Nucleosides and nucleotides having 3'-hydroxyl blocking groups and their use in polynucleotide sequencing methods

Abstract

Embodiments of the present disclosure relate to nucleotides and nucleoside molecules having acetal or thiocarbamate 3'-OH blocking groups. Also provided herein are methods for preparing such nucleotides and nucleoside molecules, and the use of fully functionalized nucleotides containing 3'-OH blocking groups in sequencing applications.

Description

background Technical Field

[0002] The present disclosure generally relates to nucleotides, nucleosides or oligonucleotides comprising a 3'-hydroxyl protecting group and their use in polynucleotide sequencing methods. Also disclosed are methods for preparing 3'-hydroxyl protected nucleotides, nucleosides or oligonucleotides.

[0003] Related technical description

[0004] Advances in molecular research have been due, in part, to improvements in techniques for characterizing molecules or their biological reactions. In particular, the study of the nucleic acids DNA and RNA has benefited from the development of techniques for sequence analysis and the study of hybridization events.

[0005] One example of a technology that has improved the study of nucleic acids is the development of fabricated arrays for immobilizing nucleic acids. These arrays typically consist of a high-density matrix of polynucleotides immobilized on a solid support material. See, for example, Fodor et al., Trends Biotech. 12: 19-26, 1994, which describes a method for assembling nucleic acids using a chemically sensitized glass surface protected by a mask but exposed in limited areas to allow phosphoramidite attachment of appropriately modified nucleotides. Fabricated arrays can also be made by techniques in which known polynucleotides are "spotted" onto predetermined locations on a solid support (e.g., Stimpson et al., Proc. Natl. Acad. Sci. 92: 6379-6383, 1995).

[0006] The method for determining the nucleotide sequence of nucleic acids bound to an array is called "sequencing by synthesis" or "SBS". This technique for determining DNA sequence ideally requires the controlled incorporation (i.e., one at a time) of the correct complementary nucleotide relative to the nucleic acid being sequenced. Since each nucleotide residue is sequenced one at a time, accurate sequencing can be performed by adding nucleotides in multiple cycles, thereby preventing a series of uncontrolled incorporations. The incorporated nucleotides are read using an appropriate label attached thereto before removal of the label moiety and subsequent next round of sequencing.

[0007] To ensure that only a single incorporation occurs, each labeled nucleotide (which is added to the growing chain to ensure that only one nucleotide is incorporated) includes a structural modification (a protecting group or blocking group). After the nucleotide with the protecting group is added, the protecting group is removed under reaction conditions that 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.

[0008] In order to be applicable to dna sequencing, nucleotide, normally nucleoside triphosphate, generally need 3 '-hydroxy blocking group, in case after adding the base on the nucleotide, be used for the polymerase that this nucleotide is mixed into the polynucleotide chain and continue to copy.Can be added on the nucleotide and still suitable group type has a lot of restrictions.Described blocking group should prevent other nucleotide molecules from being added in the polynucleotide chain, is easy to remove from sugar moiety and can not cause destruction to the polynucleotide chain simultaneously.In addition, the nucleotide of modification needs to be compatible with polymerase or for another kind of suitable enzyme that it is mixed into polynucleotide chain.Therefore, ideal blocking group must show long-term stability, can be effectively mixed by polymerase, stop the nucleotide secondary to mix or further mix, and have the ability that is removed under the mild condition that does not destroy polynucleotide structure (preferably under aqueous condition).

[0009] Reversible protecting groups have been described before. For example, Metzker et al. (Nucleic Acids Research, 22(20): 4259-4267, 1994) disclosed the synthesis and use of eight 3'-modified 2-deoxyribonucleoside 5'-triphosphates (3'-modified dNTPs) and tested their incorporation activity in two DNA template assays. WO2002 / 029003 describes a sequencing method that can include blocking the 3'-OH group on the growing DNA chain using an allyl protecting group during a polymerase reaction.

[0010] Additionally, the development of a number of reversible protecting groups and methods for their deprotection under DNA-compatible conditions were previously reported in International Application Publications WO 2004 / 018497 and WO 2014 / 139596, each of which is herein incorporated by reference in its entirety.

[0011] Overview

[0012] Some embodiments of the present disclosure relate to nucleosides or nucleotides comprising a ribose or deoxyribose sugar having a removable 3'-OH blocking group that forms a structure covalently linked to the 3'-carbon atom. in:

[0013] Each R 1a and R 1b are independently H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, cyano, halogen, optionally substituted phenyl, or optionally substituted aralkyl;

[0014] Each R 2a and R 2b are independently H, C1-C6 alkyl, C1-C6 haloalkyl, cyano or halogen;

[0015] Or, R 1a and R 2a together with the atoms to which they are attached, form an optionally substituted five- to eight-membered heterocyclyl;

[0016] R 3 is H, optionally substituted C 2- C6 alkenyl, optionally substituted C 3- C7 cycloalkenyl, optionally substituted C 2- C6 alkynyl or optionally substituted (C1-C6 alkylene)Si(R 4 )3; and

[0017] Each R 4 are independently H, C1-C6 alkyl or optionally substituted C 6- C 10 In some embodiments, when each R 1a and R 1b When R is H or C1-C6 alkyl, 2a and R 2b Both are H, then R 3 C 2- C6 alkenyl, optionally substituted C 3- C7 cycloalkenyl, optionally substituted C 2- C6 alkynyl or optionally substituted (C1-C6 alkylene)Si(R 4 )3. In some embodiments, when each R 1a 、R 1b 、R 2a and R 2b H, then R 3 Not for H.

[0018] Some embodiments of the present disclosure relate to nucleosides or nucleotides comprising a ribose or deoxyribose sugar having a removable 3'-OH blocking group that forms a structure covalently linked to the 3'-carbon atom. in:

[0019] Each R 5 and R 6 are independently H, C1-C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C1-C6 haloalkyl, C 2- C8 alkoxyalkyl, optionally substituted –(CH2) m -phenyl, optionally substituted -(CH2) n -(5- or 6-membered heteroaryl), optionally substituted -(CH2) k –C 3-C7 carbocyclyl or optionally substituted –(CH2) p –(3- to 7-membered heterocyclyl);

[0020] Each – (CH2) m –, –(CH2) n –, –(CH2) k – and – (CH2) p - is optionally substituted; and

[0021] Each of m, n, k and p is independently 0, 1, 2, 3 or 4.

[0022] Some embodiments of the present disclosure relate to oligonucleotides or polynucleotides comprising the 3'-OH blocked nucleotide molecules described herein.

[0023] Some embodiments of the present disclosure relate to methods for preparing a growing polynucleotide complementary to a target single-stranded polynucleotide in a sequencing reaction, comprising incorporating a nucleotide molecule as described herein into the growing complementary polynucleotide, wherein the incorporation of the nucleotide prevents any subsequent nucleotide from being introduced into the growing complementary polynucleotide. In some embodiments, the incorporation of the nucleotide is accomplished by a polymerase, terminal deoxynucleotidyl transferase (TdT), or reverse transcriptase. In one embodiment, the incorporation is accomplished by a polymerase (e.g., a DNA polymerase).

[0024] Some further embodiments of the present disclosure relate to methods of determining a target single-stranded polynucleotide sequence, comprising:

[0025] (a) incorporating a nucleotide comprising a 3'-OH blocking group and a detectable label as described herein into a replicating polynucleotide chain that is complementary to at least a portion of a target polynucleotide chain;

[0026] (b) detecting the identity of the nucleotide incorporated into the replicated polynucleotide chain; and

[0027] (c) Chemical removal of the label and 3'-OH blocking group from the nucleotides incorporated into the replicated polynucleotide chain.

[0028] In some embodiments, the sequencing method further comprises (d) washing away the chemically removed label and 3' blocking group from the replicated polynucleotide chain. In some embodiments, the washing step also removes unincorporated nucleotides. In some such embodiments, the 3' blocking group and detectable label of the incorporated nucleotide are first removed before introducing the next complementary nucleotide. In some further embodiments, the 3' blocking group and detectable label are removed in a single chemical reaction step. In some embodiments, the sequencing incorporation described herein is performed at least 50 times, at least 100 times, at least 150 times, at least 200 times, or at least 250 times.

[0029] Some further embodiments of the present disclosure relate to test kits, which comprise multiple nucleotides as described herein or nucleoside molecules and packaging materials thereof. The nucleotides, nucleosides, oligonucleotides or test kits set forth herein can be used for detecting, measuring or identifying a biological system (including, for example, its process or component). Exemplary techniques that can use nucleotides, oligonucleotides or test kits include order-checking, expression analysis, hybridization analysis, genetic analysis, RNA analysis, cell determination (for example, cell binding or cell function analysis) or protein determination (for example, protein binding assay or protein activity assay). Described use can be realized on an automated instrument for performing specific technology, such as an automated sequencing instrument. Described sequencing instrument can contain two or more lasers working with different wavelengths, to distinguish different detectable labels. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a line graph showing the stability of various 3'-blocked nucleotides in a buffer solution at 65°C over time.

[0031] Figure 2A A line graph showing the percentage (%) of remaining nucleotides (starting material) as a function of time comparing the deblocking rates of nucleotides with 3'-AOM blocking groups and nucleotides with 3'-O-azidomethyl (-CH2N3) blocking groups in solution.

[0032] Figure 2B FIG2 is a line graph showing the percentage (%) of 3′-unblocked nucleotides as a function of time, comparing the unblocking rates of 3′-blocked nucleotides with various acetal blocking groups in solution.

[0033] Figure 3A and 3B Shown in Illumina Sequencing results on an instrument using fully functionalized nucleotides (ffN) with 3'-AOM blocking groups in the spiking mix.

[0034] Figure 3C Shown are sequencing error rates using fully functionalized nucleotides (ffNs) with 3'-AOM blocking groups in the spiking mixture compared to standard ffNs with 3'-O-azido blocking groups.

[0035] Figure 4A and 4BEach shows a comparison of key sequencing metrics including phasing, pre-phasing, and error rates for fully functionalized nucleotides with 3'-AOM and 3'-O-azidomethyl blocking groups using two different DNA polymerases (Pol 812 and Pol 1901), respectively.

[0036] Figure 5 Graph showing the sequencing stability of fully functionalized nucleotides with 3'-AOM or 3'-O-azidomethyl blocking groups over time in a buffer solution at 45°C.

[0037] Figure 6 is a line graph showing the stability of nucleosides with various 3' blocking groups as a function of time in a buffer solution at 65°C.

[0038] Figure 7 The following is a line graph showing the percentage (%) of 3' blocked nucleotides remaining as a function of time, comparing the thiocarbamate 3' blocking group dimethylthiocarbamate (DMTC) under two different conditions ( or NaIO4) and the cleavage (deblocking) rate of the 3'-O-azidomethyl (3'-O-CH2N3) blocking group. Detailed Description of the Invention

[0040] Embodiments disclosed herein relate to nucleosides and nucleotides with 3'-OH acetal or thiocarbamate blocking groups, which are used for sequencing applications, for example, sequencing while synthesizing (SBS). These blocking groups provide better stability in solution compared to those known in the art. Particularly 3'-OH blocking groups have improved stability in the process of synthesizing fully functionalized nucleotides (ffNs), and also have good stability in solution during preparation, storage, and operation on sequencing instruments. In addition, 3'-OH blocking groups as herein described can also achieve low prephasing, lower signal attenuation to improve data quality, which makes it possible to read longer in sequencing applications.

[0041] definition

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The use of the term "include" and other forms of the term (such as "include", "includes" and "included") is not restrictive. The use of the term "have" and other forms of the term (such as "have", "has" and "had") is not restrictive. As used in this specification, whether in transitional phrases or in the body of the claims, the terms "comprise(s)" and "comprising" should be interpreted as having an open-ended meaning. That is, the above terms will be interpreted synonymously with the phrases "at least have" or "at least include". For example, when used in the context of a process, the term "comprising" means that the process includes at least the listed steps, but may include other 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 listed features or components, but may also include other features or components.

[0043] As used herein, common organic abbreviations are defined as follows:

[0044] ℃ Temperature (Celsius)

[0045] dATP deoxyadenosine triphosphate

[0046] dCTP deoxycytidine triphosphate

[0047] dGTP deoxyguanosine triphosphate

[0048] dTTP deoxythymidine triphosphate

[0049] ddNTP dideoxynucleoside triphosphate

[0050] ffN fully functionalized nucleotides

[0051] RT Room temperature

[0052] SBS Sequencing by Synthesis

[0053] SM raw materials

[0054] As used herein, the term "array" refers to a colony of different probe molecules connected to one or more substrates so that different probe molecules can be distinguished from each other according to relative position. The array can include different probe molecules, each of which is located at different addressable positions on the substrate. Alternatively or additionally, the array can include substrates separated from each other with different probe molecules, wherein different probe molecules can be identified based on the position of the substrate on the surface to which the substrate is connected or based on the position of the substrate in the liquid. Exemplary arrays wherein the mutually separated substrates are located on the surface include, but are not limited to, those arrays comprising beads in holes, such as described in U.S. Patent No. 6,355,431B1, US 2002 / 0102578, and PCT Publication WO 00 / 63437. The exemplary format (format) that can be used in the present invention for distinguishing beads in liquid arrays using, for example, a microfluidic device such as a fluorescence activated cell sorter (FACS) is described in, for example, U.S. Patent No. 6,524,793. Other examples of arrays that can be used in the present invention include, but are not limited to, those disclosed in U.S. Patents 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; as well as WO 93 / 17126; WO 95 / 11995; WO 95 / 35505; EP 742 287; and EP Those arrays described in 799 897.

[0055] As used herein, the term "covalently attached" or "covalent bonding" refers to the formation of a chemical bond characterized by the sharing of electron pairs between atoms. For example, a covalently attached polymer coating refers to a polymer coating that forms a chemical bond with the functionalized surface of a substrate, as opposed to forming a chemical bond via other means, such as adhesion or electrostatic interactions. It should be understood that a polymer covalently attached to a surface can also be bonded by other means besides covalent attachment.

[0056] As used herein, any "R" group represents a substituent that can be attached to the indicated atom. The R group can be substituted or unsubstituted. If two "R" groups are described as forming a ring or ring system "together with the atoms to which they are attached," it means that the collective unit of atoms, intervening bonds, and the two R groups is the enumerated ring. For example, when the following substructure is present:

[0057]

[0058] And R 1 and R 2 is defined as being selected from hydrogen and alkyl or R 1 and R 2 Together with the atoms to which they are attached, they form an aryl or carbocyclic group, which means that R 1 and R 2 It may be selected from hydrogen or alkyl or a substructure having the following structure:

[0059]

[0060] wherein A is an aryl ring or a carbocyclic group containing the double bond as shown.

[0061] It should be understood that some group naming conventions may include monoradicals or diradicals, depending on the context. For example, when a substituent requires two points of attachment to the rest of the molecule, it is understood that the substituent is a diradical. For example, a substituent identified as an alkyl group requiring two points of attachment includes a diradical, such as -CH2-, -CH2CH2-, -CH2CH(CH3)CH2-, etc. Other group naming conventions clearly indicate that the group is a diradical, such as "alkylene" or "alkenylene."

[0062] As used herein, the term "halogen" or "halo" refers to any radiation stable atom in column 7 of the periodic table of elements, such as fluorine, chlorine, bromine or iodine, with fluorine and chlorine being preferred.

[0063] As used herein, “C a to C b”, where “a” and “b” are integers and refer to the number of carbon atoms in an alkyl, alkenyl or alkynyl group or the number of ring atoms in a cycloalkyl or aryl group. That is, the rings of alkyl, alkenyl, alkynyl, cycloalkyl and aryl groups may contain from “a” to “b” (inclusive) carbon atoms. For example, “C1 to C4 alkyl” refers to all alkyl groups having 1 to 4 carbon atoms, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)- and (CH3)3C-; C3 to C4 cycloalkyl refers to all cycloalkyl groups having 3 to 4 carbon atoms, i.e., cyclopropyl and cyclobutyl . Similarly, "4- to 6-membered heterocyclyl" refers to all heterocyclyls having 4 to 6 total ring atoms, such as azetidine, oxetane, oxazoline, pyrrolidine, piperidine, piperazine, morpholine, and the like. If "a" and "b" are not specified for alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, it is assumed that the broadest range is described in these definitions. As used herein, the term "C1-C6" includes C1, C2, C3, C4, C5, and C6, as well as ranges defined by either of the two numbers. For example, C1-C6 alkyl includes C1, C2, C3, C4, C5, and C6 alkyl, C2-C6 alkyl, C1-C3 alkyl, and the like. Similarly, C 2- C6 alkenyl includes C2, C3, C4, C5 and C6 alkenyl, C2-C5 alkenyl, C3-C4 alkenyl, etc.; and C 2- C6 alkynyl includes C2, C3, C4, C5 and C6 alkynyl, C2-C5 alkynyl, C3-C4 alkynyl, etc. C3-C8 cycloalkyl each includes a hydrocarbon ring containing 3, 4, 5, 6, 7 and 8 carbon atoms or a range defined by either of the two numbers, such as C3-C7 cycloalkyl or C5-C6 cycloalkyl.

[0064] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., does not contain double or triple bonds). The alkyl group may have 1 to 20 carbon atoms (whenever a numerical range such as "1 to 20" appears, it refers to each integer in the given range; for example, "1 to 20 carbon atoms" means that the alkyl group may include 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although this definition also covers the occurrence of the term "alkyl" in which no numerical range is specified). The alkyl group may also be a medium-sized alkyl group having 1 to 9 carbon atoms. The alkyl group may also be a low-sized alkyl group 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" means that there are one to six carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like.

[0065] As used herein, "alkoxy" refers to the formula -OR, wherein R is an alkyl group as defined above, for example, "C1-C9 alkoxy", including but not limited to methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy and tert-butoxy, etc.

[0066] As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain containing one or more double bonds. The alkenyl group may have from 2 to 20 carbon atoms, although this definition also encompasses occurrences of the term "alkenyl" where no numerical range is specified. The alkenyl group may also be a medium-sized alkenyl group having from 2 to 9 carbon atoms. The alkenyl group may also be a lower alkenyl group having from 2 to 6 carbon atoms. The alkenyl group may be designated as "C 2- C6 alkenyl" or similar names. For example only, "C 2- "C6 alkenyl" means that there are two to six carbon atoms in the alkenyl chain, that is, the alkenyl chain is selected from ethenyl, propenyl-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methylpropen-1-yl, 2-methylpropen-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 by no means limited to, ethenyl, propenyl, butenyl, pentenyl and hexenyl, among others.

[0067] As used herein, "alkynyl" refers to a straight or branched hydrocarbon chain containing one or more triple bonds. The alkynyl group may have from 2 to 20 carbon atoms, although this definition also encompasses occurrences of the term "alkynyl" where no numerical range is specified. The alkynyl group may also be a medium-sized alkynyl group having from 2 to 9 carbon atoms. The alkynyl group may also be a lower alkynyl group having from 2 to 6 carbon atoms. The alkynyl group may be designated as "C 2- C6 alkynyl" or similar designations. For example only, "C 2- "C6 alkynyl" means that there are 2 to 6 carbon atoms in the alkynyl chain, that is, the alkynyl chain is selected from 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, etc.

[0068] As used herein, "heteroalkyl" refers to a straight or branched hydrocarbon chain containing one or more heteroatoms (i.e., elements other than carbon, including but not limited to nitrogen, oxygen, and sulfur) in the chain backbone. The heteroalkyl group may have from 1 to 20 carbon atoms, although this definition also encompasses the occurrence of the term "heteroalkyl" without specifying a numerical range. The heteroalkyl group may also be a medium-sized heteroalkyl group having from 1 to 9 carbon atoms. The heteroalkyl group may also be a low-sized heteroalkyl group having from 1 to 6 carbon atoms. The heteroalkyl group may be designated as "C1-C6 heteroalkyl" or similar designations. The heteroalkyl group may contain one or more heteroatoms. By way of example only, "C 4- "C6 heteroalkyl" means that there are four to six carbon atoms in the heteroalkyl chain and one or more additional heteroatoms in the backbone of the chain.

[0069] The term "aromatic" refers to a ring or ring system having a conjugated π electron system and includes carbocyclic aromatic groups (e.g., phenyl) and heterocyclic aromatic groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic (i.e., rings that share adjacent pairs of atoms) groups, provided that the entire ring system is aromatic.

[0070] 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) that contains only carbon in the ring backbone. When the aryl is a ring system, each ring in the system is aromatic. The aryl may have 6 to 18 carbon atoms, although this definition also encompasses occurrences of the term "aryl" where no numerical range is specified. In some embodiments, the aryl has 6 to 10 carbon atoms. The aryl may be referred to as a "C 6- C 10 Aryl", "C6 or C 10 Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl.

[0071] "Aralkyl" or "arylalkyl" is an aryl group attached as a substituent through an alkylene group, for example, "C 7-14 In some cases, the alkylene group is a lower alkylene group (i.e., C1-C6 alkylene group).

[0072] As used herein, "heteroaryl" refers to an aromatic ring or ring system (i.e., two or more fused rings sharing two adjacent atoms) containing one or more heteroatoms (i.e., elements other than carbon, including but not limited to nitrogen, oxygen, and sulfur) in the ring backbone. When the heteroaryl is a ring system, each ring in the system is aromatic. The heteroaryl may have 5-18 ring members (i.e., the number of atoms constituting the ring backbone, including carbon atoms and heteroatoms), although this definition also encompasses the occurrence of the term "heteroaryl" in which no numerical range is specified. In some embodiments, the heteroaryl has 5 to 10 ring members or 5 to 7 ring members. The heteroaryl may be designated as "5-7 yuan heteroaryl", "5-10 yuan heteroaryl" or similar names. Examples of heteroaryl rings include, but are not limited to, furanyl, 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.

[0073] "Heteroaralkyl" or "heteroarylalkyl" is a heteroaryl group attached 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., C1-C6 alkylene group).

[0074] As used herein, "carbocyclyl" refers to a non-aromatic ring or ring system that contains only carbon atoms in the ring system backbone. When the carbocyclyl is a ring system, two or more rings can be connected together in a fused, bridged or spiro manner. The carbocyclyl can have any degree of saturation, provided that at least one ring in the ring system is not aromatic. Thus, carbocyclyl includes cycloalkyl, cycloalkenyl and cycloalkynyl. The carbocyclyl group can have 3 to 20 carbon atoms, although this definition also covers the occurrence of the term "carbocyclyl" where no numerical range is specified. The carbocyclyl group can also be a medium-sized carbocyclyl group having 3 to 10 carbon atoms. The carbocyclyl group can also be a carbocyclyl group having 3 to 6 carbon atoms. The carbocyclyl group can be designated as "C 3- C6 carbocyclyl" or similar designations. Examples of carbocyclyl rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydroindane, bicyclo[2.2.2]octyl, adamantyl, and spiro[4.4]nonyl.

