleotides, the different nucleotides may be labeled with different dye compounds, or one may be dark, with no dye compounds. Where the different nucleotides are labeled with different dye compounds, it is a feature of the kits that the dye compounds are spectrally distinguishable fluorescent dyes. As used herein, the term “spectrally distinguishable fluorescent dyes” refers to fluorescent dyes that emit fluorescent energy at wavelengths that can be distinguished by fluorescent detection equipment (for example, a commercial capillary-based DNA sequencing platform) when two or more such dyes are present in one sample. When two nucleotides labeled with fluorescent dye compounds are supplied in kit form, it is a feature of some embodiments that the spectrally distinguishable fluorescent dyes can be excited at the same wavelength, such as, for example by the same light source. When four nucleotides labeled with fluorescent dye compounds are supplied in kit form, it is a feature of some embodiments that two of the spectrally distinguishable fluorescent dyes can both be excited at one wavelength and the other two spectrally distinguishable dyes can both be excited at another wavelength. Particular excitation wavelengths for the dyes are between 450–460 nm, 490–500 nm, or 520 nm or above (e.g., 523 nm or 532 nm). Post-incorporation labeling kits [0111] In other embodiments, one or more types of nucleotides (A, G, C, and T or U; dATP, dCTP, dGTP and dTTP or dUTP) are unlabeled, and wherein the first type of unlabeled nucleotides comprises a first functional moiety, and the kit further comprises a first labeling reagent, wherein the first labeling reagent comprises one or more first detectable labels and a first binding moiety that is capable of specific binding to the first functional moiety of the first type of unlabeled nucleotide. In some such embodiments, the first functional moiety of the first type of unlabeled nucleotide is bound to the first labeling reagent by either covalent bonding or noncovalent interaction via a self-immolative linker described herein. In some embodiments, two or more types of nucleotides are unlabeled. In some further embodiments, each of the four types of nucleotides is unlabeled, and wherein the second type of unlabeled nucleotides comprises a second functional moiety, and the kit further comprises a second labeling reagent, wherein the second labeling reagent comprises one or more second detectable labels and a second binding moiety that is capable of specific binding to the second functional moiety of the second type of unlabeled nucleotide. In some such embodiments, the second functional moiety of the second type of unlabeled nucleotide is bound to the second labeling reagent by either covalent bonding or noncovalent interaction via a self-immolative linker described herein. In some further embodiments, the third type of unlabeled nucleotides comprises a third functional moiety, and the kit further comprises a third labeling reagent, wherein the third labeling reagent comprises one or more third detectable labels and a third binding moiety that is capable of specific binding to the third functional moiety of the third type of unlabeled nucleotide. In some such embodiments, the third functional moiety of the third type of unlabeled nucleotide is bound to the third labeling reagent by either covalent bonding or noncovalent interaction via a self-immolative linker described herein. In other embodiments, the third type of unlabeled nucleotides comprises a mixture of the third type of unlabeled nucleotides comprising the first functional moiety and the third type of unlabeled nucleotides comprising the second functional moiety, and wherein both the first labeling reagent and the second labeling reagent are capable of specific binding to the third type of unlabeled nucleotides. In some further embodiments, the fourth type of unlabeled nucleotides is not capable of specific binding with any of the first, second, or third labeling reagent. Post-incorporation labeling kits and methods have been described in U.S. Publication No. 2023/0383342 A1, which is incorporated by reference in its entirety. Non-limiting examples of noncovalent interaction between a functional moiety of the nucleotide and a binding moiety of the labeling reagent include but are not limited to avidin (e.g., streptavidin or neutravidin) and biotin; dinitrophenyl (DNP) moiety and anti-DNP antibody; digoxigenin (DIG) and anti-DIG antibody; β-N-acetyl glucosamine (O-GlcNAc) and WGA (lectin); alkyl guanine moiety and SNAP-Tag®, alkyl chloride moiety and HaloTag®; 3-nitrotyrosine and anti-nitrotyrosine antibody; nickel or cobalt complex such as Ni-nitrilotriacetic acid (NTA) and His-Tag; zinc complex and oligo- aspartate protein. Non-limiting examples of covalent interaction between a functional moiety and a labeling reagent include but are not limited to a biorthogonal reaction selected from the group consisting of a [3+2] dipolar cycloaddition, a Diels-Alder cycloaddition, a [4+1] cycloaddition, a phosphine ligation, or condensation with 2-acylphenyl boronic acid. For example, one of the functional moiety and the binding moiety comprises or is norbornene, transcyclooctene (TCO), dibenzocyclooctyne (DBCO), or bicyclo[6.1.0]nonyne (BCN), and the other one of the functional moiety and the binding moiety comprises or is azido. In some other embodiments, one of the functional moiety and the binding moiety comprises or is TCO, and the other one of the functional moiety and the binding moiety comprises or is an optionally substituted 1,2,4,5-tetrazine moiety. Embodiments and Alternatives of Sequencing-By-Synthesis [0112] Alternatively, the sequencing methods described herein may also be carried out using unlabeled nucleotides and affinity reagents containing a fluorescent dye described herein. In some examples, the affinity reagents may bind to an incorporated nucleotide via the enzymatically cleavable linker (e.g., a self-immolative linker) of the incorporated nucleotide. For example, one, two, three or each of the four different types of nucleotides (e.g., dATP, dCTP, dGTP and dTTP or dUTP) in the incorporation mixture of step (1) may be unlabeled. Each of the four types of nucleotides (e.g., dNTPs) has a 3ʹ blocking group to ensure that only a single base can be added by a polymerase to the 3ʹ end of the primer polynucleotide. After incorporation of an unlabeled nucleotide, the remaining unincorporated nucleotides are washed away. An affinity reagent is then introduced that specifically recognizes and binds to the incorporated dNTP to provide a labeled extension product comprising the incorporated dNTP. Uses of unlabeled nucleotides and affinity reagents in sequencing by synthesis have been disclosed in WO 2018/129214 and WO 2020/097607. A modified sequencing method of the present disclosure using unlabeled nucleotides may include the following steps: (1) contacting a solid support with sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides; (2) contacting the solid support with an aqueous solution comprising DNA polymerase and one or more of four different types of unlabeled nucleotides (A, G, C and T or U) under conditions suitable for DNA polymerase-mediated primer extension, wherein each of the nucleotides comprises a 3′ blocking group and at least one type of nucleotide comprising an self-immolative linker in accordance with the present disclosure; (3) contacting the extended copy polynucleotides with a set of affinity reagents under conditions wherein one affinity reagent binds specifically to the incorporated unlabeled nucleotides to provide labeled extended copy polynucleotides; (4) imaging the solid support to determine the identity of the incorporated nucleotides (e.g., by performing one or more fluorescent measurements of the extended copy polynucleotides); and (5) contacting the solid support with an aqueous deblocking solution comprising a palladium catalyst and tris(hydroxyalkyl)phosphine under conditions suitable to chemically remove 3ʹ allyl blocking groups from incorporated nucleotides to expose a 3’- OH group for further nucleotide incorporation on the solid support; and repeating steps (2)-(5) to determine target polynucleotide sequences. [0113] In some embodiments of the modified sequencing method described herein, the method further comprises removing the affinity reagents from the incorporated nucleotides. In some such embodiments, removing the affinity reagents from the incorporated nucleotides comprises cleaving the self-immolative linkers. In still further embodiments, the 3ʹ blocking group and the affinity reagent are removed in the same reaction. In some embodiments, the method further comprises a step (6) washing the solid support with a third aqueous wash solution. In further embodiments, steps (2) through (6) are repeated at least 50, 100, 150, 200, 250 or 300 cycles to determine the target polynucleotide sequences. In some embodiments, the set of affinity reagents may comprise a first affinity reagent that binds specifically to the first type of nucleotide, a second affinity reagent that binds specifically to the second type of nucleotide, and a third affinity reagent that binds specifically to the third type of nucleotide. In some further embodiments, each of the first, second and the third affinity reagents comprises a detectable labeled that is spectrally distinguishable. In some embodiments, the affinity reagents may include protein tags, antibodies (including but not limited to binding fragments of antibodies, single chain antibodies, bispecific antibodies, and the like), aptamers, knottins, affimers, or any other known agent that binds an incorporated nucleotide with a suitable specificity and affinity. In one embodiment, at least one affinity reagent is an antibody or a protein tag. In another embodiment, at least one of the first type, the second type, and the third type of affinity reagents is an antibody or a protein tag comprising one or more detectable labels (e.g., multiple copies of the same detectable label), wherein the detectable label is or comprises a bis-boron dye moiety described herein. [0114] Some embodiments include pyrosequencing techniques. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the nascent strand (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996) “Real-time DNA sequencing using detection of pyrophosphate release.” Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) “Pyrosequencing sheds light on DNA sequencing.” Genome Res. 11(1), 3-11; Ronaghi, M., Uhlen, M. and Nyren, P. (1998) “A sequencing method based on real-time pyrophosphate.” Science 281(5375), 363; U.S. Pat. Nos. 6,210,891; 6,258,568 and 6,274,320, the disclosures of which are incorporated herein by reference in their entireties). In pyrosequencing, released PPi can be detected by being immediately converted to adenosine triphosphate (ATP) by ATP sulfurase, and the level of ATP generated is detected via luciferase-produced photons. The nucleic acids to be sequenced can be attached to features in an array and the array can be imaged to capture the chemiluminescent signals that are produced due to incorporation of a nucleotides at the features of the array. An image can be obtained after the array is treated with a particular nucleotide type (e.g., A, T, C or G). Images obtained after addition of each nucleotide type will differ with regard to which features in the array are detected. These differences in the image reflect the different sequence content of the features on the array. However, the relative locations of each feature will remain unchanged in the images. The images can be stored, processed and analyzed using the methods set forth herein. For example, images obtained after treatment of the array with each different nucleotide type can be handled in the same way as exemplified herein for images obtained from different detection channels for reversible terminator-based sequencing methods. [0115] In another exemplary type of SBS, cycle sequencing is accomplished by stepwise addition of reversible terminator nucleotides containing, for