[0075] As used herein, "cycloalkyl" refers to a fully saturated carbocyclyl ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0076] As used herein, "heterocyclyl" refers to a non-aromatic ring or ring system containing at least one heteroatom in the ring backbone. The heterocyclyl groups can be connected together in a fused, bridged or spirally connected manner. The heterocyclyl group can have any degree of saturation, provided that at least one ring in the ring system is not aromatic. The heteroatom can be present in the non-aromatic or aromatic ring of the ring system. The heterocyclyl group can have 3 to 20 ring members (i.e., the number of atoms that make up the ring backbone, including carbon atoms and heteroatoms), although this definition also covers the occurrence of the term "heterocyclyl" in which no numerical range is specified. The heterocyclyl group can also be a medium-sized heterocyclyl group having 3 to 10 ring members. The heterocyclyl group can also be a heterocyclyl group having 3 to 6 ring members. The heterocyclyl group can be designated as "3-6 membered heterocyclyl" or similar names. In preferred six-membered monocyclic heterocyclyls, the heteroatoms are selected from up to three of O, N or S, and in preferred five-membered monocyclic heterocyclyls, the heteroatoms are selected from one or two heteroatoms selected from O, N or S. Examples of heterocyclyl rings include, but are not limited to, aza acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxirane, oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl, pyrrolidindione, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxinyl, 1,4-dioxinyl, 1,4-dioxinyl, 1,3-oxathiinyl, 1,4-oxathiinyl , 1,4-oxathiolanyl, 2H-1,2-oxazinyl, trioxanyl, hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolane, 1,3-dithiolanyl, 1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolinone, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolyl, isoindolyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl, thiomorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, and tetrahydroquinoline.

[0077] "O-carboxyl" refers to "-OC(=O)R", wherein R is selected from hydrogen, C1-C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C 3- C7 carbocyclic group, C 6- C 10 aryl, 5-10 membered heteroaryl and 3-10 membered heterocyclyl.

[0078] "C-carboxyl" refers to a "-C(=O)OR" group, wherein R is selected from hydrogen, C1-C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C 3- C7 carbocyclic group, C 6- C 10 Aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl. Non-limiting examples include carboxy (ie, -C(=O)OH).

[0079] "Sulfonyl" refers to "-SO2R" where R is selected from hydrogen, C1-C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C 3- C7 carbocyclic group, C 6- C 10 aryl, 5-10 membered heteroaryl and 3-10 membered heterocyclyl.

[0080] A "sulfinyl" group refers to a "-S(=O)OH" group.

[0081] "S-sulfonamido" refers to "-SO2NR A R B ”, where R A and R B are independently selected from hydrogen, C1-C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C 3- C7 carbocyclic group, C 6- C 10 aryl, 5-10 membered heteroaryl and 3-10 membered heterocyclyl.

[0082] An "N-sulfonamido" group refers to an "-N(R A )SO2R B " group, wherein R A and R B are independently selected from hydrogen, C1-C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C 3- C7 carbocyclic group, C 6- C 10 aryl, 5-10 membered heteroaryl and 3-10 membered heterocyclyl.

[0083] A "C-amido" group refers to a "-C(=O)NR A R B " group, wherein R A and R Bare independently selected from hydrogen, C1-C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C 3- C7 carbocyclic group, C 6- C 10 aryl, 5-10 membered heteroaryl and 3-10 membered heterocyclyl.

[0084] An "N-amido" group refers to an "-N(R A )C(=O)R B " group, wherein R A and R B are independently selected from hydrogen, C1-C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C 3- C7 carbocyclic group, C 6- C 10 aryl, 5-10 membered heteroaryl and 3-10 membered heterocyclyl.

[0085] An "amino" group refers to an "-NR A R B " group, wherein R A and R B are independently selected from hydrogen, C1-C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C 3- C7 carbocyclic group, C 6- C 10 Aryl, 5-10 membered heteroaryl and 3-10 membered heterocyclyl. Non-limiting examples include free amino (ie, -NH2).

[0086] An "aminoalkyl" group refers to an amino group attached through an alkylene group.

[0087] An "alkoxyalkyl" group refers to an alkoxy group attached through an alkylene group, for example, "C 2- C8 alkoxyalkyl" and the like.

[0088] As used herein, substituents are derived from an unsubstituted parent group in which one or more hydrogen atoms have been exchanged with another atom or group. Unless otherwise specified, when a group is considered to be "substituted", it means that the group is substituted with one or more substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 heteroalkyl, C3-C7 carbocyclyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7-carbocyclyl-C1-C6-alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3-10 membered alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 heteroalkyl, C3-C7 carbocyclyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), heterocyclyl (optionally substituted by halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3-10 membered heterocyclyl-C1-C6-alkyl (optionally substituted by halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl (optionally substituted by halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl (optionally substituted by halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl (C1-C6) alkyl (optionally substituted by halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C1-C6 haloalkoxy), 5-10 membered heteroaryl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5-10 membered heteroaryl(C1-C6)alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), halogen, -CN, hydroxy, C1-C6 alkoxy, C1-C6 alkoxy(C1-C6)alkyl (i.e., ether), aryloxy, sulfhydryl (sulfhydryl and 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-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxyl, O-carboxyl, acyl, cyano, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl, -SO3H, sulfoxide, -OSO2C 1-4 Alkyl and oxo (=0). Wherever a group is described as "optionally substituted," the group may be substituted with the substituents described above.

[0089] As used herein, the term "hydroxy" refers to an -OH group.

[0090] As used herein, the term "cyano" refers to a "-CN" group.

[0091] As used herein, the term "azido" refers to a -N3 group.

[0092] As used herein, "nucleotides" include nitrogenous heterocyclic bases, sugars, and one or more phosphate groups. They are the monomeric units of nucleic acid sequences. In RNA, the sugar is ribose, while in DNA it is deoxyribose, a sugar that lacks the hydroxyl groups present in ribose. The nitrogenous heterocyclic bases can be purine or pyrimidine bases. Purine bases include adenine (A) and guanine (G), 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 the deoxyribose is bonded to the N-1 of a pyrimidine or the N-9 of a purine.

[0093] As used herein, "nucleoside" is structurally similar to a nucleotide, but lacks a phosphate moiety. An example of a nucleoside analog is an analog in which the label is attached to a base and there is no phosphate group attached to a sugar molecule. As will be appreciated by those skilled in the art, the term "nucleoside" is used in its general sense herein. Examples include, but are not limited to, ribonucleosides comprising a ribose moiety and deoxyribonucleosides comprising a deoxyribose moiety. A modified pentose moiety is a pentose moiety in which an oxygen atom has been replaced by a carbon and / or a carbon has been replaced by a sulfur or oxygen atom. A "nucleoside" is a monomer that can have a substituted base and / or sugar moiety. In addition, nucleosides can be incorporated into larger DNA and / or RNA polymers and oligomers.

[0094] As understood by those skilled in the art, the term "purine base" is used in this article with its common meaning, and includes its tautomers. Similarly, the term "pyrimidine base" is used in this article with the common meaning understood by those skilled in the art, and includes its tautomers. The non-limiting list of optionally substituted purine bases includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanines (such as 7-methylguanine), theobromine, caffeine, uric acid and isoguanine. The example of pyrimidine bases includes but is not limited to cytosine, thymine, uracil, 5,6-dihydrouracil and 5-alkylcytosine (such as 5-methylcytosine).

[0095] As used herein, when oligonucleotide or polynucleotide are described as " comprising " nucleoside as herein described or nucleotide, refer to nucleoside as herein described or nucleotide and oligonucleotide or polynucleotide formation covalent bond.Similarly, when nucleoside or nucleotide are described as a part for oligonucleotide or polynucleotide, for example, " being incorporated into " oligonucleotide or polynucleotide, this means that nucleoside as herein described or nucleotide and oligonucleotide or polynucleotide formation covalent bond.In some such embodiments, between 3 ' hydroxyl group of oligonucleotide as herein described or polynucleotide and 5 ' phosphate group of nucleotide, form covalent bond, as phosphodiester bond between 3 ' carbon atom of oligonucleotide or polynucleotide and 5 ' carbon atom of described nucleotide.

[0096] As used herein, " derivative " or " analogue " refer to synthetic nucleotides or the nucleoside derivatives of the base moiety and / or the sugar moiety with modification. Such derivatives and analogues have been discussed in, for example, Scheit at Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al. in Chemical Reviews 90:543-584,1990. Nucleotide analogues can also comprise the phosphodiester linkage of modification, including phosphorothioate, phosphorodithioate, alkyl-phosphate, aniline phosphate and phosphoramidate linkage. " derivative " used herein, " analogue " and " modified " can be used interchangeably, and are contained in the terms " nucleotide " and " nucleoside " defined herein.

[0097] As used herein, the term "phosphate" is used in its ordinary sense as understood by those skilled in the art and includes protonated forms thereof (e.g. ). As used herein, the terms "monophosphate," "diphosphate," and "triphosphate" are used with their ordinary meaning as understood by those skilled in the art and include protonated forms.

[0098] As used herein, the term "protecting group" refers to any atom or group of atoms added to a molecule to prevent an existing group in the molecule from undergoing an undesired chemical reaction. Sometimes, "protecting group" and "blocking group" are used interchangeably.

[0099] As used herein, the prefix "light" or "photo-" means relating to light or electromagnetic radiation. The term can encompass all or part of the electromagnetic spectrum, including but not limited to one or more ranges of radio, microwave, infrared, visible, ultraviolet, X-ray or gamma ray portions commonly referred to as spectrum. The portion of the spectrum can be a portion blocked by a metal region (such as those described herein) on a surface. Alternatively or additionally, the portion of the spectrum can be a portion passing through a gap region of a surface such as a region made of glass, plastic, silicon dioxide or other materials set forth herein. In a specific embodiment, radiation that can pass through metal can be used. Alternatively or additionally, radiation shielded by glass, plastic, silicon dioxide or other materials set forth herein can be used.

[0100] As used herein, the term "phasing" refers to a phenomenon in SBS that is caused by incomplete removal of the 3' terminator and fluorophore and the inability of the polymerase to complete the incorporation of a portion of the DNA chain within the cluster in a given sequencing cycle. Pre-phasing is caused by the incorporation of nucleotides without an effective 3' terminator, wherein the incorporation event is advanced by one cycle due to termination failure. Phasing and pre-phasing can cause the signal intensity measured within a specific cycle to include the signal of the current cycle and the noise of the previous and subsequent cycles. As the number of cycles increases, the sequence portion of each cluster affected by phasing and pre-phasing increases, thereby hindering the identification of the correct base. Trace amounts of unprotected or unblocked 3'-OH nucleotides exist during sequencing by synthesis (SBS) and can cause pre-phasing. The unblocked 3'-OH nucleotides may be generated during the production process or during storage and reagent handling. Therefore, it is surprising to find nucleotide analogs that reduce the incidence of pre-phasing and provide a huge advantage over existing nucleotide analogs in SBS applications. For example, the provided nucleotide analogs can result in faster SBS cycle times, lower phasing and prephasing values, and longer sequencing read lengths.

[0101] 3'-Hydroxyacetal blocking group

[0102] Some embodiments of the present disclosure relate to ribose or deoxyribose-containing nucleotides or nucleosides having a removable 3′-OH protecting or blocking group that forms a structure covalently linked to the 3′-carbon atom. in:

[0103] Each R 1a and R 1bare independently H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, cyano, halogen, optionally substituted phenyl, or optionally substituted aralkyl;

[0104] Each R 2a and R 2b are independently H, C1-C6 alkyl, C1-C6 haloalkyl, cyano or halogen;

[0105] Or, R 1a and R 2a together with the atoms to which they are attached, form an optionally substituted five- to eight-membered heterocyclyl;

[0106] R 3 is H, optionally substituted C 2- C6 alkenyl, optionally substituted C 3- C7 cycloalkenyl, optionally substituted C 2- C6 alkynyl or optionally substituted (C1-C6 alkylene)Si(R 4 )3; and

[0107] Each R 4 are independently H, C1-C6 alkyl or optionally substituted C 6- C 10 Aryl; provided that when each R 1a 、R 1b 、R 2a and R 2b When H, R 3 Not for H.

[0108] Some further embodiments of the present disclosure relate to compounds having the structure of formula (I): wherein R' is H, a monophosphate, a diphosphate, a triphosphate, a phosphorothioate, a phosphate analog, an -O- group connected to an active phosphorus group, or an -O- group protected by a protecting group; R" is H or OH; B is a nucleoside base; each R 1a 、R 1b 、R 2a 、R 2b and R 3 As defined above. In some further embodiments, B is In some further embodiments, the nucleobase is covalently bound to a detectable label (e.g., a fluorescent dye), optionally via a linker, e.g., B is In some such embodiments, R' is triphosphate. In some such embodiments, R" is H.

[0109] In some embodiments of the acetal blocking groups described herein, at least R 1a and R 1b is H. In some such embodiments, each R 1a and R 1b is H. In some other embodiments, at least R 1a and R 1b In some embodiments, each R 2a and R 2b are independently H, halogen, or C1-C6 alkyl. In some such embodiments, at least R 2a and R 2b One of is H or C1-C6 alkyl. In some such embodiments, each R 2a and R 2b is H. In some such embodiments, each R 2a and R 2b is C1-C6 alkyl, such as methyl, ethyl, isopropyl or tert-butyl. 2a and R 2b In some such embodiments, each R 2a and R 2b are independently C1-C6 alkyl or halogen. In some such embodiments, R 2a for H, and R 2b is halogen or C1-C6 alkyl.

[0110] In some embodiments of the acetal blocking groups described herein, R 3 is optionally substituted C 2- C6 alkenyl. In some such embodiments, R 3 C 2- C6 alkenyl (e.g., ethenyl, propenyl), optionally substituted by one or more substituents independently selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 haloalkyl, and combinations thereof. In some further embodiments, R 3 for

[0111] In some other embodiments, R 3 is optionally substituted C 2- C6 alkynyl. In some such embodiments, R 3 C 2-C6 alkynyl (e.g., ethynyl, propynyl), optionally substituted by one or more substituents independently selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 haloalkyl, and combinations thereof. In one embodiment, R 3 is an optionally substituted ethynyl In some other embodiments, R 3 is an optionally substituted (C1-C6 alkylene)Si(R 4 ) 3. In some such embodiments, at least one R 4 C 1-4 In some further embodiments, each R 4 is C1-C4 alkyl, for example, methyl, ethyl, isopropyl or tert-butyl. 3 is -(CH2)-SiMe3. In some optional embodiments, R 3 It is a C1-C6 alkyl group.

[0112] In some alternative embodiments, R 1a and R 2a Together with the atoms to which they are attached, they form a five- to seven-membered heterocyclyl. 1a and R 2a Together with the atoms to which they are attached, they form a six-membered heterocyclic group. In some such embodiments, the six-membered heterocyclic group has the structure In some further embodiments, at least R 1b 、R 2b and R 3 One of is H. In some other embodiments, at least R 1b 、R 2b and R 3 In one embodiment, each R 1b 、R 2b and R 3 For H.

[0113] In some further embodiments, the compound of formula (I) is also represented by formula (Ia):

[0114] Each R 2c and R2d is independently H, halogen (e.g., fluorine, chlorine), C1-C6 alkyl (e.g., methyl, ethyl, or isopropyl), or C1-C6 haloalkyl (e.g., -CHF2, -CH2F, or -CF3). In some such embodiments, R 1a and R 1b is H. In some such embodiments, each R 1a and R1b is H. In some other embodiments, at least R 1a and R 1b In some embodiments, each R 2a and R 2b is independently H, halogen, or C1-C6 alkyl. In some such embodiments, each R 2a and R 2b is H. In some such embodiments, each R 2c and R 2d is independently H, halogen, or C1-C6 alkyl. In some such embodiments, each R 2c and R 2d is C1-C6 alkyl, such as methyl, ethyl, isopropyl or tert-butyl. 2c and R 2d In some such embodiments, each R 2c and R 2d In some such embodiments, R 2c for H, and R 2d is H, halogen (fluorine, chlorine) or C1-C6 alkyl (e.g., methyl, ethyl, isopropyl or tert-butyl). In further embodiments, each R 1a and R 1b H; R 2a H; R 2b is H, halogen or methyl; R 2c is H; and R 2d is H, halogen, methyl, ethyl, isopropyl or tert-butyl.

[0115] Non-limiting embodiments of blocking groups described herein include those having a structure selected from the group consisting of:

[0116] It is covalently attached to the 3'-carbon of ribose or deoxyribose.

[0117] 3'-Hydroxythiocarbamate blocking group

[0118] Some additional embodiments of the present disclosure relate to nucleosides or nucleotides comprising ribose or deoxyribose sugars having a removable 3′-OH blocking group that forms a structure covalently linked to the 3′-carbon atom. in:

[0119] Each R 5 and R 6are independently H, C1-C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C1-C6 haloalkyl, C 2- C8 alkoxyalkyl, optionally substituted –(CH2) m -phenyl, optionally substituted -(CH2) n -(5- or 6-membered heteroaryl), optionally substituted -(CH2) k –C 3- C7 carbocyclyl or optionally substituted –(CH2) p –(3- to 7-membered heterocyclyl);

[0120] Or, R 5 and R 6 together with the atoms to which they are attached, form an optionally substituted five- to seven-membered heterocyclyl;

[0121] Each – (CH2) m –, –(CH2) n –, –(CH2) k – and – (CH2) p - is optionally substituted; and

[0122] Each of m, n, k and p is independently 0, 1, 2, 3 or 4.

[0123] Some additional embodiments relate to compounds of formula (II):

[0124] wherein R' is H, a monophosphate, a diphosphate, a triphosphate, a phosphorothioate, a phosphate analog, an -O- group connected to an active phosphorus group, or an -O- group protected by a protecting group; R" is H or OH; B is a nucleoside base; each R 5 and R 6 As defined above. In some further embodiments, B is In some further embodiments, the nucleobase is covalently bound to a detectable label (e.g., a fluorescent dye), optionally via a linker, e.g., B is In some such embodiments, R' is triphosphate. In some such embodiments, R" is H.

[0125] In some embodiments of the thiocarbamate blocking groups described herein, at least R 5 and R 6 is H. In some such embodiments, each R 5 and R 6 is H. In some such embodiments, R 5 For H and R 6is C1-C6 alkyl, for example, methyl, ethyl, isopropyl or tert-butyl. In some such embodiments, R 5 For H and R 6 C 2- C6 alkenyl (e.g., vinyl or allyl) or C 2- C6 alkynyl (e.g., ethynyl or propynyl). In some such embodiments, R 5 For H and R 2 is optionally substituted –(CH2) m -phenyl, optionally substituted -(CH2) n -(5- or 6-membered heteroaryl), optionally substituted -(CH2) k –C 3- C7 carbocyclyl or optionally substituted –(CH2) p -(3 to 7 membered heterocyclyl). In some further embodiments, the C 3- The C7 carbocyclic group may be C 3- C7 cycloalkyl or C 3- C7 cycloalkenyl. The 3 to 7 membered heterocyclic group may contain zero or one double bond in the ring structure. In a further embodiment, R 5 For H and R 6 is optionally substituted –(CH2) m -phenyl, optionally substituted -(CH2) n -6-membered heteroaryl, optionally substituted -(CH2) k -C5 or C6 carbocyclyl or optionally substituted -(CH2) p -(5 or 6 membered heterocyclyl). In some embodiments, m, n, k or p is 0. In other embodiments, m, n, k or p is 1 or 2. In some other embodiments, at least R 5 and R 6 In some further embodiments, one of R 5 and R 6 In one embodiment, R 5 and R 6 All are methyl.

[0126] In some alternative embodiments, R 5 and R 6 Together with the atoms to which they are attached, form an optionally substituted five- to seven-membered heterocyclyl. 5 and R 6 Together with the atoms to which they are attached, they form an optionally substituted piperidinyl.

[0127] Non-limiting embodiments of 3'-O-thiocarbamate blocking groups described herein include those having a structure selected from the group consisting of: (DMTC), which is covalently attached to the 3'-carbon of ribose or deoxyribose.

[0128] Additional embodiments of the present disclosure relate to oligonucleotides or polynucleotides comprising the nucleosides or nucleotides described herein.

[0129] In any embodiment of a blocking group described herein, when the group is described as "optionally substituted," it can be unsubstituted or substituted.

[0130] In any embodiment of the nucleotide or nucleoside with 3'-hydroxyl blocking group as herein described, the nucleoside or nucleotide can be covalently attached to detectable label (for example, fluorophore), optionally via a linking group.The linking group can be breakable or non-breakable.In some such embodiments, the detectable label (for example, fluorophore) is covalently attached to the nucleobase of a nucleoside or nucleotide via a breakable linking group.In some other embodiments, the detectable label (for example, fluorophore) is covalently attached to the 3' oxygen of a nucleoside or nucleotide via a breakable linking group.In some further embodiments, such a breakable linking group can include an azido moiety or a disulfide bond moiety, an acetal moiety or a thiocarbamate moiety.In some embodiments, the 3' hydroxyl blocking group and the breakable linking group (and the label connected) can be removed under identical or substantially identical chemical reaction conditions, for example, the blocking group and the detectable label can be removed in a single chemical reaction. In other embodiments, the blocking group and the detectable label are removed in two separate steps.

[0131] In some embodiments, the nucleotides or nucleosides described herein comprise a 2' deoxyribose sugar. In other aspects, the 2' deoxyribose sugar contains one, two, or three phosphate groups at the 5' position of the sugar ring. In another aspect, the nucleotides described herein are nucleoside triphosphates.

[0132] In some embodiments, the 3'-blocked nucleotides or nucleosides described herein provide superior stability in solution during storage or during reagent handling during sequencing applications compared to the same nucleotides or nucleosides protected by standard 3'-OH blocking groups disclosed in the prior art (e.g., 3'-O-azidomethyl blocking groups). For example, the acetal or thiocarbamate blocking groups disclosed herein can impart at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 2500%, or 3000% improved stability compared to azidomethyl protected 3'-OH under the same conditions for the same time period, thereby reducing prephasing values ​​and obtaining longer sequencing read lengths. In some embodiments, the stability is measured at ambient temperature or a temperature below ambient temperature (e.g., 4-10°C). In other embodiments, the stability is measured at elevated temperatures, e.g., 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. In some such embodiments, the stability is measured in a solution at an alkaline pH environment, e.g., at pH 9.0, 9.2, 9.4, 9.6, 9.8, or 10.0. In some such embodiments, the stability is measured with or without the presence of an enzyme such as a polymerase (e.g., DNA polymerase), terminal deoxynucleotidyl transferase, or reverse transcriptase.