example, a cleavable or photobleachable dye label as described, for example, in WO 04/018497 and U.S. Pat. No. 7,057,026, the disclosures of which are incorporated herein by reference. This approach is being commercialized by Solexa (now Illumina, Inc.), and is also described in WO 91/06678 and WO 07/123,744, each of which is incorporated herein by reference. The availability of fluorescently- labeled terminators in which both the termination can be reversed, and the fluorescent label cleaved facilitates efficient cyclic reversible termination (CRT) sequencing. Polymerases can also be co-engineered to efficiently incorporate and extend from these modified nucleotides. [0116] Preferably in reversible terminator-based sequencing embodiments, the labels do not substantially inhibit extension under SBS reaction conditions. However, the detection labels can be removable, for example, by cleavage or degradation. Images can be captured following incorporation of labels into arrayed nucleic acid features. In particular embodiments, each cycle involves simultaneous delivery of four different nucleotide types to the array and each nucleotide type has a spectrally distinct label. Four images can then be obtained, each using a detection channel that is selective for one of the four different labels. Alternatively, different nucleotide types can be added sequentially, and an image of the array can be obtained between each addition step. In such embodiments each image will show nucleic acid features that have incorporated nucleotides of a particular type. Different features will be present or absent in the different images due the different sequence content of each feature. However, the relative position of the features will remain unchanged in the images. Images obtained from such reversible terminator-SBS methods can be stored, processed and analyzed as set forth herein. Following the image capture step, labels can be removed, and reversible terminator moieties can be removed for subsequent cycles of nucleotide addition and detection. Removal of the labels after they have been detected in a particular cycle and prior to a subsequent cycle can provide the advantage of reducing background signal and crosstalk between cycles. Examples of useful labels and removal methods are set forth below. [0117] Some embodiments can utilize detection of four different nucleotides using fewer than four different labels. For example, SBS can be performed utilizing methods and systems described in the incorporated materials of U.S. Pub. No. 2013/0079232. As a first example, a pair of nucleotide types can be detected at the same wavelength, but distinguished based on a difference in intensity for one member of the pair compared to the other, or based on a change to one member of the pair (e.g. via chemical modification, photochemical modification or physical modification) that causes apparent signal to appear or disappear compared to the signal detected for the other member of the pair. As a second example, three of four different nucleotide types can be detected under particular conditions while a fourth nucleotide type lacks a label that is detectable under those conditions, or is minimally detected under those conditions (e.g., minimal detection due to background fluorescence, etc.). Incorporation of the first three nucleotide types into a nucleic acid can be determined based on presence of their respective signals and incorporation of the fourth nucleotide type into the nucleic acid can be determined based on absence or minimal detection of any signal. As a third example, one nucleotide type can include label(s) that are detected in two different channels, whereas other nucleotide types are detected in no more than one of the channels. The aforementioned three exemplary configurations are not considered mutually exclusive and can be used in various combinations. An exemplary embodiment that combines all three examples, is a fluorescent-based SBS method that uses a first nucleotide type that is detected in a first channel (e.g. dATP having a label that is detected in the first channel when excited by a first excitation wavelength), a second nucleotide type that is detected in a second channel (e.g. dCTP having a label that is detected in the second channel when excited by a second excitation wavelength), a third nucleotide type that is detected in both the first and the second channel (e.g. dTTP having at least one label that is detected in both channels when excited by the first and/or second excitation wavelength) and a fourth nucleotide type that lacks a label that is not, or minimally, detected in either channel (e.g. dGTP having no label). [0118] Further, as described in the incorporated materials of U.S. Pub. No. 2013/0079232, sequencing data can be obtained using a single channel. In such so-called one-dye sequencing approaches, the first nucleotide type is labeled but the label is removed after the first image is generated, and the second nucleotide type is labeled only after a first image is generated. The third nucleotide type retains its label in both the first and second images, and the fourth nucleotide type remains unlabeled in both images. [0119] Some embodiments can utilize sequencing by ligation techniques. Such techniques utilize DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides. The oligonucleotides typically have different labels that are correlated with the identity of a particular nucleotide in a sequence to which the oligonucleotides hybridize. As with other SBS methods, images can be obtained following treatment of an array of nucleic acid features with the labeled sequencing reagents. Each image will show nucleic acid features that have incorporated labels of a particular type. Different features will be present or absent in the different images due the different sequence content of each feature, but the relative position of the features will remain unchanged in the images. Images obtained from ligation-based sequencing methods can be stored, processed and analyzed as set forth herein. Exemplary SBS systems and methods which can be utilized with the methods and systems described herein are described in U.S. Pat. Nos. 6,969,488, 6,172,218, and 6,306,597, the disclosures of which are incorporated herein by reference in their entireties. [0120] Some embodiments can utilize nanopore sequencing (Deamer, D. W. & Akeson, M. “Nanopores and nucleic acids: prospects for ultrarapid sequencing.” Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D. Branton, “Characterization of nucleic acids by nanopore analysis”, Acc. Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin, and J. A. Golovchenko, “DNA molecules and configurations in a solid-state nanopore microscope” Nat. Mater. 2:611-615 (2003), the disclosures of which are incorporated herein by reference in their entireties). In such embodiments, the target nucleic acid passes through a nanopore. The nanopore can be a synthetic pore or biological membrane protein, such as α- hemolysin. As the target nucleic acid passes through the nanopore, each base-pair can be identified by measuring fluctuations in the electrical conductance of the pore. (U.S. Pat. No.7,001,792; Soni, G. V. & Meller, “A. Progress toward ultrafast DNA sequencing using solid-state nanopores.” Clin. Chem. 53, 1996-2001 (2007); Healy, K. “Nanopore-based single-molecule DNA analysis.” Nanomed. 2, 459-481 (2007); Cockroft, S. L., Chu, J., Amorin, M. & Ghadiri, M. R. “A single- molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution.” J. Am. Chem. Soc. 130, 818-820 (2008), the disclosures of which are incorporated herein by reference in their entireties). Data obtained from nanopore sequencing can be stored, processed and analyzed as set forth herein. In particular, the data can be treated as an image in accordance with the exemplary treatment of optical images and other images that is set forth herein. [0121] Some other embodiments of sequencing methods involve the use the 3ʹ blocked nucleotide described herein in nanoball sequencing technique, such as those described in U.S. Patent No. 9,222,132, the disclosure of which is incorporated by reference. Through the process of rolling circle amplification (RCA), a large number of discrete DNA nanoballs may be generated. The nanoball mixture is then distributed onto a patterned slide surface containing features that allow a single nanoball to associate with each location. In DNA nanoball generation, DNA is fragmented and ligated to the first of four adapter sequences. The template is amplified, circularized and cleaved with a type II endonuclease. A second set of adapters is added, followed by amplification, circularization and cleavage. This process is repeated for the remaining two adapters. The final product is a circular template with four adapters, each separated by a template sequence. Library molecules undergo a rolling circle amplification step, generating a large mass of concatemers called DNA nanoballs, which are then deposited on a flow cell. Goodwin et al., “Coming of age: ten years of next-generation sequencing technologies,” Nat Rev Genet. 2016;17(6):333-51. [0122] Some embodiments can utilize methods involving the real-time monitoring of DNA polymerase activity. Nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and γ- phosphate-labeled nucleotides as described, for example, in U.S. Pat. Nos. 7,329,492 and 7,211,414, both of which are incorporated herein by reference, or nucleotide incorporations can be detected with zero-mode waveguides as described, for example, in U.S. Pat. No. 7,315,019, which is incorporated herein by reference, and using fluorescent nucleotide analogs and engineered polymerases as described, for example, in U.S. Pat. No.7,405,281 and U.S. Pub. No. 2008/0108082, both of which are incorporated herein by reference. The illumination can be restricted to a zeptoliter-scale volume around a surface-tethered polymerase such that incorporation of fluorescently labeled nucleotides can be observed with low background (Levene, M. J. et al. “Zero-mode waveguides for single-molecule analysis at high concentrations.” Science 299, 682-686 (2003); Lundquist, P. M. et al. “Parallel confocal detection of single molecules in real time.” Opt. Lett.33, 1026-1028 (2008); Korlach, J. et al. “Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nano structures.” Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference in their entireties). Images obtained from such methods can be stored, processed and analyzed as set forth herein. [0123] Some SBS embodiments include detection of a proton released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons can use an electrical detector and associated techniques that are commercially available from Ion Torrent (Guilford, CT, a Life Technologies subsidiary) or sequencing methods and systems described in U.S. Pub. Nos. 2009/0026082; 2009/0127589; 2010/0137143; and 2010/0282617, all of which are incorporated herein by reference. Methods set forth herein for amplifying target nucleic acids using kinetic exclusion can be readily applied to substrates used for detecting protons. More specifically, methods set forth herein can be used to produce clonal populations of amplicons that are used to detect protons. [0124] The above SBS methods can be advantageously carried out in multiplex formats such that multiple different target nucleic acids are manipulated simultaneously. In particular embodiments, different target nucleic acids can be treated in a common reaction vessel or on a surface of a particular substrate. This allows convenient delivery of sequencing reagents, removal of unreacted reagents and detection of incorporation events in a multiplex manner. In embodiments using surface-bound target nucleic acids, the target nucleic acids can be in an array format. In an array format, the target nucleic acids can be typically bound to a surface in a spatially distinguishable manner. The target nucleic acids can be bound by direct covalent attachment, attachment to a bead or other particle or binding to a polymerase or other molecule that