[0133] In some embodiments, the 3'-blocked nucleotides or nucleosides described herein provide superior deblocking rates in solution during the chemical fragmentation step of sequencing applications compared to the same nucleotides or nucleosides protected with standard 3'-OH blocking groups disclosed in the prior art (e.g., 3'-O-azidomethyl protecting groups). For example, the acetal or thiocarbamate blocking groups disclosed herein can impart an improved deblocking rate of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, or 2000% compared to azidomethyl protected 3'-OH using standard deblocking reagents (e.g., tris(hydroxypropyl)phosphine), thereby reducing the total time for sequencing cycles. In some embodiments, the unblocking rate is measured at ambient temperature or a temperature below ambient temperature (e.g., 4-10° C.). In other embodiments, the unblocking rate is measured at an elevated temperature, e.g., 40° C., 45° C., 50° C., 55° C., 60° C., or 65° C. In some such embodiments, the unblocking rate is measured in a solution at an alkaline pH environment, e.g., at pH 9.0, 9.2, 9.4, 9.6, 9.8, or 10.0. In some such embodiments, the molar ratio of the unblocking reagent to the substrate (i.e., the 3'-blocked nucleoside or nucleotide) is about 10:1, about 5:1, about 2:1, or about 1:1.

[0134] In some embodiments, a palladium deblocking agent (e.g., Pd(0)) is used to remove a 3' acetal blocking group (e.g., an AOM blocking group). Pd may form a chelate complex with the two oxygen atoms of the AOM group and the double bond of the allyl group, placing the deblocking agent in direct proximity to the functional group to be removed and may result in an accelerated deblocking rate.

[0135] Deprotection of 3'-OH blocking group

[0136] The 3'-acetal blocking groups described herein can be removed or cleaved under various chemical conditions. For acetal blocking groups containing vinyl or alkenyl moieties Non-limiting cleavage conditions include Pd(II) complexes, such as Pd(OAc)2 or allylpalladium(II) chloride dimer, in the presence of a phosphine ligand, such as tris(hydroxymethyl)phosphine (THMP) or tris(hydroxypropyl)phosphine (THP or THPP). For those blocking groups containing alkynyl groups (e.g., ethynyl), they can also be removed by Pd(II) complexes (e.g., Pd(OAc)2 or allylpalladium(II) chloride dimer) in the presence of a phosphine ligand (e.g., THP or THMP).

[0137] Palladium fragmentation reagent

[0138] In some embodiments, the acetal blocking groups described herein can be cleaved by a palladium catalyst. In some such embodiments, the palladium catalyst is water soluble. In some such embodiments, it is a Pd(0) complex (e.g., (3,3′,3″-phosphinyltris(phenylsulfonate)palladium(0) nonasodium salt nonahydrate). In some cases, the Pd(0) complex can be generated in situ by reducing a Pd(II) complex with a reagent such as an olefin, alcohol, amine, phosphine, or metal hydride. Suitable palladium sources include Na2PdCl4, 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), and Pd(TFA)2. In one such embodiment , a Pd(0) complex is generated in situ from Na2PdCl4. In another embodiment, the palladium source is allylpalladium(II) chloride dimer [(PdCl(C3H5))2]. In some embodiments, the Pd(0) complex is generated by mixing the Pd(II) complex with a phosphine in an aqueous solution. Suitable phosphines include water-soluble phosphines such as tris(hydroxypropyl)phosphine (THP), tris(hydroxymethyl)phosphine (THMP), 1,3,5-triaza-7-phosphaadamantane (PTA, 1,3,5-triaza-7-phosphaadamantane), bis(p-sulfonatophenyl)phenylphosphine dihydratepotassium salt, tris(carboxyethyl)phosphine (TCEP) and triphenylphosphine-3,3',3"-trisulfonic acid trisodium salt.

[0139] In some embodiments, the Pd(0) is prepared in situ by mixing a Pd(II) complex [(PdCl(C3H5))2] with THP. The molar ratio of the Pd(II) complex to THP is about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. 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 an additional buffering agent, such as a primary amine, a secondary amine, a tertiary amine, a carbonate, a phosphate, or a borate, or a combination thereof. In some further embodiments, the buffer comprises ethanolamine (EA), tris (hydroxymethyl) aminomethane (Tris), glycine, sodium carbonate, sodium phosphate, sodium borate, 2-dimethylaminoethanol (DMEA), 2-diethylaminoethanol (DEEA), N,N,N',N'-tetramethylethylenediamine (TEMED) or N,N,N',N'-tetraethylethylenediamine (TEEDA), or a combination thereof. In one embodiment, the buffer is DEEA. In another embodiment, the buffer comprises one or more inorganic salts, such as carbonates, phosphates or borates, or a combination thereof. In one embodiment, the inorganic salt is a sodium salt.

[0140] Alternatively, alkynyl moieties containing blocking groups can also be cleaved in the presence of (NH4)2MoS4. Other non-limiting cleavage conditions for alkynyl moieties include Cu(II) complexes formed with THPTA ligand (tris(3-hydroxypropyltriazolylmethyl)amine) and ascorbic acid. Non-limiting cleavage conditions for blocking groups containing six-membered heterocycles (e.g., tetrahydropyran) include cyclodextrin or Ln(OTf)3 (lanthanum trifluoromethanesulfonate). Non-limiting cleavage conditions for blocking groups containing alkylsilane groups (e.g., -CH2SiMe3) include LiBF (lithium tetrafluoroborate). Other acetal blocking groups can be removed by LiBF4 or Bi(OTf)3 (bismuth trifluoromethanesulfonate), such as -O(CH2)O-C1-C6 alkyl. Non-limiting exemplary conditions for cleaving the various blocking groups described are illustrated in Scheme 1 below.

[0141] Scheme 1. Schematic illustration of 3'-deblocking conditions

[0142]

[0143] The 3'-O-thiocarbamate blocking groups described herein can be removed or cleaved under various chemical conditions. Non-limiting exemplary conditions for cleaving the thiocarbamate blocking groups described herein include NaIO4 and (potassium peroxomonosulfate).

[0144] Alternatively, the azide group in -CH2N3 can be converted to an amino group by a phosphine. Alternatively, the azide group in -CH2N3 can be converted to an amino group by contacting the molecule with a thiol, particularly a water-soluble thiol such as dithiothreitol (DTT). In one embodiment, the phosphine is THP.

[0145] Compatibility with linearization

[0146] In order to maximize the throughput of nucleic acid sequencing reactions, it is advantageous to be able to sequence multiple template molecules in parallel. Parallel processing of multiple templates can be achieved using nucleic acid array technology. These arrays are usually composed of a high-density matrix of polynucleotides fixed on a solid support.

[0147] WO 98 / 44151 and WO 00 / 18957 have all described nucleic acid amplification methods, and this method allows amplified product to be fixed on solid phase carrier, to form the array being made up of cluster or " colony (colony) ", described cluster or " colony " are formed by a plurality of identical immobilized polynucleotide chains and a plurality of identical immobilized complementary chains.This type of array is referred to as " cluster array (clustered array) " in this article.The nucleic acid molecules present in the DNA colony on the cluster array prepared according to these methods can provide the template for sequencing reaction, for example described in WO 98 / 44152.The product of the solid phase amplification reaction such as described in WO 98 / 44151 and WO 00 / 18957 is by so-called " bridged (bridged) " structure formed by annealing to immobilized polynucleotide chain and immobilized complementary chain, and two chains are all connected to solid phase carrier at 5 ' end.In order to provide the more suitable template for nucleic acid sequencing, preferably remove substantially all or at least a portion of one of the described immobilized chains in the " bridged " structure, to produce a template that is at least partially single-stranded. Thus, the portion of the template that is single-stranded is available for hybridization with a sequencing primer. The process of removing all or part of one immobilized strand in a "bridging" double-stranded nucleic acid structure is called "linearization." Linearization can be performed in a variety of ways, including but not limited to enzymatic, photochemical, or chemical cleavage. Non-limiting examples of linearization methods are disclosed in PCT Publication WO 2007 / 010251, U.S. Patent Publication 2009 / 0088327, U.S. Patent Publication 2009 / 0118128, and U.S. Application 62 / 671,816, the entire contents of which are incorporated by reference.

[0148] In some embodiments, the conditions for deprotection or removal of the 3'-OH blocking group are also compatible with the linearization process. In some further embodiments, the deprotection conditions are compatible with a chemical linearization process, which includes the use of a Pd complex and a phosphine, such as Pd(OAc)2 and THP. In some embodiments, the Pd complex is a Pd(II) complex, which generates Pd(0) in situ in the presence of a phosphine.

[0149] Unless otherwise indicated, references to nucleotides are intended to apply to nucleosides as well.

[0150] Labeled nucleotides

[0151] According to an aspect of the present disclosure, the nucleotide of described 3'-OH sealing also comprises detectable labeling, and this nucleotide is called the nucleotide of labeling.Described labeling (for example, fluorescent dye) can be combined by several methods, comprise hydrophobic attraction, ion attraction and covalent bonding, by optional linking group.In some respects, described dyestuff is bonded to substrate by covalent bonding.More particularly, described covalent bonding is by means of linking group.In some cases, the nucleotide of this labeling is also referred to as " nucleotide of modification ".

[0152] Labeled nucleosides and nucleotides can be used to label polynucleotides formed by enzymatic synthesis, such as, by way of non-limiting example, PCR amplification, isothermal amplification, solid phase amplification, polynucleotide sequencing (e.g., solid phase sequencing), nick translation reactions.

[0153] In some embodiments, the dye can be covalently attached to an oligonucleotide or nucleotide via a nucleoside base. For example, the labeled nucleotide or oligonucleotide can have a label that is attached to the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base via a linker moiety.

[0154] Unless otherwise stated, references to nucleotides are intended to apply to nucleosides as well.Unless otherwise stated, the present invention will be further described with reference to DNA, although the description also applies to RNA, PNA and other nucleic acids.

[0155] Linking group

[0156] In some embodiments described herein, the purine or pyrimidine bases of the nucleotide or nucleoside molecules described herein may be linked to a detectable label as described above. In some such embodiments, the linking group used is cleavable. The use of a cleavable linking group ensures that after the label is detected, it can be removed if necessary to avoid any interfering signal with any subsequently incorporated labeled nucleotides or nucleosides. In some embodiments, the cleavable linking group comprises an azido moiety, a -OC 2- C6 alkenyl moieties (e.g., -O-allyl), disulfide moieties, acetal moieties (same or similar to 3' acetal blocking groups described herein), or thiocarbamate moieties (same or similar to 3' acetal blocking groups described herein).

[0157] In some other embodiments, the linking group used is non-cleavable.Since in each case where a labeled nucleotide of the invention is incorporated, there is no need to subsequently incorporate any nucleotides, there is no need to remove the label from the nucleotide.

[0158] Cleavable linking groups are known in the art, and conventional chemical methods can be applied to linking groups to nucleoside bases and labels. The linking group can be broken by any suitable method, including exposure to acid, base, nucleophile, electrophile, free radical, metal, reducing agent or oxidizing agent, light, temperature, enzyme, etc. The linking groups described herein can also be broken with the same catalyst as the bond of the cleavage 3'-O-blocking group. Suitable linking groups can be adapted to standard chemical protecting groups, such as those disclosed in Greene & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons. Other suitable cleavable linking groups for solid phase synthesis are disclosed in Guillier et al. (Chem. Rev. 100: 2092-2157, 2000).

[0159] In the case where the detectable label is attached to a base, the linking group can be attached to any position of the nucleoside base as long as Watson-Crick base pairing can still be performed. In the case of purine bases, the linking group is preferably attached via the 7-position of a purine or a preferred deazapurine analog, via an 8-modified purine, via an N-6 modified adenosine or an N-2 modified guanine. For pyrimidines, attachment is preferably performed via the 5-position of cytosine, thymine or uracil and the N-4 position of cytosine.

[0160] In some embodiments, the linker group may comprise a spacer unit. The length of the linker group is not critical, as long as the label is kept at a sufficient distance from the nucleotide so as not to interfere with any interaction between the nucleotide and an enzyme (e.g., a polymerase).

[0161] In some embodiments, the linking group may be composed of a functional group similar to the 3'-OH protecting group. This will make the deprotection and unblocking process more efficient because only one treatment is required to remove the label and the protecting group.

[0162] The use of term " cleavable linking group " does not mean that suggestion needs to remove whole linking group.Fractural site can be located at the position on the linking group, and this position guarantees that after fracture, the part of linking group still keeps being connected with dyestuff and / or substrate part.As limiting examples, cleavable linking group can be the linking group of electrophilic cleavable linking group, nucleophilic cleavable linking group, the linking group of light cleavable linking group, under reducing condition, oxidizing condition cleavable (for example containing disulfide bond or azido linking group), via using safety handle (safety-catch) linking group cleavable and cleavable by elimination mechanism.Use cleavable linking group that dye compound is connected to substrate part can guarantee if necessary, described mark is removed after detection, thereby avoids any interference signal in downstream step.

[0163] Useful linking groups can be found in PCT Publication WO 2004 / 018493 (incorporated herein by reference), examples of which include linking groups that can be cleaved using a water-soluble phosphine or a water-soluble transition metal catalyst formed from a transition metal and an at least partially water-soluble ligand (e.g., a Pd(II) complex and THP). The latter forms at least a partially water-soluble transition metal complex in aqueous solution. Such cleavable linking groups can be used to attach the bases of a nucleotide to a label, such as a dye described herein.

[0164] Particular linking groups include those disclosed in PCT Publication WO 2004 / 018493 (incorporated herein by reference), such as those comprising a moiety of the formula:

[0165]

[0166] (wherein X is selected from O, S, NH and NQ, wherein Q is C 1-10 A substituted or unsubstituted alkyl group, Y is selected from O, S, NH and N (allyl), T is hydrogen or C1-C 10(a substituted or unsubstituted alkyl group, with * indicating the position at which this moiety is attached to the rest of the nucleotide or nucleoside). In some aspects, the linking group connects the base of the nucleotide to a label, such as a dye compound described herein.

[0167] Other examples of linking groups include those disclosed in U.S. Publication No. 2016 / 0040225 (incorporated herein by reference), such as those comprising a moiety of the formula:

[0168]

[0169] The linker moiety presented herein may comprise all or part of the linker structure between the nucleotide / nucleoside and the label.

[0170] Additional examples of linking groups ("L") include moieties of the formula:

[0171] Wherein B is a nucleoside base; Z is –N3 (azido), –O-C1-C6 alkyl, –OC 2- C6 alkenyl or –OC 2- C6 alkynyl; and F1 comprises a fluorescent label, which may include another linking group structure. It will be understood by those skilled in the art that the label is covalently bonded to the linking group by reacting the functional group of the label (e.g., carboxyl) with the functional group of the linking group (e.g., amino).

[0172] In certain embodiments, the length of the linker between the fluorescent dye (fluorophore) and the guanine base can be varied, for example, by inserting a polyethylene glycol spacer, so that it fluoresces more intensely than the same fluorophore attached to the guanine base via other linkages known in the art. Exemplary linkers and their properties are described in PCT Publication WO2007020457 (incorporated herein by reference). The design of the linker, and particularly its increased length, can increase the brightness of the fluorophore attached to the guanine base of a guanosine nucleotide when incorporated into a polynucleotide (e.g., DNA). Thus, when the dye is used in any analytical method requiring detection of a fluorescent dye label attached to a guanine-containing nucleotide, the linker comprises the formula –((CH2)2O) n A spacer group of -(wherein n is an integer between 2 and 50) is advantageous, as described in WO 2007 / 020457.

[0173] Nucleoside and nucleotide can be labeled at the site of sugar or core base. As known in the art, " nucleotide " is made up of nitrogenous base, sugar and one or more phosphate groups. Sugar described in RNA is ribose, and in DNA it is deoxyribose (that is, the sugar lacking the hydroxyl group present in ribose). Described nitrogenous base is the derivative of purine or pyrimidine. Described purine is adenine (A) and guanine (G), and described pyrimidine is cytosine (C) and thymine (T) or is uracil (U) in the case of RNA. The C-1 atom of deoxyribose is bonded to the N-1 of pyrimidine or the N-9 of purine. Nucleotide is also the phosphate ester of nucleoside, and described nucleoside is esterified on the hydroxyl group that the C-3 or C-5 of described sugar connects. Nucleotide is typically single, two or three phosphates.

[0174] A "nucleoside" is structurally similar to a nucleotide, but lacks the phosphate moiety. Examples of nucleoside analogs are those in which a label is attached to the base and there is no phosphate group on the sugar molecule.

[0175] Although the base is commonly referred to as purine or pyrimidine, it will be understood by those skilled in the art that derivatives and analogs that do not change the ability of nucleotides or nucleosides to perform Watson-Crick base pairing are feasible." derivative " or " analog " refers to a compound or molecule whose core structure is identical or very similar to the parent compound, but has a chemical or physical modification (such as a different or additional side group that connects the derived nucleotide or nucleoside to another molecule). For example, the base can be a deazapurine. In a specific embodiment, the derivative should be able to perform Watson-Crick pairing. " derivative " and " analog " also include, for example, synthetic nucleotides or nucleoside derivatives with modified base moieties and / or modified sugar moieties. Such derivatives and analogs are discussed in, for example, Scheit, Nucleotide analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90: 543-584, 1990. Nucleotide analogs can also contain modified phosphodiester linkages, including phosphorothioate, phosphorodithioate, alkyl-phosphate, aniline phosphate, phosphoramidate linkages, and the like.

[0176] Dye can (for example, by linking group) be connected to any position of nucleotide base.In specific embodiment, Watson-Crick base pairing can still be carried out to the analogue of gained.Specific nucleoside base labeling site comprises the C5 position of pyrimidine base or the C7 position of 7-deazapurine base.As mentioned above, linking group can be used by dye covalently attached to nucleoside or nucleotide.

[0177] In a specific embodiment, the nucleoside or nucleotide of the mark can be enzyme-mergible and enzyme-scalable. Therefore, the linking group part can have enough length to connect the nucleotide to the compound, thereby this compound can not significantly interfere with the overall combination and identification of the nucleic acid replicator to the nucleotide. Therefore, the linking group can also include a spacer. This spacer will, for example, separate the nucleotide base from the break site or the mark.

[0178] Nucleosides or nucleotides labeled with the dyes described herein can have the formula:

[0179]

[0180] Wherein dye is a dye compound; B is a nucleoside base, such as uracil, thymine, cytosine, adenine, guanine, etc.; L is an optional linking group, which may or may not be present; R' can be H, a monophosphate, a diphosphate, a triphosphate, a phosphorothioate, a phosphate analog, an -O- group connected to a reactive phosphorus-containing group, or an -O- group protected by a blocking group; R'' can be H, OH, a phosphoramidite, or a 3'-OH blocking group described herein, and R'' is H or OH. When R''' is a phosphoramidite, R' is an acid-cleavable hydroxyl protecting group, which allows subsequent monomer coupling under automated synthesis conditions.

[0181] In certain embodiments, linking group (between dyestuff and Nucleotide) and blocking group all exist and are the part that separates.In certain embodiments, linking group and blocking group can both fracture under substantially similar condition.Therefore, the blocking process of unblocking may be more effective, because only need one-time processing and can remove dye compound and blocking group.Yet, in certain embodiments, linking group and blocking group do not need to fracture under similar conditions, but can fracture independently under different conditions.

[0182] The present disclosure also includes the polynucleotides that incorporate dye compounds.Such polynucleotides can be DNA or RNA composed of deoxyribonucleotides or ribonucleotides connected by phosphodiester linkages respectively.Polynucleotides can include naturally occurring nucleotides combined with at least one modified nucleotide as described herein (e.g., labeled with dye compounds), non-naturally occurring (or modified) nucleotides or any combination thereof except the nucleotides of the labeling as described herein.Polynucleotides according to the present disclosure can also include non-natural backbone linkages and / or non-nucleotide chemical modifications.Also contemplated are chimeric structures composed of a mixture of ribonucleotides and deoxyribonucleotides comprising the nucleotides of at least one labeling.

[0183] Non-limiting exemplary labeled nucleotides described herein include:

[0184]

[0185]

[0186] wherein L represents a linking group, and R represents a sugar residue as described above, or a sugar residue in which the 5' position is substituted by one, two or three phosphate groups.

[0187] In some embodiments, non-limiting exemplary fluorescent dye conjugates are shown below:

[0188]

[0189] wherein PG represents a 3'-hydroxy blocking group as described herein. In any of the embodiments of the labeled nucleotides described herein, the nucleotide is a nucleoside triphosphate.

[0190] Reagent test kit

[0191] The present disclosure also provides a kit comprising one or more 3'-sealed nucleosides and / or nucleotides as described herein, for example, 3'-sealed nucleotides of formula (I), (Ia) or (II). Such a kit typically includes at least one 3'-sealed nucleotide or nucleoside labeled with a dye and at least one other component. The other components may be one or more components as determined by the methods described herein or in the Examples section below. Some non-limiting examples of components that can be incorporated into the kit of the present disclosure are set forth below.

[0192] In a specific embodiment, the kit can include at least one labeled 3' blocked nucleotide or nucleoside and labeled or unlabeled nucleotide or nucleoside. For example, nucleotides labeled with dyes can be provided in combination with unlabeled or natural nucleotides and / or with fluorescently labeled nucleotides or any combination thereof. The combination of nucleotides can be provided as separate individual components (e.g., one nucleotide type per container or test tube) or as a nucleotide mixture (e.g., mixing two or more nucleotides in the same container or test tube).

[0193] When the kit includes a variety of (particularly two or three, or more particularly four) 3'-blocked nucleotides labeled with a dye compound, the different nucleotides can be labeled with different dye compounds, or one nucleotide can be dark and not have a dye compound. When the different nucleotides are labeled with different dye compounds, the kit is characterized in that the dye compound is a spectrally distinguishable fluorescent dye. As used herein, the term "spectrally distinguishable fluorescent dye" refers to a fluorescent dye that emits fluorescent energy at a wavelength that can be distinguished by a fluorescence detection device (e.g., a DNA sequencing platform based on a commercial capillary) when two nucleotides labeled with a fluorescent dye compound are provided in a kit form, some embodiments are characterized in that the spectrally distinguishable fluorescent dyes can be excited at the same wavelength, for example, by the same laser excitation. When four 3'-blocked nucleotides (A, C, T, and G) labeled with a fluorescent dye compound are provided in a kit form, some embodiments are characterized in that both spectrally distinguishable fluorescent dyes can be excited at one wavelength, while the other two spectrally distinguishable dyes can be excited at another wavelength. Specific excitation wavelengths are 488 nm and 532 nm.