is attached to the surface. The array can include a single copy of a target nucleic acid at each site (also referred to as a feature) or multiple copies having the same sequence can be present at each site or feature. Multiple copies can be produced by amplification methods such as, bridge amplification or emulsion PCR as described in further detail below. [0125] The methods set forth herein can use arrays having features at any of a variety of densities including, for example, at least about 10 features/cm², 100 features/cm², 500 features/cm², 1,000 features/cm², 5,000 features/cm², 10,000 features/cm², 50,000 features/cm², 100,000 features/cm², 1,000,000 features/cm², 5,000,000 features/cm², or higher. [0126] An advantage of the methods set forth herein is that they provide for rapid and efficient detection of a plurality of target nucleic acid in parallel. Accordingly, the present disclosure provides integrated systems capable of preparing and detecting nucleic acids using techniques known in the art such as those exemplified above. Thus, an integrated system of the present disclosure can include fluidic components capable of delivering amplification reagents and/or sequencing reagents to one or more immobilized DNA fragments, the system comprising components such as pumps, valves, reservoirs, fluidic lines and the like. A flow cell can be configured and/or used in an integrated system for detection of target nucleic acids. Exemplary flow cells are described, for example, in U.S. Pub. No.2010/0111768 and US Ser. No.13/273,666, each of which is incorporated herein by reference. As exemplified for flow cells, one or more of the fluidic components of an integrated system can be used for an amplification method and for a detection method. Taking a nucleic acid sequencing embodiment as an example, one or more of the fluidic components of an integrated system can be used for an amplification method set forth herein and for the delivery of sequencing reagents in a sequencing method such as those exemplified above. Alternatively, an integrated system can include separate fluidic systems to carry out amplification methods and to carry out detection methods. Examples of integrated sequencing systems that are capable of creating amplified nucleic acids and also determining the sequence of the nucleic acids include, without limitation, the MiSeq^™ platform (Illumina, Inc., San Diego, CA) and devices described in US Ser. No.13/273,666, which is incorporated herein by reference. [0127] Arrays in which polynucleotides have been directly attached to silica-based supports are those for example disclosed in WO 00/06770 (incorporated herein by reference), wherein polynucleotides are immobilized on a glass support by reaction between a pendant epoxide group on the glass with an internal amino group on the polynucleotide. In addition, polynucleotides can be attached to a solid support by reaction of a sulfur-based nucleophile with the solid support, for example, as described in WO 2005/047301 (incorporated herein by reference). A still further example of solid-supported template polynucleotides is where the template polynucleotides are attached to hydrogel supported upon silica-based or other solid supports, for example, as described in WO 00/31148, WO 01/01143, WO 02/12566, WO 03/014392, U.S. Pat. No. 6,465,178 and WO 00/53812, each of which is incorporated herein by reference. [0128] A particular surface to which template polynucleotides may be immobilized is a polyacrylamide hydrogel. Polyacrylamide hydrogels are described in the references cited above and in WO 2005/065814, which is incorporated herein by reference. Specific hydrogels that may be used include those described in WO 2005/065814 and U.S. Pub. No. 2014/0079923. In one embodiment, the hydrogel is PAZAM (poly(N-(5-azidoacetamidylpentyl) acrylamide-co- acrylamide)). [0129] DNA template molecules can be attached to beads or microparticles, for example, as described in U.S. Pat. No. 6,172,218 (which is incorporated herein by reference). Attachment to beads or microparticles can be useful for sequencing applications. Bead libraries can be prepared where each bead contains different DNA sequences. Exemplary libraries and methods for their creation are described in Nature, 437, 376-380 (2005); Science, 309, 5741, 1728- 1732 (2005), each of which is incorporated herein by reference. Sequencing of arrays of such beads using nucleotides set forth herein is within the scope of the disclosure. [0130] Templates that are to be sequenced may form part of an “array” on a solid support, in which case the array may take any convenient form. Thus, the method of the disclosure is applicable to all types of high-density arrays, including single-molecule arrays, clustered arrays, and bead arrays. Labeled nucleotides of the present disclosure may be used for sequencing templates on essentially any type of array, including but not limited to those formed by immobilization of nucleic acid molecules on a solid support. [0131] However, labeled nucleotides of the disclosure are particularly advantageous in the context of sequencing of clustered arrays. In clustered arrays, distinct regions on the array (often referred to as sites, or features) comprise multiple polynucleotide template molecules. Generally, the multiple polynucleotide molecules are not individually resolvable by optical means and are instead detected as an ensemble. Depending on how the array is formed, each site on the array may comprise multiple copies of one individual polynucleotide molecule (e.g., the site is homogenous for a particular single- or double-stranded nucleic acid species) or even multiple copies of a small number of different polynucleotide molecules (e.g., multiple copies of two different nucleic acid species). Clustered arrays of nucleic acid molecules may be produced using techniques generally known in the art. By way of example, WO 98/44151 and WO 00/18957, each of which is incorporated herein, describe methods of amplification of nucleic acids wherein both the template and amplification products remain immobilized on a solid support in order to form arrays comprised of clusters or “colonies” of immobilized nucleic acid molecules. The nucleic acid molecules present on the clustered arrays prepared according to these methods are suitable templates for sequencing using the nucleotides labeled with dye compounds of the disclosure. [0132] The labeled nucleotides of the present disclosure are also useful in sequencing of templates on single molecule arrays. The term “single molecule array” or “SMA” as used herein refers to a population of polynucleotide molecules, distributed (or arrayed) over a solid support, wherein the spacing of any individual polynucleotide from all others of the population is such that it is possible to individually resolve the individual polynucleotide molecules. The target nucleic acid molecules immobilized onto the surface of the solid support can thus be capable of being resolved by optical means in some embodiments. This means that one or more distinct signals, each representing one polynucleotide, will occur within the resolvable area of the particular imaging device used. [0133] Single molecule detection may be achieved wherein the spacing between adjacent polynucleotide molecules on an array is at least 100 nm, more particularly at least 250 nm, still more particularly at least 300 nm, even more particularly at least 350 nm. Thus, each molecule is individually resolvable and detectable as a single molecule fluorescent point, and fluorescence from said single molecule fluorescent point also exhibits single step photobleaching. [0134] The terms “individually resolved” and “individual resolution” are used herein to specify that, when visualized, it is possible to distinguish one molecule on the array from its neighboring molecules. Separation between individual molecules on the array will be determined, in part, by the particular technique used to resolve the individual molecules. The general features of single molecule arrays will be understood by reference to published applications WO 00/06770 and WO 01/57248, each of which is incorporated herein by reference. Although one use of the nucleotides of the disclosure is in sequencing-by-synthesis reactions, the utility of the nucleotides is not limited to such methods. In fact, the nucleotides may be used advantageously in any sequencing methodology which requires detection of fluorescent labels attached to nucleotides incorporated into a polynucleotide. EXAMPLES [0135] 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. Synthesis of functionalized nucleotide containing a self-immolative linker Scheme 1. Synthesis of cAbf-TFA O O O Br NHTFA NHTFA [0136] The protected cABf linker (cABf-TFA) was synthesized in 6 steps from 4- hydroxy 2-bromo benzaldehyde, as described in Scheme 1. Briefly, 4-hydroxy 2-bromo benzaldehyde was glycosylated using tetraacetyl L-arabinofuranose and boron trifluoride diethyl etherate, to obtain the intermediate 1. This intermediate was functionalized with a 3-(N- trifluoroacetamido)propynyl group by Sonogashira cross-coupling with N- propargyltrifluoroacetamide to obtain compound 2. Next, the aldehyde was reduced to hydroxyl group with sodium borohydride to obtain intermediate 3. This intermediate was treated with carbonyl diimidazole (CDI), followed by glycine methyl ester to introduce the carbamate group on the benzylic hydroxyl group (compounds 4). Full deprotection of intermediate 4 was accomplished by treating with aqueous LiOH. cAbf-TFA was obtained by coupling compound 5 with 6-(trifluoroacetamido)hexanoic acid with TSTU as the coupling agent. The final product cABf-TFA was characterized by ¹H NMR and MS.¹H NMR (400 MHz, D2O): δ (ppm) 7.34 (d, J = 8.7 Hz, 1H, Ar-CH), 7.13 (t, J = 2.4 Hz, 1H, Ar-CH), 7.03 (dt, J = 8.6, 2.2 Hz, 1H, Ar-CH), 5.61 (t, J = 1.7 Hz, 1H, 1-CH Abf), 5.08 (s, 2H, O-CH2-Ar), 4.27 (m, 1H, 2-CH Abf), 4.09 (m, 3H, 4-CH Abf, CH2 Gly), 4.00 (dd, J = 5.2, 2.4, 1H, 3-CH Abf), 3.74 (dd, J = 12.4, 3.4, 1H, 5- CHH Abf), 3.65 (dd, J = 12.4, 5.3 Hz, 1H, 5-CHH Abf), 3.60 (s, 2H, Alkenyl-CH2-NHTFA), 3.18 (t, J = 7.0 Hz, 2H, CH2-CH2-NHTFA), 2.66 (m, 20H, TEAB salt), 2.21 (t, J = 7.3 Hz, 2H, CH2- CH2-CONHTFA), 1.60 – 1.44 (m, 4H, CH2-CH2-CH2), 1.27 (m, 2H, CH2-CH2-CH2), 1.01 (m, 27H, TEAB salt). ¹⁹F NMR (376 MHz, D₂O): δ (ppm) -75.82. LC-MS (ESI): (positive ion) m/z 620 (M+H⁺), 637 (M+NH4⁺), 642 (M+Na⁺); (negative ion) m/z 618 (M-H⁺).

Scheme 2. Synthesis of a green dye labeled ffT O O O O NHTFA NH HO HN HO HN ₂ N O N O 3- was aqueous to obtain the cAbf intermediate, which was coupled with the green dye AF550POPO using TSTU as the coupling agent. The intermediate cAbf-AF550POPO was then coupled to 5’-triphosphate 3’- azidomethyl 5-(3-aminopropynyl) 2’-deoxythymidine, using TSTU as the coupling agent. [0138] Characterization of cAbf-AF550POPO: Yield: 4.9 µmol, (33%). LC-MS (ES): (positive ion) m/z 1179 (M-H⁺); (negative ion) m/z 1177 (M-H⁺), 588 (M-2H⁺). RP-HPLC (YMC- Packpro C18, 250x4.6 mm, gradient from 5% to 50% acetonitrile in 0.1 M TEAB, 20 minutes, flow rate 1 mL/min): tR 19.1 min. [0139] Characterization of ffT-AZM-cAbf-AF550POPO: Yield: 2.75 µmol, (76%). LC-MS (ES): (negative ion) m/z 1735 (M-H⁺), 867 (M-2H⁺), 578 (M-3H⁺). RP-HPLC (YMC- Packpro C18, 250x4.6 mm, gradient from 5% to 50% acetonitrile in 0.1 M TEAB, 20 minutes, flow rate 1 mL/min): t_R 18.0 min. Example 2. Enzymatic Cleavage of c-Abf Linker [0140] A 3ʹ-O-azidomethyl ffT with a self-immolative linker containing carbamate ABF moiety (c-Abf) conjugated to a dye was synthesized according to Example 1. at 60°C. was so final reaction contained 50 μM c-ABF ffT and 5 µM enzyme in 100 mM sodium citrate pH 5.5. The reaction was incubated for 0 or 1 minute at 60°C and 20 µl of sample was combined with 20 µl of 500 mM self-immolation buffer (CAPS pH 11) and incubated for 5 minutes at 60°C prior to analysis by HPLC (YMC Triart C18, 250 X 4.5 mL, S-5 μm, 12 nm, 0.1M TEAB/100% Acetonitrile) to monitor the production of the self-immolation product and depletion of intact c- Abf ffT.