[0194] In one embodiment, the kit comprises a first 3'-blocked nucleotide labeled with a first dye and a second nucleotide labeled with a second dye, wherein the difference in absorbance maxima of the dyes is at least 10 nm, particularly 20 nm to 50 nm. More particularly, the two dye compounds have a Stokes shift of between 15 and 40 nm, wherein "Stokes shift" is the distance between the peak absorption wavelength and the peak emission wavelength.

[0195] In an optional embodiment, test kit of the present disclosure can comprise the nucleotide of 3 ' sealing, and wherein identical base is used two or more different dye marks.Can use the first dye mark first nucleotide (for example, the T nucleoside triphosphate of 3 ' sealing or the G nucleoside triphosphate of 3 ' sealing).The second nucleotide (for example, the C nucleoside triphosphate of 3 ' sealing) can use the second dye mark different on the first dye spectrum, for example the absorption of " green " dye is less than 600nm, and the absorption of " blue " dye is less than 500nm, for example 400nm to 500nm, particularly 450nm to 460nm.The third nucleotide (for example, the A nucleoside triphosphate of 3 ' sealing) can be labeled as the mixture of the first and second dye, or the mixture of the first, second and third dye, and the fourth nucleotide (for example, the G nucleoside triphosphate of 3 ' sealing or the T nucleoside triphosphate of 3 ' sealing) may be " dark " and do not have mark.In an example, described nucleotide 1-4 can be labeled as " blue ", " green ", " blue / green " and dark. To further simplify the instrument, the four nucleotides can be labeled with two dyes excited by a single laser, so that nucleotides 1-4 can be labeled "blue 1," "blue 2," "blue 1 / blue 2," and dark.

[0196] In certain embodiments, the kit may comprise four labeled 3'-blocked nucleotides (e.g., A, C, T, G), wherein each type of nucleotide comprises the same 3' blocking group and fluorescent label, and wherein each fluorescent label has a different fluorescence maximum and each fluorescent label can be distinguished from the other three labels. The kit may comprise two or more fluorescent labels having similar maximum absorbances but different Stokes shifts. In some other embodiments, one of the nucleotides is unlabeled.

[0197] Although the kit is exemplified herein for configurations with different nucleotides labeled with different dye compounds, it should be understood that the kit may include 2, 3, 4 or more different nucleotides with the same dye compound. In some embodiments, the kit further includes an enzyme and a buffer suitable for the action of the enzyme. In some such embodiments, the enzyme is a polymerase, a terminal deoxynucleotidyl transferase, or a reverse transcriptase. In specific embodiments, the enzyme is a DNA polymerase, such as DNA polymerase 812 (Pol 812) or DNA polymerase 1901 (Pol 1901). The amino acid sequences of Pol 812 and Pol 1901 polymerases are described in, for example, U.S. patent application Ser. No. 16 / 670,876, filed October 31, 2019, and U.S. patent application Ser. No. 16 / 703,569, filed December 4, 2019, which are incorporated herein by reference.

[0198] Other components included in such kits may include buffers, etc. The nucleotides of the present disclosure and any other nucleotide components including mixtures of different nucleotides may be provided in the kit in a concentrated form that is diluted before use. In such embodiments, a suitable dilution buffer may also be included. Similarly, one or more components determined in the methods set forth herein may be included in the kit of the present disclosure.

[0199] Sequencing methods

[0200] Nucleotides or nucleosides according to the labeling of the present disclosure can be used in any analytical method, for example, including a method for detecting a fluorescent label attached to a nucleotide or nucleoside, whether the nucleotide or nucleoside of the labeling exists alone or is incorporated into or is connected to a larger molecular structure or conjugate. In this article, the term "incorporation into a polynucleotide" can mean that 5' phosphate participates in constituting a phosphodiester bond connected to the 3'-OH group of a second (modified or unmodified) nucleotide, which itself can form a part of a longer polynucleotide chain. The 3' end of the nucleotide described herein may or may not participate in constituting a phosphodiester bond connected to the 5' phosphate of other (modified or unmodified) nucleotides. Therefore, in a non-limiting embodiment, the present disclosure provides a method for detecting the nucleotides incorporated into a polynucleotide, the method comprising: (a) incorporating at least one nucleotide of the present disclosure into a polynucleotide, and (b) detecting the nucleotides incorporated into the polynucleotide by detecting the fluorescent signal of a dye compound connected to the nucleotide.

[0201] The method may comprise: a synthesis step (a) of incorporating one or more nucleotides according to the present invention into a polynucleotide; and a detection step (b) of detecting the one or more nucleotides incorporated into the polynucleotide by detecting or quantitatively measuring the fluorescence of the one or more nucleotides incorporated into the polynucleotide.

[0202] Some embodiments of the present application relate to sequencing methods, comprising: (a) incorporating at least one labeled nucleotide described herein into a polynucleotide; and (b) detecting the labeled nucleotide incorporated into the polynucleotide by detecting a fluorescent signal of a new fluorescent dye attached to the nucleotide.

[0203] Some embodiments of the present disclosure relate to a method of determining a target single-stranded polynucleotide sequence, comprising:

[0204] (a) incorporating a nucleotide comprising a 3'-OH blocking group and a detectable label as described herein into a replicating polynucleotide chain that is complementary to at least a portion of a target polynucleotide chain;

[0205] (b) detecting the identity of the nucleotide incorporated into the replicated polynucleotide chain; and

[0206] (c) Chemical removal of the label and 3' blocking group from the nucleotides incorporated into the replicated polynucleotide chain.

[0207] In some embodiments, the sequencing method further comprises (d) washing away the chemically removed mark and 3' blocking group from the replicated polynucleotide chain. In some such embodiments, before introducing the next complementary nucleotide, the 3' blocking group and the detectable label are first removed. In some further embodiments, the 3' blocking group and the detectable label are removed in a single chemical reaction step. In some embodiments, the washing step (d) also removes unincorporated nucleotides. In some further embodiments, a palladium scavenger is also used in the washing step after the mark and 3' blocking group chemical rupture.

[0208] In some embodiments, steps (a) to (d) are repeated until the sequence of the portion of the template polynucleotide chain is determined. In some such embodiments, steps (a) to (d) are repeated at least 50 times, at least 75 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, or at least 300 times.

[0209] In some embodiments, the label and the 3' blocking group are removed in two separate chemical reactions. In some such embodiments, removing the label from the nucleotides incorporated into the replicated polynucleotide chain comprises contacting the replicated chain including the incorporated nucleotides with a first fragmentation solution. In some such embodiments, the first fragmentation solution comprises a phosphine, such as a trialkylphosphine. Non-limiting examples of trialkylphosphines include tris(hydroxypropyl)phosphine (THP), tris(2-carboxyethyl)phosphine (TCEP), tris(hydroxymethyl)phosphine (THMP) or tris(hydroxyethyl)phosphine (THEP). In one embodiment, the first fragmentation solution contains THP. In some such embodiments, removing the 3' blocking group from the nucleotides incorporated into the replicated polynucleotide chain comprises contacting the replicated chain including the incorporated nucleotides with a second fragmentation solution. In some such embodiments, the second fragmentation solution comprises a palladium (Pd) catalyst. In some further embodiments, the Pd catalyst is a Pd(0) catalyst. In some such embodiments, the Pd(0) is prepared in situ by mixing a Pd(II) complex [(PdCl(C3H5))2] with THP. The molar ratio of the Pd(II) complex to THP is approximately 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In one embodiment, the molar ratio of Pd:THP is 1:5. 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 second cleavage solution may include one or more buffering agents, such as primary amines, secondary amines, tertiary amines, carbonates, phosphates, or borates, or combinations thereof. In some further embodiments, the buffering agent includes ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, sodium carbonate, sodium phosphate, sodium borate, 2-dimethylaminoethanol (DMEA), 2-diethylaminoethanol (DEEA), N,N,N′,N′-tetramethylethylenediamine (TEMED), or N,N,N′,N′-tetraethylethylenediamine (TEEDA), or combinations thereof. In one embodiment, the buffering agent is DEEA. In another embodiment, the buffer comprises one or more inorganic salts, such as carbonates, phosphates, or borates, or a combination thereof. In one embodiment, the inorganic salt is a sodium salt. In some other embodiments, the second fracture solution contains NaIO4 or In some further embodiments, the 3' blocked nucleotide contains an AOM group, and the second fragmentation solution contains a palladium (Pd) catalyst and one or more buffers described herein (eg, a tertiary amine such as DEEA) and has a pH of about 9.0 to 10.0 (eg, 9.6 or 9.8).

[0210] In some alternative embodiments, the label and the 3'-OH blocking group are removed in a single chemical reaction. In some such embodiments, the label is attached to the nucleotide via a cleavable linker that comprises the same moiety as the 3' blocking group, for example, both the linker and the 3' blocking group may comprise an acetal moiety as described herein. or thiocarbamate moiety In some such embodiments, the single chemical reaction is performed in a cleavage solution containing the Pd catalyst described above.

[0211] In some further embodiments, the nucleotides used in the incorporation step (a) are fully functionalized A, C, T, and G nucleoside triphosphates, each of which comprises a 3' blocking group as described herein. In some such embodiments, the nucleotides herein provide superior stability in solution during sequencing compared to the same nucleotides protected with a standard 3'-O-azidomethyl blocking group. For example, compared to an azidomethyl protected 3'-OH under the same conditions for the same period of time, the acetal or thiocarbamate blocking groups disclosed herein can impart at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 2500% or 3000% improved stability, thereby reducing the pre-phase value and obtaining a longer sequencing read length. In some embodiments, the stability is measured at ambient temperature or a temperature below ambient temperature (e.g., 4-10°C). In other embodiments, the stability is measured at an elevated temperature, such as 40°C, 45°C, 50°C, 55°C, 60°C or 65°C. In some such embodiments, the stability is measured in a solution at an alkaline pH environment, for example, at pH 9.0, 9.2, 9.4, 9.6, 9.8, or 10.0. In some further embodiments, the predetermined phase value of the nucleotides having a 3' block as described herein is less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles of SBS. In some further embodiments, the phasing value of the nucleotide of described 3 ' sealing is less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06 or 0.05 after 50, 100 or 150 SBS cycles.In one embodiment, each ffN comprises the 3 '-AOM group.

[0212] In some embodiments, the 3'-blocked nucleotides described herein provide superior deblocking rates in solution during the chemical fragmentation step of a sequencing run compared to the same nucleotides protected with a standard 3'-O-azidomethyl blocking group. For example, an acetal (e.g., AOM) or thiocarbamate blocking group disclosed herein can impart an improved deblocking rate of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, or 2000% compared to an azidomethyl-protected 3'-OH using a standard deblocking reagent (e.g., tris(hydroxypropyl)phosphine), thereby reducing the total time for sequencing cycles. In some embodiments, the unblocking time of each nucleotide is reduced by about 5%, 10%, 20%, 30%, 40%, 50% or 60%. For example, under certain chemical reaction conditions, the unblocking time of 3'-AOM and 3'-O-azidomethyl is about 4-5 seconds and about 9-10 seconds, respectively. In some embodiments, the half-life (t 1 / 2 ) is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times faster than the azidomethyl blocking group. In some such embodiments, the t of AOM is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times faster than the azidomethyl blocking group. 1 / 2 The t of azidomethyl is about 1 minute. 1 / 2 About 11 minutes. In some embodiments, the unblocking rate is measured at ambient temperature or below ambient temperature (e.g., 4-10°C). In other embodiments, the unblocking rate is measured at an elevated temperature, such as 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. In some such embodiments, the unblocking rate is measured in a solution in an alkaline pH environment, for example, at pH 9.0, 9.2, 9.4, 9.6, 9.8, or 10.0. In some such embodiments, the molar ratio of the unblocking reagent to the substrate (i.e., 3'-protected nucleoside or nucleotide) is about 10:1, about 5:1, about 2:1, about 1:1, about 1:2, about 1:5, or about 1:10. In one embodiment, each ffN comprises the 3'-AOM group.

[0213] In any of the embodiments of the methods described herein, the labeled nucleotides are nucleoside triphosphates.In any of the embodiments of the methods described herein, the target polynucleotide chain is attached to a solid support, such as a flow cell.

[0214] In one embodiment, during the synthesis step, at least one nucleotide is incorporated into the polynucleotide by the action of a polymerase. In some such embodiments, the polymerase can be DNA polymerase Pol 812 or Pol 1901. However, other methods of linking nucleotides to polynucleotides can be used, such as chemical oligonucleotide synthesis or ligation of labeled oligonucleotides to unlabeled oligonucleotides. Thus, when referring to nucleotides and polynucleotides, the term "incorporation" can encompass polynucleotide synthesis by chemical methods as well as enzymatic methods.

[0215] In a specific embodiment, the synthesis step is performed and optionally comprises incubating the template polynucleotide chain with a reaction mixture comprising labeled 3'-blocked nucleotides of the present disclosure. The polymerase can also be provided under conditions that allow for the formation of phosphodiester linkages between free 3'-OH groups on the polynucleotide chain annealed to the template polynucleotide chain and 5' phosphate groups on the nucleotides. Thus, the synthesis step can comprise the formation of the polynucleotide chain guided by complementary base pairing of the nucleotides with the template chain.

[0216] In all embodiments of the method, the detection step can be performed while the polynucleotide chain incorporating the nucleotides of the label is annealed to the template chain, or after a denaturation step in which the two chains are separated. Other steps, such as chemical or enzymatic reaction steps or purification steps, may be included between the synthesis step and the detection step. In particular, the target chain incorporating the nucleotides of the label can be separated or purified and then further processed or used in subsequent analysis. For example, the target polynucleotide labeled with the nucleotides described herein in the synthesis step can be subsequently used as a labeled probe or primer. In other embodiments, the products of the synthesis steps described herein can be subjected to further reaction steps, and if desired, the products of these subsequent steps can be purified or separated.

[0217] Those familiar with standard molecular biology techniques will be familiar with suitable conditions for the synthesis step. In one embodiment, the synthesis step can be similar to a standard primer extension reaction using nucleotide precursors (including nucleotides described herein) to form an extended target strand complementary to the template strand in the presence of a suitable polymerase. In other embodiments, the synthesis step itself can constitute part of the amplification reaction, thereby producing a labeled double-stranded amplification product consisting of annealed complementary strands derived from the replication of the target and template polynucleotide strands. Other exemplary synthesis steps include nick translation, strand displacement polymerization, random primed DNA labeling, and the like. A particularly useful polymerase for the synthesis step is an enzyme that can catalyze the incorporation of nucleotides described herein. A variety of naturally occurring or modified polymerases can be used. For example, a thermostable polymerase can be used for a synthesis reaction performed using thermal cycling conditions, while a thermostable polymerase may not be required for an isothermal primer extension reaction. Suitable thermostable polymerases capable of incorporating nucleotides according to the present disclosure include those described in WO 2005 / 024010 or WO 06 / 120433, each of which is incorporated herein by reference. In synthesis reactions carried out at lower temperatures (e.g., 37° C.), the polymerase does not necessarily need to be a thermostable polymerase, and thus the choice of polymerase depends on many factors, such as reaction temperature, pH, strand displacement activity, etc.

[0218] In specific non-limiting embodiments, the present disclosure encompasses methods for nucleic acid sequencing, resequencing, whole genome sequencing, single nucleotide polymorphism scoring, and any other application involving detection of the labeled nucleotides or nucleosides described herein when incorporated into polynucleotides. Any of a variety of other applications that facilitate the use of polynucleotides labeled with nucleotides comprising fluorescent dyes can use the dye-labeled nucleotides or nucleosides described herein.

[0219] In a specific embodiment, the present disclosure provides the purposes of the nucleotide of labeling according to the present disclosure in polynucleotide sequencing by synthesis (SBS) reaction. Sequencing by synthesis generally involves using a polymerase or a ligase to sequentially add one or more nucleotides or oligonucleotides to the growing polynucleotide chain in the 5' to 3' direction to form an extended polynucleotide chain complementary to the template nucleic acid to be sequenced. The identity of the base present in one or more added nucleotides can be determined in a detection or "imaging" step. The identity of the base added can be determined after each nucleotide incorporation step. Conventional Watson-Crick base pairing rules can then be used to infer the sequence of the template. For example, when single nucleotide polymorphisms are scored, it may be useful to use the nucleotide of labeling as herein described to determine the identity of a single base, and this single base extension reaction is within the scope of the present disclosure.

[0220] In one embodiment of the present disclosure, the sequence of a template polynucleotide is determined by detecting the incorporation of one or more 3'-blocked nucleotides described herein into a nascent chain complementary to the template polynucleotide to be sequenced, the incorporation of said nucleotide being performed by detecting a fluorescent label attached to the incorporated nucleotide. The sequencing of the template polynucleotide can be initiated with a suitable primer (or prepared as a hairpin construct, which will include a primer as part of the hairpin), and the nascent chain is extended in a stepwise manner by adding nucleotides to the 3' end of the primer in a polymerase-catalyzed reaction.

[0221] In a specific embodiment, each different nucleoside triphosphate (A, T, G and C) can be marked by fluorophores that are different from each other, and also a blocking group is included in the 3' position to prevent uncontrolled polymerization. Optionally, one of four kinds of nucleotides may be unlabeled (dark). Described polymerase incorporates nucleotide into the nascent chain complementary to the template polynucleotide, and described blocking group stops the further incorporation of nucleotide. Any unbound nucleotide can be washed off, and can be optically " read " from the fluorescent signal of each nucleotide incorporated by suitable means (such as charge coupled device, it uses laser excitation and suitable emission filter). Then can simultaneously or sequentially remove (deprotection) 3'-blocking group and fluorescent dye compound, to expose nascent chain to further incorporate nucleotide. Usually, the identity of the nucleotide incorporated will be determined after each incorporation step, but this is not strictly necessary. Similarly, United States Patent (USP) 5,302,509 (it is incorporated herein by reference) discloses the method that the polynucleotide being fixed on solid support is ordered sequence.

[0222] As described above, the method utilizes the incorporation of fluorescently labeled 3'-blocked nucleotides A, G, C, and T into a growing chain complementary to a fixed polynucleotide in the presence of a DNA polymerase. The polymerase incorporates bases complementary to the target polynucleotide, but is prevented from further addition by the 3'-blocking group. The label of the incorporated nucleotide can then be determined, and the blocking group can be removed by chemical cleavage to allow further polymerization to occur. The nucleic acid template to be sequenced in the sequencing-by-synthesis reaction can be any polynucleotide desired to be sequenced. The nucleic acid template used for the sequencing reaction will typically contain a double-stranded region with a free 3'-OH group, which serves as a primer or as a starting point for further addition of other nucleotides in the sequencing reaction. The region of the template to be sequenced will overhang the free 3'-OH group on the complementary chain. The overhang region of the template to be sequenced can be single-stranded or double-stranded, provided that there is a "gap" on the chain complementary to the template chain to be sequenced to provide a free 3'-OH group for the initiation of the sequencing reaction. In such an embodiment, sequencing can be performed by strand displacement. In some embodiments, a primer with a free 3'-OH group can be added as a separate component (e.g., short oligonucleotide) hybridized with the single-stranded region of the template to be sequenced. Optionally, the primer and the template chain to be sequenced can each form a part for a partial self-complementary nucleic acid chain that can form an intramolecular duplex (e.g., hairpin loop structure). Hairpin polynucleotides and the method thereof that can be connected with a solid phase carrier are disclosed in PCT Publication WO 01 / 57248 and WO 2005 / 047301, which are each incorporated herein by reference. Nucleotide can be continuously added to the growing primer, thereby causing the synthesis of the polynucleotide chain in the 5' to 3' direction. The property of the base added can be determined (particularly but not necessarily, determining after each addition of nucleotide) to provide the sequence information of the nucleic acid template. Therefore, via forming a phosphodiester bond with the 5' phosphate group of the nucleotide, the nucleotide is combined with the free 3'-OH group of the nucleic acid chain, thereby nucleotide is incorporated into the nucleic acid chain (or polynucleotide).

[0223] The nucleic acid template to be sequenced can be DNA or RNA, or even a hybrid molecule composed of deoxynucleotides and ribonucleotides. The nucleic acid template can contain naturally occurring and / or non-naturally occurring nucleotides and natural or non-natural backbone linkages, provided that they do not prevent the replication of the template in the sequencing reaction.

[0224] In some embodiments, the nucleic acid template to be sequenced can be connected to a solid support via any suitable connection method known in the art, such as by covalent attachment. In some embodiments, the template polynucleotide can be directly attached to a solid support (e.g., a silica-based support). However, in other embodiments of the present disclosure, the surface of the solid support can be modified in some manner to allow direct covalent attachment of the template polynucleotide, or the template polynucleotide can be fixed by a hydrogel or polyelectrolyte multilayer, which itself can be non-covalently attached to a solid support.

[0225] Sequencing by Synthesis Implementation and Alternatives

[0226] Some embodiments include pyrosequencing technology. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) when a specific nucleotide is incorporated into a nascent chain (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. Patents 6,210,891; 6,258,568 and 6,274,320, the disclosures of which are incorporated herein by reference in their entireties). In pyrophosphate sequencing, the released PPi can be detected by being immediately converted into adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP generated is detected via the photons generated by luciferase. The nucleic acid to be sequenced can be connected to the features in the array, and the array can be imaged to capture the chemiluminescent signals generated due to the incorporation of nucleotides on the features of the array. After treating the array with a specific nucleotide type (e.g., A, T, C or G), an image can be obtained. The image obtained after adding each nucleotide type will be different depending on the features detected in the array. These differences in the image reflect the different sequence contents of the features on the array. However, the relative position of each feature will remain unchanged in the image. The method described herein can be used to store, process and analyze images. For example, the image obtained after treating the array with each different nucleotide type can be processed in the same manner as the reversible terminator-based sequencing method exemplified herein to obtain images from different detection channels.

[0227] In another exemplary SBS type, cycle sequencing is accomplished by gradually adding reversible terminator nucleotides, and the reversible terminator nucleotides include, for example, breakable or photobleachable dye labels, such as described in WO 04 / 018497 and U.S. Patent No. 7,057,026, which are incorporated herein by reference. The method is 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 (both terminators can be reversed) and the fluorescent labeling of the break contribute to effective cyclic reversible termination (CRT) sequencing. Polymerases can also be co-engineered to effectively incorporate these modified nucleotides and extend from these modified nucleotides.