O H O O N O H SO N N H N N HN O H O SO [0142] FIGs. 1A and 1B are HPLC analysis of the resulting test samples. FIG. 1B illustrates the resulting self-immolation product relative to the starting material after the c-ABF ffT was incubated in the presence of ABFase GH51 for 1 minute. FIG. 1A illustrates the HPLC peak of the starting material c-ABF ffT as a negative control. The HPLC analysis showed that the c-ABF ffT was subjected to enzymatically triggered self-immolation. Similar experiments were conducted in various buffer solutions such as Tris (pH 8.6), CAPS (pH 10), Tris-HCl (pH 8), Tris- HCl (pH 7) and sodium citrate (pH 5.5) at 60 °C and it was observed that the c-Abf linker fully self-immolates efficiently and quickly at various pH conditions. Example 3. Stability testing of c-Abf Linker [0143] In this experiment, the stability of a c-Abf linker was tested at pH 9.9 (incorporation mix conditions) and pH 5.5 (ABFase enzymatic cleavage conditions) and compared to that of nucleotide containing AOL linker and LN3 linker (under incorporation mix conditions). 1 mL solutions of each linker test substrate (AOL, LN3 and c-ABF) at 0.1 mM were made in a solution of 50 mM buffer (glycine pH 9.9 or sodium citrate pH 5.5), 50 mM NaCl, 6 mM MgSO4 and 1 mM EDTA (in glycine buffer solutions only). These were incubated in a heating block at 60°C in the dark for 16 days. At set time points, 40 µL aliquots were taken and analyzed by UPLC (Agilent Poroshell HPH C18, 2.7 µm, 4.6 x 50mm, 0.5mL/min, 0.1M TEAB / acetonitrile) to determine the percentage of linker substrate remaining and the rate of degradation. The stability of the c-ABF was tested at pH 9.9 (incorporation mix conditions) and pH 5.5 (ABFase enzymatic cleavage conditions) and compared to that of known linkers AOL and LN3 (each under incorporation mix conditions of pH 9.9). the experiment. Regardless of pH conditions, c-ABF degradation was 4 to 5-fold slower than LN3 at 60°C. c-ABF degradation did not exceed 20% over 16 days under these conditions. Example 4. Enzymatic Cleavage of Acetal-ABF Linker [0145] A linker including an acetal leaving group with the ABF group (referred to herein as acetal-ABF or a-ABF) was investigated. [_{0146] ), intended to model an} acetal leaving similar conditions as in Example 1 followed by HPLC analysis were performed to monitor the reaction of interest of the model substrate. [0147] FIGs.3A–3C plot HPLC analysis of the resulting test samples. FIG.3A is an HPLC plot of the substrate at t = 0. FIG. 3B is an HPLC plot of the substrate at t = 30 seconds. FIG.3C is an HPLC plot of the substrate at t = 30 minutes at pH 11 and 60 °C. The results show that the model substrate was capable of enzyme-triggered self-immolation.

Claims

WHAT IS CLAIMED IS: 1. A nucleotide comprising a nucleobase, a ribose or 2´ deoxyribose, and a detectable moiety, wherein the detectable moiety is covalently attached to the nucleotide via a self- immolative linker, and wherein the self-immolative linker comprises an optionally substituted benzyl moiety, a leaving group comprising a carbamate moiety or an acetyl moiety, and an enzymatically cleavable moiety. 2. The nucleotide of claim 1, wherein the self-immolative linker has the structure of formula (I): (I), wherein an optionally present spacer moiety; X is O or NH; R^EC is the enzymatically cleavable moiety; R^BZ is H, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C6- C₁₀ aryl, or unsubstituted or substituted 5 to 10 membered heteroaryl; the phenylene moiety is optionally substituted with one or more electron withdrawing groups or one or more electron donating groups; the asterisk indicates the point of connection to the nucleobase; and the detectable moiety is covalently attached to the leaving group, the benzyl moiety (if L² is present), each optionally via a spacer L³. of claim 2, wherein the self-immolative linker has the structure of formula (Ia-1) or (Ia-2): (Ia- each R is independently H or an electron donating group; each R² is independently H or an electron withdrawing group; and R³ is H, an electron donating group, or an electron withdrawing group. 4. The nucleotide of claim 2, wherein the self-immolative linker has the structure of formula (Ib-1), (Ib-2) or (Ib-3): or each R² is independently H or an electron withdrawing group; and each R³ is independently H, an electron donating group, or an electron withdrawing group. 5. The nucleotide of any one of claims 2 to 4, wherein R^BZ is H. 6. The nucleotide of any one of claims 2 to 5, wherein X is O. 7. The nucleotide of claim 6, wherein –X–R^EC is –O–glycoside. 8. The nucleotide of claim 7, wherein R^EC is an arabinofuranosidyl (ABF) group.

9. The nucleotide of claim 8, wherei . 10. The nucleotide of any one mprises . claims 2 to 10, wherein L³ is a 2 to 10 membered one is optionally replaced by a triazole moiety. 12. The nucleotide of any one of claims 1 to 11, wherein the nucleotide comprises a 3´ blocking group. 13. The nucleotide of claim 12, wherein the 3´ blocking group is enzymatically cleavable. 14. The nucleotide of claim 12 or 13, wherein the 3´ blocking group and the detectable moiety are removable by a single enzymatic reaction. 15. The nucleotide of any one of claims 12 to 14, wherein the 3´ blocking group is – O–glycoside, forming an –O–glycosidic bond with the 3ʹ carbon atom of the nucleotide. 16. The nucleotide of claim 15, wherein the 3ʹ blocking group has a structure: the squiggle line indicates the point of 2´ deoxyribose. 17. The nucleotide of claim any one of claims 1 to 16, wherein the detectable moiety is a fluorescent dye. 18. The nucleotide of any one of claims 1 to 16, wherein the detectable moiety is a functional group that is capable of attaching to a labeling reagent. 19. The nucleotide of any one of claims 1 to 18, wherein the nucleotide is a nucleotide triphosphate comprising 2´ deoxyribose. 20. An oligonucleotide or polynucleotide comprising a nucleotide of claim 19 incorporated therein. 21. The oligonucleotide or polynucleotide of claim 20, wherein the oligonucleotide or polynucleotide is at least partially complementary and hybridized to a target polynucleotide immobilized on a surface of a solid support.

22. The oligonucleotide or polynucleotide of claim 21, wherein the solid support comprises an array of a plurality of target polynucleotides immobilized thereon. 23. A kit comprising a nucleotide according to any one of claims 1 to 19. 24. The kit of claim 23, further comprising a first enzyme, wherein the first enzyme is a polymerase, a terminal deoxynucleotidyl transferase, or a reverse transcriptase. 25. The kit of claim 24, wherein the polymerase is a DNA polymerase. 26. The kit of claims 24 or 25, further comprising a second enzyme for removing the detectable moiety of the nucleotide. 27. The kit of claim 26, wherein the second enzyme is a glycoside hydrolase or glycosidase. 28. The kit of claim 27, wherein the second enzyme is an arabinofuranosidase, a glucosidase, a mannosidase, a xylosidase, a galactosidase, an N-acetyl-glucosaminidase, or a glucuronidase. 29. A method of preparing a growing polynucleotide complementary to a target single- stranded polynucleotide, comprising incorporating a nucleotide of any one of claims 1 to 19 into a growing complementary polynucleotide. 30. The method of claim 29, wherein the incorporation of the nucleotide is accomplished by a polymerase, a terminal deoxynucleotidyl transferase, or a reverse transcriptase. 31. A method of determining the sequences of a plurality of target polynucleotides, comprising: (a) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides; (b) contacting the solid support with an aqueous 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, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides, wherein at least one type of nucleotide is a nucleotide of claim 19 carrying a fluorescent label through the self-immolative linker, and wherein each of the one or more of four different type of nucleotides comprises a 3´ blocking group; (c) imaging the solid support and performing one or more fluorescent measurements of the extended copy polynucleotides; and (d) removing the fluorescent label and the 3´ blocking group from nucleotides incorporated into the extended copy polynucleotides.

32. The method of claim 31, wherein step (d) comprises enzymatically removing the fluorescent label from the incorporated nucleotides. 33. The method of claim 31 or 32, wherein the fluorescent label and the 3´ blocking group are removed in a single reaction. 34. The method of any one of claims 31 to 33, further comprising: (e) washing the solid support after the removal of the 3ʹ blocking group and the fluorescent label from the incorporated nucleotides. 35. The method of claim 34, further comprising repeating steps (b) to (e) until the sequences of at least a portion of the target polynucleotides are determined. 36. The method of claim 35, wherein steps (b) to (e) are repeated at least 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 cycles. 37. A method of determining the sequences of a plurality of target polynucleotides, comprising: (a’) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides; (b’) contacting the solid support with an aqueous 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, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides, wherein at least one type of nucleotide is an unlabeled nucleotide of claim 19 having a first functional group attached via the self-immolative linker, and wherein each of the one or more of four different type of nucleotides comprises a 3´ blocking group; (c’) contacting the extended copy polynucleotides with an aqueous labeling mixture comprising a first labeling reagent, wherein the first labeling reagent comprises one or more first fluorescent labels and a first binding moiety that is capable of specific binding to the first functional group of the unlabeled nucleotide; (d’) imaging the solid support and performing one or more fluorescent measurements of the extended copy polynucleotides; and (e’) removing the one or more fluorescent labels and the 3´ blocking group from the nucleotides incorporated into the extended copy polynucleotides. 38. The method of claim 37, wherein step (e’) comprises enzymatically removing the one or more fluorescent labels from the incorporated nucleotides.

39. The method of claim 37 or 38, wherein the one or more fluorescent labels and the 3´ blocking group are removed in a single reaction. 40. The method of any one of claims 37 to 39, further comprising: (f’) washing the solid support after the removal of the 3ʹ blocking group and the one or more fluorescent labels from the incorporated nucleotides. 41. The method of claim 40, further comprising repeating steps (b’) to (e’) until the sequences of at least a portion of the target polynucleotides are determined. 42. The method of claim 41, wherein steps (b’) to (e’) are repeated at least 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 cycles. 43. The method of any one of claims 31 to 42, wherein the fluorescent label is removed by contacting the solid support with an aqueous cleavage solution comprising a glycoside hydrolase or glycosidase that is capable of catalyzing the degradation of the self-immolative linker. 44. The method of claim 43, wherein the glycoside hydrolase or glycosidase is an arabinofuranosidase, a glucosidase, a mannosidase, a xylosidase, a galactosidase, an N-acetyl- glucosaminidase, or a glucuronidase. 45. The method of claim 43 or 44, wherein the concentration of the glycoside hydrolase or glycosidase in the aqueous cleavage solution is at least about 0.1 µM. 46. The method of any one of claims 43 to 45, wherein the removal of the fluorescent label is conducted at a temperature of at least about 30°C. 47. The method of any one of claims 43 to 46, wherein the removal of the fluorescent label is conducted at a pH between about 5 and about 10.