[0228] Preferably, in reversible terminator-based sequencing embodiments, the label does not substantially inhibit extension under SBS reaction conditions. However, the detection label can be removed by fragmentation or degradation. After the label is incorporated into the nucleic acid features of the array, an image can be captured. In a specific embodiment, each cycle involves the simultaneous delivery of four different nucleotide types to the array, with each nucleotide type having a spectrally distinct label. Four images can then be acquired, each using a detection channel selective for one of the four different labels. Alternatively, different nucleotide types can be added sequentially, and images of the array can be acquired between each addition step. In such an embodiment, each image will display the nucleic acid features incorporating a specific nucleotide type. Due to the different sequence content of each feature, different features will be present or absent in different images. However, the relative positions of the features will remain unchanged in the images. As described herein, images obtained from this reversible terminator-SBS method can be stored, processed, and analyzed. After the image capture step, the label can be removed, and the reversible terminator portion can be removed for subsequent nucleotide addition and detection cycles. Detection of the removal of the label in a specific cycle and before subsequent cycles can provide the advantage of reducing background signal and crosstalk between cycles. Examples of useful marking and removal methods follow.

[0229] Some embodiments can use less than four different marks to utilize the detection of four different nucleotides.For example, SBS can utilize the method and system described in the incorporation material of U.S. Patent Publication 2013 / 0079232 to perform.As a first example, a pair of nucleotide types can be detected under the same wavelength, but based on the intensity difference of a member relative to another member in the pair, or based on the change (such as via chemical modification, photochemical modification or physical modification) of a member in the pair to distinguish, the change causes the obvious signal of the signal detected by other members compared to the pair to appear or disappear.As a second example, three of the four different nucleotide types can be detected under specific conditions, and the fourth nucleotide type lacks the mark detectable under those conditions, or is detected to a minimum (such as, due to the minimum detection caused by background fluorescence, etc.) under those conditions. The incorporation of the first three nucleotide types into nucleic acid can be determined based on the existence of their respective signals, and the incorporation of the fourth nucleotide type into nucleic acid can be determined according to the absence or minimum detection of any signal.As a third example, a nucleotide type can include the mark detected in two different channels, and other nucleotide types are detected in no more than one channel. The three exemplary configurations described above are not considered mutually exclusive and can be used in various combinations. An exemplary embodiment combining all three examples is a fluorescence-based SBS method that uses a first nucleotide type detected in a first channel (e.g., dATP with a label detected in the first channel when excited by a first excitation wavelength), a second nucleotide type detected in a second channel (e.g., dCTP with a label detected in the second channel when excited by a second excitation wavelength), a third nucleotide type detected in both the first and second channels (e.g., dTTP with at least one label detected in both channels when excited by the first and / or second excitation wavelengths), and a fourth nucleotide type (e.g., dGTP without a label) that lacks a label (is not detected or is minimally detected in either channel).

[0230] Furthermore, as described in the incorporated materials in U.S. Patent Publication 2013 / 0079232, sequencing data can be obtained using a single channel. In this so-called single-dye sequencing approach, 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 the first image is generated. The third nucleotide type retains its label in both the first and second images, while the fourth nucleotide type is unlabeled in both images.

[0231] Some embodiments can utilize the sequencing by linking (ligation) technology. Such technology utilizes DNA ligase to incorporate oligonucleotides and identify the incorporation of these oligonucleotides. The oligonucleotides generally have different marks related to the identity of the specific nucleotides in the sequence of oligonucleotide hybridization. Like other SBS methods, after the nucleic acid features of an array are processed with a labeled sequencing reagent, an image can be obtained. Each image will show the nucleic acid features that have been combined with a specific type of mark. Because the sequence content of each feature is different, different features will exist or not in different images, but the relative position of the feature will remain unchanged in the image. As described herein, the image obtained from the sequencing method based on the link can be stored, processed and analyzed. The exemplary SBS system and method that can be used together with the method and system described herein are discussed in U.S. Patents 6,969,488, 6,172,218 and 6,306,597, and its disclosure is incorporated herein by reference in its entirety.

[0232] Some embodiments can utilize nanopore sequencing (Deamer, DW & 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 a biological membrane protein, such as α-hemolysin. As the target nucleic acid passes through the nanopore, each base pair can be identified by measuring the fluctuations in the pore's electrical conductivity. (U.S. Patent 7,001,792; Soni, GV & 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, SL, Chu, J., Amorin, M. & Ghadiri, MR. "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). As described herein, data obtained from nanopore sequencing can be stored, processed, and analyzed. In particular, the data may be processed as images according to the exemplary processing of optical and other images set forth herein.

[0233] Some other embodiments of the sequencing method relate to the use of 3' unblocked nucleotides described herein in nanoball sequencing technology, such as those described in U.S. Patent No. 9,222,132, the disclosure of which is incorporated herein by reference. By rolling circle amplification (RCA) method, a large number of discrete DNA nanoballs may be produced. The nanoball mixture is then distributed onto a patterned sliding surface containing features that allow a single nanoball to be associated with each position. During the production of DNA nanoballs, DNA is fragmented and linked to the first of four adapter sequences. The template is amplified, circularized and cut with a type II endonuclease. A second set of adapters is added, followed by amplification, circularization and cutting. The process is repeated for the remaining two adapters. The final product is a circular template with four adapters, each of which is separated by a template sequence. The library molecules undergo a rolling circle amplification step to generate a large number of concatemers called DNA nanoballs, which are then deposited in a flow cell. Goodwin et al., “Coming of age: ten years of next-generation sequencing technologies,” Nat Rev Genet. 2016;17(6):333-51.

[0234] Some embodiments can utilize methods involving real-time monitoring of DNA polymerase activity. Incorporation of nucleotides can be detected by fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and a gamma-phosphate-labeled nucleotide, such as described in U.S. Patent No. 5,426,038, e.g., U.S. Patent Nos. 7,329,492 and 7,211,414, the entire contents of which are incorporated herein by reference, or incorporation of nucleotides can be detected using zero-mode waveguides, such as described in U.S. Patent No. 7,315,019, which is incorporated herein by reference, and using fluorescent nucleotide analogs and engineered polymerases, such as described in U.S. Patent No. 7,405,281 and U.S. Patent No. 2008 / 0108082, which are incorporated herein by reference. Illumination can be confined to a zeptoliter-scale volume around the surface-bound polymerase, allowing observation of the incorporation of fluorescently labeled nucleotides with low background (Levene, MJ et al., "Zero-mode waveguides for single-molecule analysis at high concentrations." Science 299, 682-686 (2003); Lundquist, PM 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 nanostructures." Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference in their entireties). Images obtained from this method can be stored, processed, and analyzed as described herein.

[0235] Some SBS embodiments include detecting protons released after nucleotides are incorporated into extension products. For example, sequencing based on detecting released protons can use electrical detectors and related technologies, which are commercially available from Ion Torrent (Guilford, CT, a subsidiary of Life Technologies), or can use the sequencing methods and systems described in U.S. Patent Publication Nos. 2009 / 0026082; 2009 / 0127589; 2010 / 0137143; and 2010 / 0282617, the entire contents of which are incorporated herein by reference. The method of using kinetic repulsion to amplify target nucleic acids, as described herein, can be easily applied to substrates for detecting protons. More specifically, the method described herein can be used to generate clonal populations of amplicons for detecting protons.

[0236] Above-mentioned SBS method can advantageously be carried out with multiple forms, makes to operate a plurality of different target nucleic acids simultaneously.In specific embodiment, can in common reaction vessel or on the surface of specific substrate, handle different target nucleic acids.This makes it possible to easily deliver sequencing reagent, remove unreacted reagent and detect incorporation event in multiple ways.In the embodiment using the target nucleic acid of surface binding, described target nucleic acid can be in array form.In array form, described target nucleic acid can be attached to surface in a spatially distinguishable manner usually.Described target nucleic acid can be by direct covalent attachment, be connected to pearl (bead) or other particles or be attached to polymerase or be connected to other molecules on surface and combine.Described array can comprise single copy of target nucleic acid at each site (also referred to as feature) or can have multiple copies with identical sequence at each site or feature.Can produce multiple copies by amplification method such as bridge amplification or emulsion PCR, as described in further detail below.

[0237] The methods described herein can be used with arrays having various densities of features, for example, at least about 10 features / cm 2 , 100 features / cm 2 , 500 features / cm 2 , 1,000 features / cm 2 , 5,000 features / cm 2 , 10,000 features / cm 2 , 50,000 features / cm 2 , 100,000 features / cm 2 , 1,000,000 features / cm 2 5,000,000 features / cm 2 or higher.

[0238] The advantage of the methods described herein is that they provide rapid and efficient parallel detection of multiple target nucleic acids. Therefore, the present disclosure provides an integrated system capable of preparing and detecting nucleic acids using techniques known in the art, such as the techniques illustrated above. Therefore, the integrated system of the present disclosure may include a fluid assembly capable of delivering amplification reagents and / or sequencing reagents to one or more fixed DNA fragments, the system including components such as pumps, valves, containers, fluid lines, etc. A flow cell can be configured and / or used to detect target nucleic acids in the integrated system. Exemplary flow cells are described in, for example, U.S. Patent Publication Nos. 2010 / 0111768 and 13 / 273,666, each of which is incorporated herein by reference. As illustrated for the flow cell, one or more fluid assemblies of the integrated system can be used for amplification methods and detection methods. Taking the nucleic acid sequencing embodiment as an example, one or more fluid assemblies of the integrated system can be used for the amplification methods described herein and for the delivery of sequencing reagents used in sequencing methods, such as those illustrated above. Alternatively, the integrated system can include separate fluid systems to perform the amplification method and the detection method. Examples of integrated sequencing systems that can produce amplified nucleic acids and also determine nucleic acid sequences include, but are not limited to, MiSeq TM The platform (Illumina, Inc., San Diego, CA) and the devices described in US Patent No. 13 / 273,666, which is incorporated herein by reference.

[0239] The array that wherein polynucleotide has been directly connected on the carrier based on silica is for example disclosed those arrays in WO 00 / 06770 (incorporating this paper into by reference), and wherein polynucleotide is fixed on the glass carrier by the reaction of the side chain epoxide group on glass and the internal amino on polynucleotide.In addition, polynucleotide can be connected to solid phase support by the reaction based on the nucleophilic reagent of sulphur and solid phase support, for example, as described in WO 2005 / 047301 (incorporating this paper into by reference).The further example of the template polynucleotide of solid phase load is that wherein template polynucleotide is connected to be loaded on the hydrogel on solid phase support based on silica or other solid phase support, for example, WO 00 / 31148, WO 01 / 01143, WO02 / 12566, WO 03 / 014392, United States Patent (USP) 6,465, described in 178 and WO 00 / 53812, it is incorporated into this paper into by reference separately.

[0240] The specific surface on which the template polynucleotide can be fixed is a polyacrylamide hydrogel. Polyacrylamide hydrogels are described in the references cited above and in WO 2005 / 065814, which are incorporated herein by reference. Specific hydrogels that can be used are included in WO 2005 / 065814 and U.S. Patent Publication 2014 / 0079923. In one embodiment, the hydrogel is PAZAM (poly-N-(5-azidoacetamidopentyl) acrylamide-co-acrylamide).

[0241] The DNA template molecules can be attached to beads or microparticles, for example, as described in U.S. Patent No. 6,172,218 (incorporated herein by reference). Attachment to beads or microparticles can be used for sequencing applications. Bead libraries can be prepared, wherein each bead contains a different DNA sequence. Exemplary libraries and methods for creating them are described in Nature, 437, 376-380 (2005); Science, 309, 5741, 1728-1732 (2005), each of which is incorporated herein by reference. Sequencing arrays of such beads using the nucleotides described herein is within the scope of the present disclosure.

[0242] The template to be sequenced can form part of an "array" on a solid support, in which case the array can take any convenient form. Thus, the methods of the present disclosure are applicable to all types of high-density arrays, including single-molecule arrays, cluster arrays, and bead arrays. The labeled nucleotides of the present disclosure can be used for sequencing templates on essentially any type of array, including but not limited to those formed by immobilizing nucleic acid molecules on a solid support.

[0243] In some embodiments, the nucleotide sequence of the present invention is preferably amplified by a plurality of nucleic acid molecules. However, the nucleotide sequence of the present invention is particularly advantageous in the case of the order-checking of cluster arrays. In cluster arrays, the different regions on the array (commonly referred to as sites or features) comprise a plurality of polynucleotide template molecules. Generally, the plurality of polynucleotide molecules can not be distinguished separately by optical means, but are detected as a whole. Depending on the formation mode of the array, each site on the array can comprise a plurality of copies of an independent polynucleotide molecule (for example, this site is homologous for specific single-stranded or double-stranded nucleic acid species) or even a plurality of copies of a small amount of different polynucleotide molecules (for example, multiple copies of two different nucleic acid species). The cluster arrays of nucleic acid molecules generally known in the art can be produced using technology. For example, WO 98 / 44151 and WO 00 / 18957 (each of which is incorporated herein) describe the amplification method of nucleic acid, wherein template and amplification product all keep fixed on a solid support to form the array consisting of the cluster or " cluster " of fixed nucleic acid molecules. The nucleic acid molecules present on the cluster arrays prepared according to these methods are suitable templates for using the nucleotides labeled by the dye compounds of the present invention to carry out order-checking.

[0244] The labeled nucleotides disclosed herein can also be used for sequencing templates on single molecule arrays. As used herein, the term "single molecule array" or "SMA" refers to a population of polynucleotide molecules distributed (or arranged) on a solid support, wherein any single polynucleotide is spaced apart from all other molecules of the population at a distance that allows the resolution of a single polynucleotide molecule. Thus, in some embodiments, the target nucleic acid molecules immobilized on the surface of the solid support can be resolved optically. This means that one or more different signals, each representing a polynucleotide, will occur within the resolvable area of ​​the specific imaging device being used.

[0245] Single molecule detection can be achieved where the spacing between adjacent polynucleotide molecules on the array is at least 100 nm, more particularly at least 250 nm, even more particularly at least 300 nm, and even more particularly at least 350 nm. Thus, each molecule can be individually resolved and detected as a single molecule fluorescent spot, and the fluorescence of the single molecule fluorescent spot also exhibits single-step photobleaching.

[0246] The terms "individual resolution" and "individual resolution" are used herein to specify that, when visualized, a molecule on an array can be distinguished from its adjacent molecules. The separation between individual molecules on the array will be determined, in part, by the specific techniques used to resolve the individual molecules. The general characteristics of single molecule arrays will be understood by reference to disclosed applications WO 00 / 06770 and WO 01 / 57248, each of which is incorporated herein by reference. Although one use of the nucleotides disclosed herein is in sequencing-by-synthesis reactions, the use of the nucleotides is not limited to such methods. In fact, the nucleotides can be advantageously used in any sequencing method requiring detection of fluorescent labels attached to the nucleotides incorporated into the polynucleotides.

[0247] In particular, the labeled nucleotides disclosed herein can be used in automated fluorescent sequencing protocols, particularly fluorescent dye-terminator cycle sequencing based on the chain termination sequencing method of Sanger and colleagues. Such methods typically incorporate fluorescently labeled dideoxynucleotides into primer extension sequencing reactions using enzymes and cycle sequencing. The so-called Sanger sequencing method and related protocols (Sanger-type) utilize random chain termination with labeled dideoxynucleotides.

[0248] Thus, the present disclosure also encompasses labeled nucleotides that are dideoxynucleotides lacking a hydroxyl group at both the 3' and 2' positions, such dideoxynucleotides being suitable for use in Sanger-type sequencing methods, among other things.

[0249] It will be appreciated that the labeled nucleotides of the present disclosure incorporating 3'-blocking groups can also be used in Sanger methods and related protocols, since the same effect obtained by using dideoxynucleotides can be achieved by using nucleotides having 3'-OH blocking groups: both prevent the incorporation of subsequent nucleotides. When using nucleotides according to the present invention having 3' blocking groups in Sanger-type sequencing methods, it will be appreciated that the dye compound or detectable label attached to the nucleotide does not need to be attached via a cleavable linker, since each instance of incorporating a labeled nucleotide of the present disclosure does not require subsequent incorporation of a nucleotide, and thus does not require removal of the label from the nucleotide.

[0250] In any embodiment of the methods described herein, the nucleotide used in the sequencing application is a 3' blocked nucleotide described herein, for example, a nucleotide of formula (I), (Ia) or (II). In any embodiment, the 3' blocked nucleotide is a nucleoside triphosphate. Example

[0251] Additional embodiments are disclosed in further detail in the following examples, which are in no way intended to limit the scope of the claims.

[0252] Example 1. Preparation of 3'-acetal-blocked nucleosides

[0253] In this example, various 3'-acetal protected T nucleosides were prepared according to Scheme 2.

[0254]

[0255] Option 2

[0256]

[0257] Preparation of T1: 5-iodo-2'-deoxyuridine (5.0 g, 14.12 mmol) was added to a 100 mL flask purged with oven-dried nitrogen. It was co-evaporated three times with 30 mL of pyridine and then placed under nitrogen. Anhydrous pyridine (25 mL) was added and the reaction was stirred at room temperature until a homogeneous solution was obtained (~15 minutes). The mixture was cooled to 0°C in an ice-water bath and tert-butyldiphenylsilyl chloride (4.04 mL, 15.5 mmol) was slowly added dropwise with vigorous stirring (~1 hour). The reaction was kept at 0°C for 8 hours until all the starting materials were consumed (TLC). Saturated aqueous ammonium chloride solution (~15 mL) was added and the reaction was allowed to warm to room temperature. The mixture was diluted with ethyl acetate (100 mL) and washed with saturated aqueous ammonium chloride solution (200 mL). The organic layer was separated and the aqueous layer was extracted with ethyl acetate (4x50 mL). The organic layers were combined, dried (MgSO4), and concentrated in vacuo. Residual solvent was removed under high vacuum to yield ~8 g of a clear yellow oil. The crude product, T1, was purified by silica gel flash column chromatography to give a white crystalline solid. The yield was 6.94 g (83%). LC-MS (electrospray negative ionization) 591.08 [MH].

[0258] Preparation of T2: T1 (6.23 g, 10.5 mmol), copper(I) iodide (200 mg, 1.05 mmol), and bis(triphenylphosphine)palladium(II) dichloride (369 mg, 0.526 mmol) were placed in an oven-dried, nitrogen-purged, brown 500 mL three-necked flask under nitrogen. The flask was protected from light and anhydrous, degassed DMF (200 mL) was added. To this solution was added 2,2,2-trifluoro-N-prop-2-ynylacetamide (4.74 g, 31.6 mmol) followed by degassed triethylamine (2.92 mL, 21.0 mmol). When no more starting material was observed by TLC analysis, the reaction was stirred at room temperature under nitrogen for 6 hours. Volatiles were removed in vacuo (~15 minutes) and the DMF was removed under high vacuum (~1 hour) to give a brown residue. This was dissolved in ethyl acetate (200 mL) and extracted with 0.1 M EDTA aqueous solution (2x200 mL). The aqueous layers were combined and further extracted with ethyl acetate (200 mL). The organic phases were combined, dried (MgSO4) and volatiles removed in vacuo (~30 minutes) and further dried under high vacuum (~1 hour) to give approximately 8 g of a crude brown / yellow oil. The mixture was purified by flash column chromatography on silica gel to give an off-white solid. Yield: 6.0 g (85%). LC-MS (electrospray negative ionization) 614.19 [MH].

[0259] Preparation of T3: Under nitrogen, to a 100 mL flask dried in an oven-dried, nitrogen-purged container containing the starting nucleoside T2 (2.0 g, 3.25 mmol), anhydrous DMSO (6.9 mL, 97.5 mmol) was added in one portion at room temperature and stirred until a homogeneous solution formed. Acetic acid (11.1 mL, 195 mmol) and acetic anhydride (15.1 mL, 162.09 mmol) were added dropwise (approximately 5 minutes each). The mixture was warmed to 50°C and stirred until the starting nucleoside was completely consumed as determined by TLC (EtOAc / petroleum ether 3:2) (~5 hours). The reaction was then concentrated to half volume and cooled to approximately 0.5°C in an ice bath. Workup was initiated by slowly adding cold (~0.5°C) NaHCO3 (saturated aqueous solution) (45 mL), followed by further stirring until no further foaming was observed (~15 minutes). The solution was allowed to warm to room temperature, and the aqueous phase was then extracted into EtOAc (3 x 100 mL). The combined organic layers were dried over MgSO4, filtered, and the volatiles were evaporated under reduced pressure and further under high vacuum. The crude product T3 was purified by flash chromatography on silica gel as an off-white solid. Yield: 1.79 g (82%). LC-MS (electrospray negative ionization) 674.20 [MH] - .

[0260] Preparation of T4: To a solution of the starting nucleoside T3 (1.79 g, 2.649 mmol) in anhydrous CH2Cl2 (50 mL) under N2 was added cyclohexene (1.34 mL, 13.2 mmol). The mixture was cooled to 0°C with an ice bath and distilled sulfuryl chloride (322 μL, 3.97 mmol) was slowly added dropwise (approximately 20 minutes) under a nitrogen atmosphere. After stirring at this temperature for 20 minutes, TLC (EtOAc:petroleum ether = 3:2 v / v) indicated complete consumption of the starting nucleoside. The chloride intermediate was then quenched by the direct dropwise addition of freshly distilled corresponding unsaturated alcohol (5 equivalents) as shown in Scheme 3. The resulting solution was stirred at room temperature for 2 hours, after which the volatiles were evaporated under reduced pressure. The oily residue was partitioned between EtOAc:brine (3:2) (125 mL). The organic layer was separated and the aqueous layer was further extracted into EtOAc (2 x 50 mL). The combined organic extracts were dried over MgSO4, filtered, and the volatiles were evaporated under reduced pressure. The oily residue was partitioned between EtOAc:brine (3:2) (125 mL). The organic layer was separated, and the aqueous layer was further extracted into EtOAc (2 x 50 mL). The combined organic extracts were dried over MgSO4, filtered, and the volatiles were evaporated under reduced pressure. The crude product T4 was purified by flash chromatography on silica gel to give the final product as a yellow oil. Yield: AOM: 1.20 g (69%); PrOM: 1.29 g (71%); DPrOM: 1.34 g (71%).

[0261] 3'-AOM: yellow oil. LC-MS (electrospray negative ionization) [MH] 684.24.

[0262] 3'-PrOM: yellow oil. LC-MS (electrospray negative ionization) [MH] 682.22.

[0263] 3'-DPrOM: yellow oil. LC-MS (electrospray negative ionization) [MH] 710.25.

[0264]

[0265] Option 3.

[0266] Preparation of T5: At room temperature, anhydrous THF (9 mL) was added to the raw material T4 (1.04 g, 1.516 mmol) in a 50 mL round-bottom flask under nitrogen. TBAF (1.0 M in THF, 1.7 mL, 1.70 mmol) was then added dropwise and the solution was stirred until all the raw materials were consumed (TLC) (~ 2 hours). The solution turned orange during the reaction. Volatiles were removed in vacuo to obtain an orange residue, which was dissolved in EtOAc (100 mL) and separated with NaHCO (saturated aqueous solution) (60 mL). It was separated into two layers and the aqueous layer was extracted with EtOAc (60 mL). The organic layers were combined, dried (MgSO ), filtered and evaporated to obtain a crude product as a yellow oil. The crude product was purified by flash chromatography on silica gel to obtain a clear yellow oil. Yield: AOM: 637 mg (94%); PrOM: 526 mg (78%); DPrOM: 617 mg (86%).

[0267] 3'-AOM: clear yellow oil. LC-MS (electrospray negative ionization) [MH] 446.12.

[0268] 3'-PrOM: clear yellow oil (526 mg 78%). LC-MS (electrospray negative ionization): [MH] 444.10.

[0269] 3'-DPrOM: clear yellow oil (617 mg 86%). LC-MS (electrospray negative ionization): [MH] 472.13.

[0270] In addition, two other 3'-blocked T nucleosides (3'-eAOM T and 3'-iAOM T) were prepared in a similar manner as described above. 3'-iAOM T: LC-MS (ES): (negative ion) m / z 325.5 (MH + ), (positive ion) 327.3 (M+H +)。 3'-eAOM T: LC-MS (ES): (positive ion) m / z 341.3 (M+1H + ).

[0271]

[0272] Example 2. 3'-OH blocking group stability test

[0273] In this example, stability testing of 5'-mP 3'-AOM T nucleotides was performed in parallel with standard 5'-mP 3'-O-azidomethyl T nucleotides in spiking buffer solution.

[0274]

[0275] Preparation of buffer solution

[0276] 1 mL of 0.1 mM of each 5'-monophosphate 3' protected T nucleotide in 100 mL of ethanolamine buffer (pH 9.8), 100 mM NaCl and 2.5 mM EDTA solution was incubated for 2 weeks in a heat block at 65° C. At set time points, 40 μL aliquots were taken and analyzed by HPLC to determine the percentage of remaining blocked nucleotides and the final unblocked nucleotides formed.

[0277] The results of stability tests on 5'-monophosphate 3'-blocked nucleotides with AOM, PrOM, DPrOM acetal protecting groups and the standard azidomethyl blocking group are shown in Figure 1 3'-blocked nucleoside monophosphates with AOM, PrOM, and DPrOM blocking groups were observed to provide a 30-50-fold or greater improvement in reducing the deblocking rate in solution. This experiment simulated the performance of the corresponding fully functionalized nucleotides (ffN) when stored in an incorporation mixture in a sequencing instrument cartridge. The improved stability provided by these acetal protecting groups will also result in lower prephasing rates in sequencing runs. Finally, it improves the shelf life of the incorporation mixture.

[0278] Example 3. 3'-AOM deblocking assay

[0279] In this example, deblocking experiments for 5'-mP 3'-AOM T and standard 5'-mP 3'-O-azidomethyl T nucleotides were performed separately in unique solutions for each blocking group. Conditions were designed to closely mimic Illumina's standard deblocking reagents and followed the same methodology. The concentrations of active deblocking reagent, buffer, and nucleoside remained the same across all experiments, but each component was uniquely identified. Thus, observed differences in rates between the individual deblocking chemistries could not be attributed to differences in formulation concentrations.

[0280]

[0281] Standard azidomethyl deblocking conditions

[0282] Nucleotide: 5'-monophosphate 3'-O-azidomethyl T. Active deblocking reagent: tris(hydroxypropyl)phosphine (THP) (1 M in 18 mΩ water). (Optional) Additive: Sodium ascorbate (0.1 mM in 18 mΩ water). Final concentration = 1 mM. Buffer: Ethanolamine pH 9.8 (2 M in 18 mΩ water). Quencher: H2O2.

[0283] AOM unblocking conditions

[0284] Nucleotide: 5'-monophosphate 3'-O-azidomethyl T. In a glass vial under nitrogen, a stock solution of 3'-AOM T was diluted to 0.1 mM in 100 mM ethanolamine buffer (pH 9.8). A stock solution of sodium ascorbate additive was added to a final concentration of 0.1 mM and the solution was stirred for 5 minutes. To initiate the assay, a deblocking reagent (Pd / THP = 1 / 5; sodium ascorbate; ethanolamine) was added to the stirred solution at room temperature to a final concentration of 1 mM THP. At designated time points, a 40 μL aliquot was removed and quenched with 6 μL of a 1:3 mixture of EDTA / H2O2 (0.025:0.075 M). HPLC analysis was performed by measuring the areas of the starting nucleoside peak, the 3'-OH peak, and any other nucleotide peaks present in the HPLC chromatogram. No other nucleotide-based side products were observed.

[0285] The comparison results are shown in Figure 2AIt was observed that AOM provided a 10-fold rate improvement in the deblocking rate in solution compared to the standard azidomethyl blocking group. The purpose of this experiment was to simulate the performance of the corresponding ffN in sequencing during the deblocking step. The significant increase in deblocking speed will allow the use of a flush-through deblocking step instead of the 10 to 20 second incubation time commonly used by some Illumina sequencing platforms. Therefore, the deblocking rate will have a significant impact on sequencing by synthesis (SBS) cycle time.

[0286] Similar experimental conditions were used for the deblocking assays of 3'-eAOM T and 3'-iAOM T. As a separate change, the ratio of Pd catalyst to substrate was reduced to 5:1 in order to observe smaller differences in deblocking rates. 3'-AOM T was used as a reference and the results are presented in Figure 2B These results indicate that at this specific concentration of Pd catalyst deblocking reagent, the deblocking rates of eAOM and iAOM are 2-3 times slower than those of AOM. It is expected that the difference in deblocking rates between substituted and unsubstituted versions of the AOM blocking group will be smaller when the Pd catalyst to substrate ratio is higher.

[0287] Example 4. Optimization of palladium fragmentation mixture in sequencing

[0288] The Pd / THP catalyst used in the deblocking reaction described in Example 2 is very air sensitive. It exhibits a substantial loss of activity when exposed to air. In this example, an oxidation stress assay was developed to evaluate the air sensitivity of different formulations of palladium cleavage mixtures.

[0289] 0.5 mL of the Pd fragmentation mixture was aliquoted into 5 mL glass vials and exposed to air at room temperature for 3 hours. The residual activity of the oxidized fragmentation mixture was assessed by measuring the fragmentation of 3'-AOM T as follows. A stock solution of 3'-AOM T was diluted to 0.1 mM in 100 mM fragmentation mixture buffer. A stock solution of sodium ascorbate was added to a final concentration of 1 mM, and the oxidized fragmentation mixture was then diluted to a final concentration of 1 / 20. After 1 hour, 40 μL of the solution was immediately quenched with 10 μL of a 1:1 EDTA / H2O2 mixture (0.25:0.25 M) and analyzed by HPLC. In this experiment, various buffering agents were screened, including: primary amines (e.g., ethanolamine, Tris, and glycine); tertiary amines (e.g., 2-dimethylaminoethanol (DMEA), 2-diethylaminoethanol (DEEA), N,N,N′,N′-tetramethylethylenediamine (TEMED), or N,N,N′,N′-tetraethylethylenediamine (TEEDA)); and various inorganic salts (e.g., borates, carbonates, phosphates). It was observed that inorganic buffers (e.g., sodium borate, sodium carbonate, sodium phosphate) provided the best air stability, and the palladium complex retained a high % activity. In addition, tertiary amines also significantly improved the stability of the Pd fragmentation mixture compared to primary amines.

[0290] Based on these findings, two kinds of palladium fragmentation mixtures were prepared. In first example, the stock solution of 250mM borate buffered aqueous solution was diluted with water (14mL) (pH 9.6, 20mL), then THP (1M in 100mM Tris, pH 9,5mL, 5.0mmol) and allylpalladium chloride (II) dimer (183mg, 0.5mmol) were added. This mixture was stirred vigorously at room temperature for several minutes, then 1M sodium ascorbate aqueous solution (0.5mL, 0.5mmol), 5M NaCl aqueous solution (10mL) and 10%v / v Tween 20 (0.5mL) were added. In a second example, a stock solution of 2M DEEA buffered water (pH 9.6, 0.6 mL) was diluted with water (7.6 mL) and then THP (1 M in 100 mM Tris, pH 9, 1.2 mL, 1.2 mmol) and solid allylpalladium (II) chloride dimer (43.9 mg, 0.12 mmol) were added. The mixture was stirred vigorously at room temperature for several minutes and then 1M sodium ascorbate aqueous solution (0.12 mL, 0.12 mmol), 5M NaCl aqueous solution (2.4 mL) and 10% v / v Tween 20 (0.12 mL) were added.

[0291] Example 5. Preparation of fully functionalized nucleotides and their use in sequencing applications

[0292] In this example, the preparation of various fully functionalized nucleotides (ffNs) with a 3′-AOM blocking group is described in detail. These ffNs were also used in Illumina Sequencing by synthesis on the platform.

[0293] Scheme 4. Synthesis of 3'-AOM-ffC-LN3-SO7181

[0294]

[0295] Synthesis of intermediate AOM C2: Under N2, nucleoside C1 (0.5g, 0.64mmol) was dissolved in anhydrous DCM (12mL), and the mixture was cooled to 0°C. Cyclohexene (0.32mL, 3.21mmol) was added, followed by dropwise addition of SO2Cl2 (1.0M in DCM, 1.27mL, 1.27mmol). Additional cyclohexene (0.32mL, 3.21mmol) was added, and the reaction was quickly transferred to a rotary evaporator to remove all volatiles under reduced pressure. The solid residue was dried under high vacuum for 10 minutes and then dissolved in anhydrous DCM (5mL) under N2. The mixture was cooled to 0°C, and ice-cold allyl alcohol (5mL) was added dropwise. The reaction was stirred at 0°C for 2h, then quenched by adding saturated NaHCO3 aqueous solution (50mL) and DCM (30mL). The two phases were separated and the aqueous layer was extracted with EtOAc (2 x 50 mL). The organic layers were combined, dried over MgSO4, filtered, and the volatiles were evaporated under reduced pressure. The crude product was purified by flash chromatography on silica gel using EtOAc / petroleum ether to afford AOM C2 as a white solid (264 mg, 52% yield). LC-MS (electrospray negative ionization): [MH] 787, [M+Cl] 823.

[0296] Synthesis of intermediate AOM C3: Under N2, AOM C2 (246 mg, 0.31 mmol) was dissolved in anhydrous THF (9.5 mL), and the mixture was cooled to 0 ° C. Acetic acid (0.054 mL, 0.94 mmol) was added, followed by dropwise addition of TBAF (1.0 M in THF, 5 wt.% water, 0.99 mL, 0.94 mmol). The reaction was stirred at 0 ° C for 5 h, then diluted with EtOAc (20 mL), and then poured into a 0.05 M aqueous HCl solution (20 mL). The mixture was separated into two layers, and the aqueous layer was extracted with EtOAc (2x20 mL). The organic layers were combined, dried over MgSO4, filtered, and the volatiles were evaporated under reduced pressure. The crude product was purified by flash chromatography on silica gel using DCM / EtOAc to obtain AOM C3 as a light yellow solid (114 mg, 66% yield). LC-MS (negative electrospray ionization): [MH] 549, [M+H2O-H] 567, [M+Cl] 585, (positive ion electrospray ionization): [M+H] 551, [M+H2O+H] 569.

[0297] Synthesis of intermediate AOM C4: AOM C3 (0.114 g, 0.21 mmol), freshly activated Molecular sieves, a proton sponge (0.066 g, 0.31 mmol) and a magnetic stirrer were placed under N2, and anhydrous trimethyl phosphate (1.0 mL) was added. The reaction mixture was cooled to -10°C, and freshly distilled POCl3 (23 μL, 0.25 mmol) was added dropwise. The reaction was stirred at -10°C for 1 hour. A solution of di-tri-n-butylammonium salt of pyrophosphate (0.5 M in DMF, 1.7 mL, 0.85 mmol) and anhydrous tri-n-butylamine (0.41 mL, 1.74 mmol) were premixed and added to the ice-cold activated nucleoside solution at once. The mixture was stirred vigorously at room temperature for 5 minutes. The reaction mixture was poured into a separate flask containing a vigorously stirred 2M TEAB aqueous solution (~10 mL). The reaction flask was rinsed with a small amount of H2O, and the washings were added to the 2M TEAB solution. The combined mixture was then stirred at room temperature for 4 hours, and the solvent was then evaporated under reduced pressure. The residue was dissolved in aqueous NH3 (35%, 10 mL) and stirred at room temperature overnight. The reaction was concentrated in vacuo and purified by flash chromatography on DEAE-Sephadex. The product was further purified by preparative HPLC to give pure AOM C4 (62 μmol, 30% yield, as determined by UV-Vis spectroscopy, λ max =294nm,ε=8600M -1 cm -1 ). LC-MS (electrospray negative ionization): [MH] 589.

[0298] 3'-AOM-WC-LN3-SO7181 synthesis: under N2, LN3-SO7181 (0.0205mmol) is dissolved in anhydrous DMA (4mL).N, N-diisopropylethylamine (28.6 μ L, 0.164mmol) is added, followed by TSTU (0.1M in DMA, 234 μ L, 0.0234mmol). The reaction is stirred at room temperature for 1 hour under N2. Meanwhile, the aqueous solution of AOM C4 (0.0101mmol) is evaporated to dryness under reduced pressure, resuspended in 0.1M TEAB aqueous solution (400 μ L) and added to LN3-SO7181 solution. The reaction is stirred at room temperature for 17.5 hours, then quenched with 0.1M TEAB aqueous solution (4mL). The crude product is purified by flash chromatography on DEAE-Sephadex. The product was further purified by preparative HPLC to give pure 3'-AOM-ffC-LN3-SO7181 (6.81 μmol, 67% yield, as determined by UV-Vis spectroscopy, λ max =644nm,ε=200000M -1 cm- 1 ). LC-MS (electrospray negative ionization): [MH] 1561, [M-2H] 781, [M-3H] 520.

[0299] Scheme 5. Synthesis of 3'-AOM-ffA

[0300]

[0301]

[0302] Synthesis of intermediate AOM A2: under N2 atmosphere, nucleoside A1 (716mg, 0.95mmol) is dissolved in 10mL anhydrous dichloromethane, cyclohexene (481 μ L, 4.75mmol) is added, and the solution is cooled to about-15 ℃. Sulfuryl chloride (distilled, 92 μ L, 1.14mmol) is added dropwise, and the reaction is stirred for 20 minutes. After all raw materials are consumed, additional cyclohexene (481 μ L, 4.75mmol) is added, and the reaction mixture is evaporated to dryness under reduced pressure. The residue is quickly purged with nitrogen, and allyl alcohol (5mL,~100mmol) is then added under stirring at 0 ℃. The reaction is stirred at 0 ℃ for 1 hour, then quenched with 50mL saturated NaHCO3 aqueous solution. The mixture is extracted with 2x100mL of ethyl acetate. The combined organic phase is washed with 100mL water and 100mL salt water, then dried over MgSO4, filtered and evaporated to dryness. The residue was purified by flash chromatography on silica gel using petroleum ether / EtOAc. 60% yield (435 mg, 0.57 mmol). LC-MS (ES and CI): (positive ion) m / z 763 (M+H + ); (negative ion) m / z761(MH + ).

[0303] Synthesis of intermediate AOM A3: Under N2 atmosphere, nucleoside AOM A2 (476 mg, 0.62 mmol) was dissolved in anhydrous THF (5 mL), and then a THF solution of 1.0 M TBAF (750 μL, 0.75 mmol) was added. The solution was stirred at room temperature for 1.5 hours. The solution was diluted with 50 mL of EtOAc, then washed with 100 mL of a saturated solution of NaH2PO4 (pH=3), and washed with 100 mL of brine. The organic phase was dried over MgSO4, filtered and evaporated to dryness. The residue was purified by flash chromatography on silica gel using EtOAc / MeOH. 90% yield (292 mg, 0.55 mmol). LC-MS (ES and CI): (positive ion) m / z 525 (M+H + ); (negative ion) m / z 523 (MH + ).

[0304] Synthesis of intermediate AOM A4: Nucleoside AOM A3 (285 mg, 0.544 mmol) was dried over P2O5 under reduced pressure for 18 h. Anhydrous triethyl phosphate (2 mL) and some freshly activated Molecular sieves were added, and the reaction flask was cooled to 0° C. in an ice bath. Freshly distilled POCl 3 (61 μL, 0.65 mmol) was added dropwise, followed by the addition of Proton (175mg, 0.816mmol). After addition is complete, the reaction is further stirred at 0 ℃ for 15 minutes.Then, the anhydrous DMF solution of di-tri-n-butylammonium salt (5.4mL, 2.72mmol) of 0.5M pyrophosphoric acid is added quickly, then tri-n-butylamine (540 μ L, 2.3mmol) is added immediately. The reaction is kept for another 10 minutes in an ice-water bath, then poured into 1M triethylammonium bicarbonate aqueous solution (TEAB, 20mL) and quenched, and stirred at room temperature for 4 hours. All solvents are evaporated under reduced pressure. 35% ammonia solution (20mL) is added to the above-mentioned residue, and the mixture is stirred at room temperature for at least 5 hours. The solvent is then evaporated under reduced pressure. First, the crude product is purified on DEAE-Sephadex A25 (100g) by ion exchange chromatography. Use the post with triethylammonium bicarbonate aqueous solution gradient elution. Merge the parts containing triphosphate, and under reduced pressure, the solvent is evaporated to dryness. The crude material was further purified by preparative-scale HPLC using a YMC-Pack-Pro C18 column, eluting with 0.1 M TEAB and acetonitrile. Compound AOM A4 was obtained as a triethylammonium salt in 56% yield (306 μmol). LC-MS (ES and CI): (negative ion) m / z 612 (MH + ); (positive ion) m / z 614 (M+H + ), 715(M+Et3NH + ).

[0305] The general procedure of ffA synthesis: dye linker (0.020mmol) is dissolved in 2mL anhydrous N, N'-dimethylacetamide (DMA). Add N, N'-diisopropylethylamine (28.4 μ L, 0.163mmol), then add 0.1M anhydrous DMA (TSTU, 232 μ L, 0.023mmol) solution of N, N, N', N'-tetramethyl-O-(N-succinimidyl) uronium tetrafluoroborate. The reaction was stirred at room temperature under nitrogen for 1 hour. Meanwhile, the aqueous solution of triphosphoric acid AOM A4 (0.01mmol) was evaporated to dryness under reduced pressure, and resuspended in the 0.1M triethylammonium bicarbonate (TEAB) aqueous solution of 200 μ L. Activated dye-linker solution was added to triphosphate, and the reaction was stirred at room temperature for 18 hours. First, the crude product was purified by ion exchange chromatography on DEAE-Sephadex A25 (25g). The fractions containing the triphosphate were combined and the solvent was evaporated to dryness under reduced pressure. The crude material was further purified by preparative-scale RP-HPLC using a YMC-Pack-Pro C18 column. 3'-AOM-ffA-LN3-NR7180A: 38% yield (3.8 μmol). LC-MS (ES): (negative ion) m / z 1459 (MH+ ), 729(M-2H + ), 486(M-3H + ). 3'-AOM-ffA-LN3-BL-NR550S0: 37% yield (3.7 μmol). LC-MS (ES): (negative ion) m / z 1771 (MH + ), 885(M-2H + ), 589(M-3H + ). 3'-AOM ffA-LN3-BL-NR 6 50C5: 51% yield (51 μmol). LC-MS (ES): (negative ion) m / z 1917 (MH + ), 958(M-2H + ), 645(M-3H + ).

[0306] Scheme 6. Synthesis of 3'-AOM-pppG

[0307]

[0308] Synthesis of intermediate AOM G4: Under N2 atmosphere, known nucleoside dG3 (100 mg, 0.143 mmol) was dissolved in 10 mL of anhydrous dichloromethane, cyclohexene (72 μL, 0.714 mmol) was added, and the solution was cooled to -12 ° C. Sulfuryl chloride (distilled, (1 M, in DCM), 171 μL, 0.171 mmol) was added dropwise, and the reaction was stirred for 10 minutes. Additional cyclohexene (72 μL, 0.714 mmol) was added, and the reaction was stirred at -12 ° C for 30 minutes. The reaction was evaporated to dryness under reduced pressure, the residue was purged with nitrogen, and ice-cold neat allyl alcohol (distilled, 0.8 mL, 12 mmol) was added with stirring at -12 ° C. The reaction was stirred at -12 ° C for 60 minutes and then quenched with 2 mL of saturated NaHCO3 aqueous solution. The mixture was separated with ethyl acetate (2 mL), and the aqueous layer was extracted with ethyl acetate. The combined organic phases were washed with 4 mL of water and 4 mL of brine, dried over MgSO₄, filtered, and evaporated to a crude oil. The residue was purified by flash chromatography on silica gel to afford AOM G4 as a clear oil. 36% yield (50.9 mg, 0.072 mmol). LC-MS (ES and CI): (positive ion) m / z 710 [M+H] + ; (negative ion) m / z 708[MH] - .

[0309] Synthesis of intermediate AOM G5: Nucleoside AOM-G4 (111 mg, 0.156 mmol) was dissolved in anhydrous THF (5 mL) under N2 atmosphere. Acetic acid (27 μL, 0.468 mmol) was added, followed by a 1.0 M THF solution of TBAF (296 μL, 0.296 mmol). The solution was stirred at room temperature for 5 hours. The solution was diluted with 10 mL of EtOAc, washed with 10 mL of 0.05 M HCl aqueous solution, and the organic phase was separated. The aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over MgSO4, filtered, and evaporated to dryness. The residue was purified by flash chromatography on silica gel to obtain AOM G5 as a white solid. 44% yield (32.4 mg, 0.068 mmol). LC-MS (ES and CI): (positive ion) m / z 472 [M+H] + ; (negative ion) m / z 470[MH] - .

[0310] Synthesis of 3'-AOM-pppG: Nucleoside AOM-G5 (79 mg, 0.168 mmol) and freshly activated The molecular sieves were dried under reduced pressure with P2O5 for 18 hours. Proton was added under nitrogen. To the 4-thiazolinone methyl ester (175mg, 0.816mmol) and anhydrous triethyl phosphate (0.8mL), and stirred at room temperature for 1 hour.The reaction flask was cooled to 0 ℃ in an ice bath, freshly distilled POCl was added dropwise (19 μ L, 0.202mmol), and the reaction was stirred at 0 ℃ for 15 minutes.Then, the anhydrous DMF solution of the di-tri-n-butylammonium salt of 0.5M pyrophosphoric acid (1.68mL, 0.84mmol) was added quickly, and then tri-n-butylamine (168 μ L, 0.705mmol) was added immediately.The reactant was removed from the ice / water bath and stirred vigorously for 5 minutes, then poured into 1M triethylammonium bicarbonate aqueous solution (TEAB, 6mL) and quenched, and stirred at room temperature for 18 hours.All solvents were evaporated under reduced pressure.Residue was dissolved in 35% ammonia solution (10mL), and stirred at room temperature for at least 5 hours.Then the solvent was evaporated under reduced pressure, and further co-evaporated with water. The crude product was first purified by ion exchange chromatography on DEAE-Sephadex A25 (50 g). The column was eluted with a linear gradient of aqueous triethylammonium. Fractions containing the triphosphate were collected, and the solvent was evaporated to dryness under reduced pressure. The crude material was further purified by preparative-scale HPLC using a YMC-Pack-Pro C18 column. The triethylammonium salt of 3'-AOM-pppG was obtained in 24% yield (39.7 μmol). LC-MS (ES and CI): (negative ion) m / z 576 [MH] - ; (positive ion) m / z 578[M+H]+ .

[0311] 3'-AOM-ffT-LN3-NR550S0 was synthesized in a similar manner as described in the preparation of 3'-AOM ffA and ffC.

[0312] Scheme 7. Synthesis of 3'-AOM-fft-LN3'-NR550S0

[0313]

[0314] Synthesis of intermediate T1: 5-iodo-2'-deoxyuridine (3 g, 8.4 mmol) and palladium (II) acetate (1.6 g, 7.14 mmol) were dissolved in dry, degassed DMF, followed by the addition of N-allyltrifluoroacetamide (6.4 mL, 42 mmol). The solution was placed under vacuum, then purged with nitrogen three times, and then degassed triethylamine (2.3 mL, 16.8 mmol) was added. The solution was heated to 80°C for 2 hours. The black mixture was cooled to room temperature and then diluted with 50 mL of methanol. About 0.5 g of activated carbon was added, and the solution was filtered on celite and then evaporated under reduced pressure to give a brown thick oil. The crude product was purified by chromatography on silica gel using EtOAc / MeOH as eluent. Yield: (2.27 g, 5.99 mmol). LC-MS (ES and CI): (negative ion) m / z 378 (MH + ).

[0315] Synthesis of intermediate T2: 5-[3-(2,2,2-trifluoroacetamido)-allyl]-2'-deoxyuridine (T1) (2.55 g, 6.72 mmol) was dissolved in anhydrous DMF. Imidazole (1.37 g, 20.1 mmol) was added, followed by 4-(dimethylamino)pyridine (410 mg, 3.36 mmol). The reaction was cooled to 0°C, and then tert-butyl(chloro)diphenylsilane (1.92 mL, 7.39 mmol) was slowly added in three portions at intervals of 30 minutes. The reaction was stirred at 0°C for 6 hours. The solvent was then evaporated, and the residue was resuspended in 200 mL of EtOAc and washed with 2x200 mL of saturated NaHCO3 aqueous solution and 200 mL of water, followed by 100 mL of brine. The organic phase was dried over MgSO4, filtered, and evaporated to dryness. The crude product was purified by flash chromatography on silica gel using DCM / EtOAc. 68% yield (2.806 g, 4.54 mmol). LC-MS (ES and CI): (positive ion) m / z 618 (M+H + ); (negative ion) m / z 616 (MH + ).

[0316] Synthesis of intermediate T3: 5'-O-(tert-butyldiphenylsilyl)-5-[3-(2,2,2-trifluoroacetamido)-allyl]-2'-deoxyuridine (T2) (2.8 g, 4.53 mmol) was dissolved in 10 mL of anhydrous DMSO (136 mmol), followed by addition of glacial acetic acid (16 mL, 272 mmol) and acetic anhydride (16 mL, 158 mmol). The reaction was heated to 50°C for 6 hours and then quenched with 200 mL of saturated NaHCO3 aqueous solution. After the solution stopped bubbling, it was extracted with 2 x 150 mL of EtOAc. The organic phases were combined and washed with 2 x 200 mL of saturated NaHCO3 aqueous solution, 200 mL of water, and 100 mL of brine. The organic phase was dried over MgSO4, filtered, and evaporated to dryness. The crude product was purified by flash chromatography on silica gel using DCM / EtOAc. 77% yield (2.375 g, 3.51 mmol). LC-MS (ES and CI): (positive ion) m / z 678 (M+H + ); (negative ion) m / z 676 (MH + ).

[0317] Synthesis of Intermediate T4: 5'-O-(tert-Butyldiphenylsilyl)-3'-O-methylmethylthio-5-[3-(2,2,2-trifluoroacetamido)-allyl]-2'-deoxyuridine (T3) (310 mg, 0.45 mmol) was dissolved in 5 mL of anhydrous dichloromethane under N2 atmosphere, cyclohexene (228 μL, 2.25 mmol) was added, and the solution was cooled to approximately -15°C. Sulfuryl chloride (distilled, 55 μL, 0.675 mmol) was added dropwise, and the reaction was stirred for 20 minutes. After all the starting material was consumed, additional cyclohexene (228 μL, 2.25 mmol) was added, and the reaction was evaporated to dryness under reduced pressure. The residue was quickly purged with nitrogen, and then ice-cold allyl alcohol (2.5 mL) was added with stirring at 0°C. The reaction was stirred at 0°C for 35 minutes, then quenched with 25 mL of saturated NaHCO3 aqueous solution, and then further diluted with 100 mL of saturated NaHCO3 aqueous solution. The mixture was extracted with 2 x 50 mL of ethyl acetate. The combined organic phase was dried over MgSO4, filtered and evaporated to dryness. The residue was purified by flash chromatography using DCM / EtOAc on silica gel. 69% yield (214 mg, 0.311 mmol). LC-MS (ES and CI): (positive ion) m / z 688 (M+H + ); (negative ion) m / z 686 (MH + ).

[0318] Synthesis of intermediate T5: Under N2 atmosphere, 5'-O-(tert-butyldiphenylsilyl)-3'-O-allyloxymethyl-5-[3-(2,2,2-trifluoroacetamido)-allyl]-2'-deoxyuridine (T4) (210 mg, 0.305 mmol) was dissolved in anhydrous THF (3 mL). A 1.0 M THF solution of TBAF (367 μL, 0.367 mmol) was added. The solution was stirred at room temperature for 3 hours. The solution was diluted with 50 mL of EtOAc and then washed with 50 mL of saturated NaH2PO4 (pH = 3) and 50 mL of water. The organic phase was dried over MgSO4, filtered and evaporated to dryness. The residue was purified by flash chromatography on silica gel using DCM / EtOAc. 95% yield (130 mg, 0.289 mmol). LC-MS (ES and CI): (negative ion) m / z 448 (MH + ),484(M+Cl - ).

[0319] Synthesis of intermediate T6: 3'-O-allyloxymethyl-5-[3-(2,2,2-trifluoroacetamido)-allyl]-2'-deoxyuridine (T5) (120 mg, 0.267 mmol) was dried over P2O5 under reduced pressure for 18 hours. Anhydrous triethyl phosphate (1 mL) and some freshly activated Molecular sieves, and then the reaction bottle was cooled to 0 ° C. Freshly distilled POCl3 (30 μL, 0.32 mmol) was added dropwise, and then Proton (85mg, 0.40mmol). After addition was complete, the reaction was further stirred at 0 ℃ for 15 minutes. Then, the anhydrous DMF solution of di-tri-n-butylammonium salt (2.7mL, 1.33mmol) of 0.5M pyrophosphate was quickly added, followed by immediate addition of tri-n-butylamine (270 μL, 1.2mmol). The reaction was kept for another 10 minutes in an ice-water bath, then poured into 1M triethylammonium bicarbonate aqueous solution (TEAB, 10mL) and quenched, and stirred at room temperature for 4 hours. All solvents were evaporated under reduced pressure. 35% ammonia solution (10mL) was added to the above-mentioned residue, and the mixture was stirred at room temperature for 18 hours. The solvent was then evaporated under reduced pressure, and the residue was resuspended in 10mL of 0.1M TEAB and filtered. First, the filtrate was purified on DEAE-Sephadex A25 (100g) by ion exchange chromatography. The post was eluted with triethylammonium bicarbonate aqueous solution (TEAB). The fractions containing the triphosphate were combined and the solvent was evaporated to dryness under reduced pressure. The crude material was further purified by preparative-scale HPLC using a YMC-Pack-Pro C18 column. Compound T6 was obtained as the triethylammonium salt in 33% yield (89 μmol). LC-MS (ES and CI): (negative ion) m / z 592 (MH + ), 295(M-2H + ).

[0320] Synthesis of 3'-AOM-fft-LN3'-NR550S0: Under N2, dry known compound LN3-NR550S0 (0.015mmol) was dissolved in anhydrous DMA (2mL). N, N-diisopropylethylamine (17 μL, 0.1mmol) was added, followed by TSTU (0.1M in DMA, 180 μL, 0.018mmol). The reaction was stirred at room temperature for 1 hour under N2. Meanwhile, the T6 aqueous solution (0.01mmol) was evaporated to dryness under reduced pressure, resuspended in a 0.1M TEAB aqueous solution (200 μL), and added to a LN3-NR550S0 solution. The reaction was stirred at room temperature for 18 hours, then quenched with a 0.1M TEAB aqueous solution (4mL). The crude product was purified by flash chromatography on DEAE-Sephadex. The product was further purified by preparative HPLC to give pure 3'-AOM-ffT-LN3'-NR550S0 in 67% yield (41 μmol, determined by UV-Vis spectroscopy, λ max =550nm,ε=125000M -1 cm -1 )LC-MS(ES):(negative ion)m / z1521(MH + ), 761(M-2H + ), 507(M-3H+ ).

[0321] Sequencing by synthesis experiments

[0322] Illumina The instrument was used to perform sequencing experiments on ffNs. All standard commercial reagents were used, except for the new spike-in mix containing these ffNs. A standard 2x150 formulation was used. In addition to the standard sequencing-by-synthesis (SBS) protocol, a palladium fragmentation mix (Pd:THP = 1 / 5 in DEEA, as described in Example 4) was added with a 5-second incubation to deblock the 3'-AOM.

[0323] In the first experiment, the following ffNs were used to be incorporated into the mixture: 3'-AOM-ff-LN3-NR550S0, 3'-AOM-ffA-LN3-BL-NR550S0, 3'-AOM-ffA-LN3-BL-NR650C5, 3'-AOM-ffA-LN3-NR7180A, 3'-AOM-ffC-LN3-SO7181 and 3'-AOM-pppG (dark G). And the sequencing results of read 1 are summarized as follows.

[0324]

[0325] %PF: Percentage of clusters passing the filter after 26 cycles

[0326] In the second experiment, similar to the preparation of 3'-AOM-pppG described above, unlabeled 3'-AOM-pppT (LC-MS(ES): (negative ion) m / z 551 (MH + )). It was used for sequencing in the presence of commercial green WG-LN3-PEG12-ATTO532 (used on an Illumina 4-channel system) and the same WA and WA were used as described in the first experiment above. The results are summarized below. There was a significant improvement in the phasing and prephasing values, and no signal attenuation was observed ( Figure 3A ). In addition, the error rates of both read segment 1 and read segment 2 are also reduced.

[0327]

[0328]

[0329] In another experiment, the incorporation mixture included 3'-AOM-ff-LN3'-NR550S0, 3'-AOM-ffA-LN3-BL-NR550S0, 3'-AOM-ffA-LN3-BL-NR650C5, 3'-AOM-ffA-LN3-NR7180A, 3'-AOM-ffC-LN3-SO7181 and 3'-AOM-pppG (dark G). Similar to the previous run, the fragmentation mixture containing palladium catalyst (Pd / THP=1:10; 100mM DEEA described in Example 4) incubated for 5 seconds was added to the standard SBS cycle. Using standard DNA polymerase, but the incorporation time was 2 times longer. No signal attenuation phenotype was observed ( Figure 3B ). In addition, these sequencing results were compared with commercial products of ffNs with standard azidomethyl blocking groups. Runs (average of 3; N=3) were compared. It was observed that the error rates were almost the same ( Figure 3C The sequencing results are summarized as follows.

[0330]

[0331] In addition, the main sequencing indicators of ffN with 3'-AOM blocking group were compared with the standard DNA polymerase Pol 812. The main sequencing indicators produced by commercial kits were compared, and the comparison results are shown in Figure 4A Due to the improved stability of 3'-AOM-ffNs, very low prephasing was observed. However, even with a 2-fold increase in incorporation time, phasing was still improved.

[0332] In another experiment, a different DNA polymerase (Pol 1901) was used instead of the DNA polymerase in the commercial kit (Pol 812). Pol 1901 allowed the use of a standard 1x incorporation time during sequencing, rather than the 2x incorporation time described above. In addition, the incubation in the Pd fragmentation mix was reduced by half compared to the standard run. This resulted in a 10% time saving in the overall SBS chemistry cycle. Sequencing metrics were significantly improved and exceeded those obtained from the standard commercial kit containing a 3'-O-azidomethyl blocking group ( Figure 4B ).

[0333] Stability test of 3' blocking group during sequencing

[0334] To demonstrate the improved stability of ffNs with 3'-AOM, they were compared with standard ffNs with 3'-O-azidomethyl groups. ffN were compared in parallel. The two groups of ffN were incubated at 45°C for several days in a standard spike-in mixture formulation (excluding only DNA polymerase). Fresh polymerase was added directly before the incorporation into the mixture. The sequencing conditions described previously were used. The pre-phase % is a direct indicator of the percentage of 3'OH-ffN in the mixture and is therefore directly related to the stability of the 3' blocking group. The pre-phase values ​​of the two groups of ffN were recorded and plotted ( Figure 5 At 45°C, it was observed that ffN containing a 3'-AOM group appeared to be six times more stable than the standard ffN with a 3'-O-azidomethyl group. Sequencing metrics also confirmed the trend observed during stability assays in solution – the 3'-AOM blocking group was significantly more stable than the 3'-O-azidomethyl group.

[0335] Example 6. Preparation of 3'-O-thiocarbamate-blocked nucleosides

[0336] In this example, various 3'-O-thiocarbamate protected T nucleosides were prepared according to Scheme 8.

[0337] Scheme 8. Synthesis of 3'-O-dimethylthiocarbamate T nucleoside

[0338]

[0339] Preparation of T-7: To an oven-dried, nitrogen-purged 100 mL flask was added 5'-O-(4,4'-dimethoxytrityl)thymidine (1.0 g, 1.836 mmol). This was co-evaporated with anhydrous DMF (3 x 20 mL) and placed under nitrogen. Anhydrous DCM (9.2 mL) and 4-dimethylaminopyridine (224 mg, 0.184 mmol) were added and stirred at room temperature until a homogeneous solution was formed. 1,1'-Thiocarbonyldiimidazole (360 mg, 2.02 mmol) was then quickly added under a stream of nitrogen, the reaction was resealed and stirred at room temperature for 2 hours until all the starting materials were consumed. The reaction mixture was filtered through a pad of silica gel, and the filter cake was washed with EtOAc (10 mL). The volatiles were removed in vacuo, and the crude residue was used without further purification.

[0340] Preparation of T-8: Compound T-7 from the previous step was used immediately after vacuum drying. The residue was placed in a 25 mL round-bottom flask under nitrogen, and dimethylamine (2M in THF, 7.3 mL, 14.6 mmol) was added, and the reaction was stirred for 2 hours until all the starting material was consumed according to TLC. All volatiles were removed in vacuo to form a clear crude residue, which was purified by flash column chromatography on silica gel to give T-8 as a white solid. Yield: 1.15 g (99%). LC-MS (electrospray negative ionization) 630.23 [MH].

[0341] Preparation of T-9: The starting nucleoside T-8 (320 mg, 0.504 mmol) was dissolved in a small amount of acetonitrile in a 50 mL round-bottom flask under air. A 5:1 AcOH / H2O solution (12.5 mL:2.5 mL) was added in one portion and the reaction was stirred at room temperature until all the starting material was consumed (2-4 hours). All volatiles were evaporated under vacuum and the residue was co-evaporated in toluene (2 x 60 mL) to give the crude product as an off-white solid. The crude product was purified by flash column chromatography to give T-9 as a white solid. Yield: 123 mg (74%). LC-MS (electrospray negative ionization) [MH] 328.10.

[0342] Nucleosides with two other thiocarbamate protecting groups were also prepared using the corresponding MeNH2 or NH3 following similar synthetic steps. The general reaction scheme is shown below:

[0343]

[0344] 3'-O-Thiocarbamate blocking group stability test

[0345] Stability assays of 5'-mP3'-DMTCT nucleotides were performed in parallel with standard 5'-mP 3'-O-azidomethyl T nucleotides in incorporation buffer.

[0346]

[0347] For both 5'-mP 3'-DMTC T and 5'-mP 3'-O-azidomethyl T, the final solution volume was 1 mL, and the final concentration of the corresponding nucleotide was 0.1 mM. The other components of the aqueous buffer solution included ethanolamine (EA), ethanolamine HCl, NaCl (100 mM), and EDTA (2.5 mM). The concentrations of the buffer solution were as follows: 0.5 M EA buffer, 0.5 M NaCl, and 0.01 M EDTA.

[0348] Stability testing methodology

[0349] 200 μL of 10x buffer solution was added to a 1.7 mL polypropylene snap lock microtube and diluted with an accurate volume of 18 mΩ water. The corresponding nucleoside was then added, the vial sealed and mixed by inversion, gentle stirring, or pumping with a micropipette. A 40 μL aliquot was taken and analyzed by HPLC to serve as the starting (or t=0) value. The vial was then placed in a preheated heating mantle set to 65°C, covered with a thick layer of aluminum foil, and heated for one month. 40 μL aliquots were taken regularly (week 1: once a day. Weeks 2-4: once every 2 days) and analyzed by HPLC to determine the percentage of raw material and the percentage of unblocked (3'-OH) nucleotides in the sample. HPLC analysis was performed by measuring the area of ​​the starting nucleotide peak and the 3'OH peak. These values ​​were used to calculate the percentage of unblocked nucleotides, which was presented graphically and used to compare the stability between samples spiked in buffer. Figure 6 Comparison of the stability of three different thiocarbamate 3'-blocked nucleotides at 65°C with nucleotides blocked with a 3'-O-azidomethyl blocking group is shown. It was observed that while nucleotides with 3'-OC(=S)NH2 or 3'-OC(=S)NHCH3 were less stable than nucleotides protected with a standard 3'-O-azidomethyl group, nucleotides with 3'-DMTC conferred improved stability over the 9-day experimental period. Thus, DMTC exhibited superior stability to the standard azidomethyl blocking group.

[0350] Example 7. 3'-O-thiocarbamate blocking group deprotection test

[0351] In this example, deblocking or unblocking assays for 5'-mP 3'-DMTC T and standard 5'-mP 3'-O-azidomethyl T nucleotides were performed separately in unique solutions for each blocking group. Conditions were designed to closely mimic Illumina's standard unblocking reagents and followed the same methodology. The concentrations of active unblocking reagent, buffer, and nucleoside remained the same across all assays, but each component was uniquely labeled. Thus, observed differences in rates between the individual unblocking chemistries could not be attributed to differences in formulation concentrations.

[0352]

[0353] General Methodology for Deblocking Tests

[0354] Prepare each reaction component individually as a concentrated stock solution in 18 mΩ water, store appropriately, and combine aliquots in the order specified below. Initiate the reaction by adding the pre-prepared deblocking reagent. Final concentrations: nucleoside (0.1 mM), active deblocking reagent (1 mM), additive (specific to the deblocking reagent), buffer (100 mM). Final volume: 2000 μL.

[0355] A pre-formulated buffer solution was added to a 3 mL glass vial, followed by a pre-formulated additive solution. Dilute with an accurate volume of 18 mΩ water and stir for 10 minutes. An aliquot of the nucleotide solution was then added and stirred for 5 minutes. A 40 μL aliquot was then taken, a quencher was added and analyzed by HPLC as a reference peak (or t = 0 minutes). The deblocking reagent was then added to the stirred solution once and the timing was started. At the specified time point, a 40 μL aliquot was taken and immediately quenched with an appropriate quencher and then analyzed by HPLC to determine the amount of deblocked nucleotides that occurred at these specified time points. The results were plotted graphically and used to compare deblocking efficiency and efficacy.

[0356] DMTC unblocking

[0357] Nucleotide: 5'-mP 3'-DMTCT. Activity deblocking reagent: NaIO4 (0.1M in 18mΩ water) or (0.1 M in 18 mΩ water). Additives: None. NaIO4 buffer: pH 6.75 phosphate buffer (1 M in 18 mΩ water). Buffer: pH 8.65 phosphate buffer (1 M in 18 mΩ water). Quencher: Sodium thiosulfate. 3'-O-azidomethyl deblocking conditions were the same as described in Example 3.

[0358] HPLC analysis is performed by measuring the areas of the initial nucleoside peak, the 3'-OH peak, and any other nucleotide peaks present in the HPLC chromatogram. These values ​​are used to calculate the percentage of starting and deblocked nucleotides, displayed graphically, and used to compare deblocking rates, efficiencies, and potencies between samples. Comparative results are shown in Figure 7 It was observed that the deblocking of DMTC by NaIO4 was ineffective. When DMTC is broken, the percentage of the remaining starting material with the nucleotides having the DMTC blocking group is significantly less. In summary, it has been shown that the DMTC unblocking rate (using ) is better than the deblocking rate of the standard azidomethyl blocking group.

Claims

1. A nucleotide containing ribose or deoxyribose having a structure selected from the group consisting of: wherein L is a linking group, the dye is a fluorescent dye, and R is said ribose or deoxyribose sugar having a removable 3'-OH blocking group covalently attached to the 3'-carbon atom wherein the linking group is selected from: Wherein B is a nucleoside base; Z is -N3 (azido), -O-C1-C6 alkyl, -OC 2- C6 alkenyl or -OC 2- C6 alkynyl; and F1 comprises a fluorescent dye, which may comprise additional linker structures.

2. The nucleotide of claim 1, comprising a 2' deoxyribose sugar.

3. The nucleotide of claim 1, wherein the nucleotide comprises a triphosphate at the 5' position of the ribose or deoxyribose sugar.

4. The nucleotide of claim 2, wherein the nucleotide comprises a triphosphate at the 5' position of the ribose or deoxyribose sugar.

5. The nucleotide of any one of claims 1 to 4, wherein the 3'-OH blocking group and the cleavable linking group are removed under the same chemical reaction conditions.

6. The nucleotide of any one of claims 1 to 4, wherein the removable 3'-OH blocking group confers at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 2500% or 3000% improved stability compared to an azidomethyl protected 3'-OH under the same conditions for the same period of time.

7. The nucleotide of claim 5, wherein the removable 3'-OH blocking group confers at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 2500% or 3000% improved stability compared to an azidomethyl protected 3'-OH under the same conditions for the same period of time.

8. An oligonucleotide incorporating a nucleotide according to any one of claims 1 to 7, wherein a phosphodiester bond is formed between the 3' carbon atom of the oligonucleotide and the 5' carbon atom of the nucleotide.

9. The oligonucleotide of claim 8, which is immobilized on the surface of an array.

10. A method of preparing a growing polynucleotide that is complementary to a target single-stranded polynucleotide in a sequencing reaction, comprising incorporating a nucleotide according to any one of claims 1 to 7 into the growing complementary polynucleotide, wherein incorporation of the nucleotide prevents the incorporation of any subsequent nucleotide into the growing complementary polynucleotide.

11. A method according to claim 10, comprising incorporating a nucleotide according to any one of claims 3 to 7 into a growing complementary polynucleotide.

12. The method of claim 10 or 11, wherein the incorporation of the nucleotide is accomplished by a polymerase.

13. A method for determining a target single-stranded polynucleotide sequence, comprising: (a) incorporating a nucleotide according to any one of claims 1 to 7 into a replicating polynucleotide chain that is complementary to at least a portion of a target polynucleotide chain; (b) detecting the identity of the nucleotide incorporated into the replicated polynucleotide chain; as well as (c) Chemical removal of the label and 3'-OH blocking group from the nucleotides incorporated into the replicated polynucleotide chain.

14. The method of claim 13, wherein the nucleotide incorporated in step (a) is a nucleoside triphosphate.

15. The method of claim 13, further comprising (d) washing the chemically removed label and 3'-OH blocking group from the replicated polynucleotide strand.

16. The method of claim 14, further comprising (d) washing the chemically removed label and 3'-OH blocking group from the replicated polynucleotide strand.

17. The method of claim 15, wherein a palladium scavenger is used in step (d).

18. The method of claim 16, wherein a palladium scavenger is used in step (d).

19. The method of any one of claims 15-18, further comprising repeating steps (a) to (d) until the sequence of the portion of the template polynucleotide chain is determined.

20. The method of claim 19, wherein Steps (a) to (d) are repeated at least 50 times, at least 75 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, or at least 300 times.

21. The method of any one of claims 15 to 18, wherein Steps (a) to (d) are repeated at least 50 times, at least 75 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, or at least 300 times.

22. The method of any one of claims 13 to 18 or 20, wherein the label and 3'-OH blocking group are removed from the nucleotide incorporated into the replicated polynucleotide chain in a single chemical reaction.

23. The method of claim 19, wherein the label and 3'-OH blocking group are removed from the nucleotide incorporated into the replicated polynucleotide chain in a single chemical reaction.

24. The method of claim 21, wherein the label and 3'-OH blocking group are removed from the nucleotide incorporated into the replicated polynucleotide chain in a single chemical reaction.

25. The method of claim 22, wherein step (c) comprises contacting the incorporated nucleotide with a fragmentation solution comprising a palladium catalyst.

26. The method of claim 23, wherein step (c) comprises contacting the incorporated nucleotide with a fragmentation solution comprising a palladium catalyst.

27. The method of claim 24, wherein step (c) comprises contacting the incorporated nucleotide with a fragmentation solution comprising a palladium catalyst.

28. The method of any one of claims 13 to 18 or 20, wherein the label and 3'-OH blocking group are removed from the nucleotide incorporated into the replicated polynucleotide chain in two separate chemical reactions.

29. The method of claim 19, wherein the label and 3'-OH blocking group are removed from the nucleotide incorporated into the replicated polynucleotide chain in two separate chemical reactions.

30. The method of claim 21, wherein the label and 3'-OH blocking group are removed from the nucleotide incorporated into the replicated polynucleotide chain in two separate chemical reactions.

31. The method of claim 28, wherein step (c) comprises contacting the incorporated nucleotide with a fragmentation solution comprising a phosphine and a palladium catalyst.

32. The method of claim 29, wherein step (c) comprises contacting the incorporated nucleotide with a fragmentation solution comprising a phosphine and a palladium catalyst.

33. The method of claim 30, wherein step (c) comprises contacting the incorporated nucleotide with a fragmentation solution comprising a phosphine and a palladium catalyst.

34. The method of claim 31, wherein the phosphine is tris(hydroxymethyl)phosphine, tris(hydroxyethyl)phosphine, or tris(hydroxypropyl)phosphine.

35. The method of claim 32, wherein the phosphine is tris(hydroxymethyl)phosphine, tris(hydroxyethyl)phosphine, or tris(hydroxypropyl)phosphine.

36. The method of claim 33, wherein the phosphine is tris(hydroxymethyl)phosphine, tris(hydroxyethyl)phosphine, or tris(hydroxypropyl)phosphine.

37. The method of any one of claims 25-27, 31-36, wherein the cleavage solution comprising a palladium catalyst further comprises one or more buffering agents selected from the group consisting of primary amines, secondary amines, tertiary amines, carbonates, phosphates, borates, and combinations thereof.

38. The method of claim 37, wherein the buffer is selected from the group consisting of ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, carbonates, phosphates, borates, 2-dimethylaminoethanol (DMEA), 2-diethylaminoethanol (DEEA), N,N,N′,N′-tetramethylethylenediamine (TEMED) and N,N,N′,N′-tetraethylethylenediamine (TEEDA), and combinations thereof.

39. The method of claim 37, wherein the cleavage solution further comprises ascorbic acid or a salt thereof.

40. The method of any one of claims 25-27, 31-36, or 38, wherein the cleavage solution further comprises ascorbic acid or a salt thereof.

41. The method of any one of claims 13-18, 20, 23-27, 29-36, 38-39, wherein the method is performed in a multiplex format.

42. The method of claim 19, wherein the method is performed in a multiplex format.

43. The method of claim 21, wherein the method is performed in a multiplex format.

44. The method of claim 22, wherein the method is performed in a multiplex format.

45. The method of claim 28, wherein the method is performed in a multiplex format.

46. ​​The method of claim 37, wherein the method is performed in a multiplex format.

47. The method of claim 40, wherein the method is performed in a multiplex format.

48. The method of any one of claims 13-18, 20, 23-27, 29-36, 38-39, 42-47, wherein the method is performed in an array format using surface-bound target nucleic acids that are bound to the surface in a spatially distinguishable manner.

49. The method of claim 19, wherein the method is performed in an array format using surface-bound target nucleic acids that are bound to the surface in a spatially distinguishable manner.

50. The method of claim 21, wherein the method is performed in an array format using surface-bound target nucleic acids that are bound to the surface in a spatially distinguishable manner.

51. The method of claim 22, wherein the method is performed in an array format using surface-bound target nucleic acids that are bound to the surface in a spatially distinguishable manner.

52. The method of claim 28, wherein the method is performed in an array format using surface-bound target nucleic acids that are bound to the surface in a spatially distinguishable manner.

53. The method of claim 37, wherein the method is performed in an array format using surface-bound target nucleic acids that are bound to the surface in a spatially distinguishable manner.

54. The method of claim 40, wherein the method is performed in an array format using surface-bound target nucleic acids that are bound to the surface in a spatially distinguishable manner.

55. The method of claim 41, wherein the method is performed in an array format using surface-bound target nucleic acids that are bound to the surface in a spatially distinguishable manner.

56. The method of claim 48, wherein the array comprises multiple copies of a target nucleic acid having the same sequence at each site.

57. The method of any one of claims 49-55, wherein the array comprises multiple copies of a target nucleic acid having the same sequence at each site.

58. The method of claim 56, wherein the multiple copies of the target nucleic acid are formed by bridge amplification.

59. The method of claim 57, wherein multiple copies of the target nucleic acid are formed by bridge amplification.

60. The method of claim 56, wherein the multiple copies of the target nucleic acid are present in concatemers.

61. The method of claim 57, wherein the multiple copies of the target nucleic acid are present in concatemers.

62. The method of any one of claims 49-56, 58-61, wherein the array has at least about 10 features / cm 2 , 100 features / cm 2 , 500 features / cm 2 , 1,000 features / cm 2 , 5,000 features / cm 2 , 10,000 features / cm 2 , 50,000 features / cm 2 , 100,000 features / cm 2 , 1,000,000 features / cm 2 or 5,000,000 features / cm 2 density.

63. The method of claim 48, wherein the array has at least about 10 features / cm 2 , 100 features / cm 2 , 500 features / cm 2 , 1,000 features / cm 2 , 5,000 features / cm 2 , 10,000 features / cm 2 , 50,000 features / cm 2 , 100,000 features / cm 2 , 1,000,000 features / cm 2 or 5,000,000 features / cm 2 density.

64. The method of claim 57, wherein the array has at least about 10 features / cm 2 , 100 features / cm 2 , 500 features / cm 2 , 1,000 features / cm 2 , 5,000 features / cm 2 , 10,000 features / cm 2 , 50,000 features / cm 2 , 100,000 features / cm 2 , 1,000,000 features / cm 2 or 5,000,000 features / cm 2 density.

65. The method of any one of claims 13-18, 20, 23-27, 29-36, 38-39, 42-47, 49-56, 58-61, 63, or 64, wherein the method uses a first nucleotide type detected in a first channel, a second nucleotide type detected in a second channel, a third nucleotide type detected in both the first channel and the second channel, and a fourth nucleotide type that lacks labeling and is not detected or is minimally detected in either channel.

66. The method of claim 19, wherein the method uses a first nucleotide type detected in a first channel, a second nucleotide type detected in a second channel, a third nucleotide type detected in both the first channel and the second channel, and a fourth nucleotide type that lacks labeling and is not detected or is minimally detected in either channel.

67. The method of claim 21, wherein the method uses a first nucleotide type detected in a first channel, a second nucleotide type detected in a second channel, a third nucleotide type detected in both the first channel and the second channel, and a fourth nucleotide type that lacks labeling and is not detected or is minimally detected in either channel.

68. The method of claim 22, wherein the method uses a first nucleotide type detected in a first channel, a second nucleotide type detected in a second channel, a third nucleotide type detected in both the first channel and the second channel, and a fourth nucleotide type that lacks labeling and is not detected or is minimally detected in either channel.

69. The method of claim 28, wherein the method uses a first nucleotide type detected in a first channel, a second nucleotide type detected in a second channel, a third nucleotide type detected in both the first channel and the second channel, and a fourth nucleotide type that lacks labeling and is not detected or is minimally detected in either channel.

70. The method of claim 37, wherein the method uses a first nucleotide type detected in a first channel, a second nucleotide type detected in a second channel, a third nucleotide type detected in both the first channel and the second channel, and a fourth nucleotide type that lacks labeling and is not detected or is minimally detected in either channel.

71. The method of claim 40, wherein the method uses a first nucleotide type detected in a first channel, a second nucleotide type detected in a second channel, a third nucleotide type detected in both the first channel and the second channel, and a fourth nucleotide type that lacks labeling and is not detected or is minimally detected in either channel.

72. The method of claim 41, wherein the method uses a first nucleotide type detected in a first channel, a second nucleotide type detected in a second channel, a third nucleotide type detected in both the first channel and the second channel, and a fourth nucleotide type that lacks labeling and is not detected or is minimally detected in either channel.

73. The method of claim 48, wherein the method uses a first nucleotide type detected in a first channel, a second nucleotide type detected in a second channel, a third nucleotide type detected in both the first channel and the second channel, and a fourth nucleotide type that lacks labeling and is not detected or is minimally detected in either channel.

74. The method of claim 57, wherein the method uses a first nucleotide type detected in a first channel, a second nucleotide type detected in a second channel, a third nucleotide type detected in both the first channel and the second channel, and a fourth nucleotide type that lacks labeling and is not detected or is minimally detected in either channel.

75. The method of claim 62, wherein the method uses a first nucleotide type detected in a first channel, a second nucleotide type detected in a second channel, a third nucleotide type detected in both the first channel and the second channel, and a fourth nucleotide type that lacks labeling and is not detected or is minimally detected in either channel.

76. The method of any one of claims 13-18, 20, 23-27, 29-36, 38-39, 42-47, 49-56, 58-61, 63, or 64, wherein the method uses four different nucleotide types, wherein each nucleotide type has a spectrally distinct label.

77. The method of claim 19, wherein the method uses four different nucleotide types, wherein each nucleotide type has a spectrally distinct label.

78. The method of claim 21, wherein the method uses four different nucleotide types, wherein each nucleotide type has a spectrally distinct label.

79. The method of claim 22, wherein the method uses four different nucleotide types, wherein each nucleotide type has a spectrally distinct label.

80. The method of claim 28, wherein the method uses four different nucleotide types, wherein each nucleotide type has a spectrally distinct label.

81. The method of claim 37, wherein the method uses four different nucleotide types, wherein each nucleotide type has a spectrally distinct label.

82. The method of claim 40, wherein the method uses four different nucleotide types, wherein each nucleotide type has a spectrally distinct label.

83. The method of claim 41, wherein the method uses four different nucleotide types, wherein each nucleotide type has a spectrally distinct label.

84. The method of claim 43, wherein the method uses four different nucleotide types, wherein each nucleotide type has a spectrally distinct label.

85. The method of claim 48, wherein the method uses four different nucleotide types, wherein each nucleotide type has a spectrally distinct label.

86. The method of claim 57, wherein the method uses four different nucleotide types, wherein each nucleotide type has a spectrally distinct label.

87. The method of claim 62, wherein the method uses four different nucleotide types, wherein each nucleotide type has a spectrally distinct label.

88. The method of any one of claims 20, 23-27, 29-36, 38-39, 42-47, 49-56, 58-61, 63-64, 66-75, or 77-87, which provides a predetermined phase value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

89. The method of claim 21 , providing a prephase value of less than about 0.25, 0.24, 0.23, 0.22, 0.21 , 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

90. The method of claim 22, which provides a prephase value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

91. The method of claim 28, which provides a prephase value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

92. The method of claim 37, which provides a prephase value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

93. The method of claim 40, which provides a prephase value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

94. The method of claim 41 , providing a prephase value of less than about 0.25, 0.24, 0.23, 0.22, 0.21 , 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

95. The method of claim 48, which provides a prephase value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

96. The method of claim 57, which provides a prephase value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

97. The method of claim 62, which provides a prephase value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

98. The method of claim 65, which provides a prephase value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

99. The method of claim 76, which provides a prephase value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

100. The method of any one of claims 20, 23-27, 29-36, 38-39, 42-47, 49-56, 58-61, 63-64, 66-75, 77-87, or 89-99, which provides a phasing value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

101. The method of claim 21 , providing a phasing value of less than about 0.25, 0.24, 0.23, 0.22, 0.21 , 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

102. The method of claim 22, which provides a phasing value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

103. The method of claim 28, which provides a phasing value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

104. The method of claim 37, which provides a phasing value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

105. The method of claim 40, which provides a phasing value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

106. The method of claim 41 , providing a phasing value of less than about 0.25, 0.24, 0.23, 0.22, 0.21 , 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

107. The method of claim 48, which provides a phasing value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

108. The method of claim 57, which provides a phasing value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

109. The method of claim 62, which provides a phasing value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

110. The method of claim 65, which provides a phasing value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

111. The method of claim 76, which provides a phasing value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

112. The method of claim 88, which provides a phasing value of less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after 50, 100, or 150 cycles.

113. The method of any one of claims 13-18, 20, 23-27, 29-36, 38-39, 42-47, 49-56, 58-61, 63-64, 66-75, 77-87, 89-99, or 101-112, wherein incorporation of the nucleotide is accomplished by a DNA polymerase.

114. The method of claim 19, wherein the incorporation of the nucleotide is accomplished by a DNA polymerase.

115. The method of claim 21, wherein the incorporation of the nucleotide is accomplished by a DNA polymerase.

116. The method of claim 22, wherein the incorporation of the nucleotide is accomplished by a DNA polymerase.

117. The method of claim 28, wherein the incorporation of the nucleotide is accomplished by a DNA polymerase.

118. The method of claim 37, wherein the incorporation of the nucleotide is accomplished by a DNA polymerase.

119. The method of claim 40, wherein the incorporation of the nucleotide is accomplished by a DNA polymerase.

120. The method of claim 41, wherein the incorporation of the nucleotide is accomplished by a DNA polymerase.

121. The method of claim 48, wherein the incorporation of the nucleotide is accomplished by a DNA polymerase.

122. The method of claim 57, wherein the incorporation of the nucleotide is accomplished by a DNA polymerase.

123. The method of claim 62, wherein the incorporation of the nucleotide is accomplished by a DNA polymerase.

124. The method of claim 65, wherein the incorporation of the nucleotide is accomplished by a DNA polymerase.

125. The method of claim 76, wherein the incorporation of the nucleotide is accomplished by a DNA polymerase.

126. The method of claim 88, wherein the incorporation of the nucleotide is accomplished by a DNA polymerase.

127. The method of claim 100, wherein the incorporation of the nucleotide is accomplished by a DNA polymerase.

128. A kit comprising one or more nucleotides according to any one of claims 1 to 7.

129. The kit of claim 128, wherein at least one nucleotide is a nucleotide 5'-triphosphate having a structure covalently linked to the 3'-carbon of a 2' deoxyribose sugar. The 3'-OH blocking group.

130. The kit of claim 128, further comprising an enzyme and a buffer suitable for the action of the enzyme.

131. The kit of claim 129, further comprising an enzyme and a buffer suitable for the action of the enzyme.

132. The kit of claim 130, wherein The enzyme is a polymerase.

133. The kit of claim 131, wherein The enzyme is a polymerase.

134. The kit of claim 132, wherein the polymerase is a DNA polymerase.

135. The kit of claim 133, wherein the polymerase is a DNA polymerase.

136. The kit of any one of claims 128 to 135, comprising at least one nucleotide labeled with a detectable label according to any one of claims 1 to 7.

137. The kit of any one of claims 128 to 135, comprising two, three or four types of nucleotides labeled with a detectable label according to any one of claims 1 to 7.

138. The kit of claim 137, further comprising dark nucleotides that do not have a detectable label.

139. The kit of claim 137, wherein the different types of labeled nucleotides are labeled with spectrally distinguishable dye compounds.

140. The kit of claim 138, wherein the different types of labeled nucleotides are labeled with spectrally distinguishable dye compounds.

141. The kit of claim 137, wherein at least one type of nucleotide is labeled with two or more different dyes at the nucleoside bases.

142. The kit of claim 138, wherein at least one type of nucleotide is labeled with two or more different dyes at the nucleoside bases.

143. The kit of claim 139, wherein at least one type of nucleotide is labeled with two or more different dyes at the nucleoside bases.

144. The kit of claim 140, wherein at least one type of nucleotide is labeled with two or more different dyes at the nucleoside bases.

145. The kit of claim 137, wherein a first type of nucleotide is labeled with a first dye, a second type of nucleotide is labeled with a second dye that is spectrally different from the first dye, a third type of nucleotide is labeled with a mixture of the first and second dyes or a mixture of the first, second, and third dyes, and a fourth nucleotide contains no label.

146. The kit of any of claims 138-144, wherein a first type of nucleotide is labeled with a first dye, a second type of nucleotide is labeled with a second dye that is spectrally different from the first dye, a third type of nucleotide is labeled with a mixture of the first and second dyes or a mixture of the first, second, and third dyes, and a fourth nucleotide contains no label.

147. The kit of claim 137, comprising four types of nucleotides labeled with a detectable label, wherein the nucleotides labeled with a detectable label are A, C, T, and G, wherein each type of nucleotide contains a fluorescent label with a different fluorescence maximum, and each fluorescent label is distinguishable from the other three fluorescent labels.

148. The kit of any one of claims 128-135, 138-145, or 147, further comprising a cleavage solution comprising tris(hydroxypropyl)phosphine, a palladium (Pd) catalyst, ascorbic acid or a salt thereof, and a buffer selected from the group consisting of ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, carbonates, phosphates, borates, 2-dimethylaminoethanol (DMEA), 2-diethylaminoethanol (DEEA), N,N,N′,N′-tetramethylethylenediamine (TEMED), or N,N,N′,N′-tetraethylethylenediamine (TEEDA), and combinations thereof.

149. The kit of claim 136, further comprising a cleavage solution comprising tris(hydroxypropyl)phosphine, a palladium (Pd) catalyst, ascorbic acid or a salt thereof, and a buffer selected from the group consisting of ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, carbonate, phosphate, borate, 2-dimethylaminoethanol (DMEA), 2-diethylaminoethanol (DEEA), N,N,N′,N′-tetramethylethylenediamine (TEMED) or N,N,N′,N′-tetraethylethylenediamine (TEEDA), and combinations thereof.

150. The kit of claim 137, further comprising a cleavage solution comprising tris(hydroxypropyl)phosphine, a palladium (Pd) catalyst, ascorbic acid or a salt thereof, and a buffer selected from the group consisting of ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, carbonate, phosphate, borate, 2-dimethylaminoethanol (DMEA), 2-diethylaminoethanol (DEEA), N,N,N′,N′-tetramethylethylenediamine (TEMED) or N,N,N′,N′-tetraethylethylenediamine (TEEDA), and combinations thereof.

151. The kit of claim 146, further comprising a cleavage solution comprising tris(hydroxypropyl)phosphine, a palladium (Pd) catalyst, ascorbic acid or a salt thereof, and a buffer selected from the group consisting of ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, carbonates, phosphates, borates, 2-dimethylaminoethanol (DMEA), 2-diethylaminoethanol (DEEA), N,N,N′,N′-tetramethylethylenediamine (TEMED) or N,N,N′,N′-tetraethylethylenediamine (TEEDA), and combinations thereof.

152. The kit of any one of claims 128-135, 138-145, 147, or 149-151, further comprising a wash solution comprising a palladium scavenger.

153. The kit of claim 136, further comprising a wash solution comprising a palladium scavenger.

154. The kit of claim 137, further comprising a wash solution comprising a palladium scavenger.

155. The kit of claim 146, further comprising a wash solution comprising a palladium scavenger.

156. The kit of claim 148, further comprising a wash solution comprising a palladium scavenger.

157. The kit of any one of claims 128-135, 138-145, 147, 149-151, or 153-156, for multiplex sequencing by synthesis.

158. The kit of claim 136, which is used for multiplex sequencing by synthesis.

159. The kit of claim 137, which is used for multiplex sequencing by synthesis.

160. The kit of claim 146, which is used for multiplex sequencing by synthesis.

161. The kit of claim 148, which is used for multiplex sequencing by synthesis.

162. The kit of claim 152, which is used for multiplex sequencing by synthesis.

163. Use of the nucleotide sequence of any one of claims 1 to 7 in multiplex sequencing by synthesis.

164. The use of claim 163, wherein the sequencing step is repeated at least 50 times, at least 75 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, or at least 300 times.

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