Nucleosides and nucleotides with 3' acetal end groups

Abstract

The present invention relates to a nucleoside or nucleotide comprising a nucleobase attached to a detectable label via a cleavable linker, wherein the nucleoside or nucleotide comprises a ribose or 2'deoxyribose moiety and a 3'-OH capping group, and wherein the cleavable linker comprises a moiety having structure (I), wherein X and Y are each independently O or S; and R la , R 2b , R 2 , R 3a and R 3b are each independently H, halogen, unsubstituted or substituted C 1 ‑C 6 Alkyl, or C 1 ‑C 6 Also provided herein are methods for preparing such nucleotide molecules and nucleoside molecules, and the use of fully functionalized nucleotides containing 3' acetal capping groups for sequencing applications.

Description

[0001] Priority Applications Incorporated by Reference

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 042,240, filed on June 22, 2020, which is incorporated by reference in its entirety. Background of the Invention Technical Field

[0004] The present disclosure generally relates to nucleotides, nucleosides or oligonucleotides comprising a 3' acetal blocking group, and their use in polynucleotide sequencing methods. Also disclosed are methods of preparing 3' blocked nucleotides, nucleosides or oligonucleotides. Background Art

[0006] Progress in molecular research has always been driven, in part, by improvements in techniques used to characterize molecules or their biological reactions. In particular, the study of nucleic acids DNA and RNA has benefited from the development of techniques for sequence analysis and the study of hybridization events.

[0007] An example of the technology of improved nucleic acid research is to develop an assembly array of immobilized nucleic acids. These arrays are usually composed of a high-density matrix of polynucleotides fixed to 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, which is protected by a mask but exposed in a limited area to allow attachment of appropriately modified nucleotide phosphoramidites. Assembly arrays can also be manufactured by "spotting" known polynucleotides to predetermined positions on a solid support (e.g., Stimpson et al., Proc. Natl. Acad. Sci. 92: 6379-6383, 1995).

[0008] One method of determining the nucleotide sequence of nucleic acids bound to an array is called "sequencing by synthesis" or "SBS". Ideally, this technique for determining the nucleotide sequence of DNA requires the controlled incorporation (i.e., one at a time) of the correct complementary nucleotide opposite to the nucleic acid being sequenced. This enables accurate sequencing by adding nucleotides in multiple cycles, since only one nucleotide residue is sequenced at a time, thereby preventing uncontrolled incorporation of nucleotide sequences. The incorporated nucleotides are read using an appropriate label attached to the incorporated nucleotides, after which the label portion is removed and the next round of sequencing is performed.

[0009] To ensure that only a single nucleotide is incorporated, a structural modification (a "protecting group" or "capping group") is included in each labeled nucleotide that is added to the growing chain to ensure that only one nucleotide is incorporated. After the nucleotide with the protecting group 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.

[0010] In order to be used for DNA sequencing, nucleotides (usually nucleotide triphosphates) generally require 3'-hydroxyl protecting groups, so that once a base is added to the nucleotide, the polymerase used to incorporate it into the polynucleotide chain is prevented from continuing to replicate. There are many restrictions on the type of groups that can be added to the nucleotide and are still suitable. Protective groups should prevent additional nucleotide molecules from being added to the polynucleotide chain, while being able to be easily removed from the sugar part without damaging the polynucleotide chain. In addition, modified nucleotides need to be compatible with polymerase or another suitable enzyme for incorporating it into the polynucleotide chain. Therefore, ideal protecting groups must show long-term stability, can be effectively incorporated by polymerase, block the second or further incorporation of nucleotides, and can be removed under mild conditions (preferably under aqueous conditions) without damaging the polynucleotide structure.

[0011] 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. WO 2002 / 029003 describes a sequencing method that may include the use of an allyl protecting group to cap the 3'-OH group on the growing DNA chain in a polymerase reaction.

[0012] In addition, the development of a number of reversible protecting groups and methods for removing these protecting groups under DNA-compatible conditions were previously reported in International Application Publication Nos. WO 2004 / 018497 and WO 2014 / 139596, each of which is hereby incorporated by reference in its entirety. Summary of the invention

[0013] Some embodiments of the present disclosure relate to a nucleotide or nucleoside comprising a nucleobase attached to a detectable label via a cleavable linker, wherein the nucleoside or nucleotide comprises a ribose or 2'deoxyribose moiety and a 3'-OH capping group, and wherein the cleavable linker comprises a moiety having the structure:

[0014]

[0015] wherein X and Y are each independently O or S; and R 1a , R 1b , R 2 , R 3a and R 3b Each is independently H, halogen, unsubstituted or substituted C1-C6 alkyl, or C1-C6 haloalkyl.

[0016] Some embodiments of the present disclosure relate to oligonucleotides or polynucleotides comprising a 3'-OH terminated labeled nucleotide described herein.

[0017] Some embodiments of the present disclosure relate to a method for preparing a growing polynucleotide complementary to a target single-stranded polynucleotide in a sequencing reaction, the method comprising incorporating a nucleotide molecule as described herein into the growing complementary polynucleotide, wherein incorporation of the nucleotide prevents any subsequent nucleotide from being introduced into the growing complementary polynucleotide. In some embodiments, incorporation of nucleotides 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).

[0018] Some other embodiments of the present disclosure relate to a method for determining the sequence of a target single-stranded polynucleotide, the method comprising:

[0019] (a) incorporating a nucleotide as described herein into a copy polynucleotide chain complementary to at least a portion of a target polynucleotide chain;

[0020] (b) detecting the identity of the nucleotide incorporated into the copied polynucleotide chain; and

[0021] (c) chemically removing the label and 3'-OH blocking group from the nucleotides incorporated into the copy polynucleotide chain.

[0022] In some embodiments, the detection step includes determining the identity of the nucleotides incorporated into the copy polynucleotide chain by measuring the fluorescent signal from the detectable label one or more times. In some embodiments, the sequencing method also includes (d) using a post-cleavage washing solution to wash the chemically removed label and 3'-OH blocking group from the copy polynucleotide chain. In some embodiments, this washing step also removes unincorporated nucleotides. In other embodiments, the method may include a separate washing step for washing unincorporated nucleotides from the copy polynucleotide chain before step (b). In some such embodiments, the 3'-OH blocking group and detectable label of the incorporated nucleotide are removed before the next complementary nucleotide is introduced. In other embodiments, the 3'-OH blocking group and detectable label are removed in a single step of the chemical reaction. In some embodiments, the sequential 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.

[0023] Other embodiments of the present disclosure relate to a kit comprising a plurality of nucleotides or nucleoside molecules as described herein and packaging materials thereof. The nucleotides, nucleosides, oligonucleotides or kits set forth herein can be used to detect, measure or identify a biological system (including, for example, its process or component). Exemplary techniques that can employ these nucleotides, oligonucleotides or kits include sequencing, 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 determination or protein activity determination). The purposes can be on an automated instrument (such as an automated sequencing instrument) for performing a particular technique. The sequencing instrument can include two or more lasers operating at different wavelengths to distinguish different detectable labels. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a line graph comparing the 3′-OH deblocking efficiency of [(allyl)PdCl]2 and Na2PdCl4 when using various ratios of tri(hydroxypropyl)phosphine (THP).

[0025] Figure 2 is a line graph showing the change in prephasing percentage values ​​over time for a standard fully functionalized nucleotide (ffN) having a LN3 linker moiety and a 3'-O-azidomethyl capping group compared to ffN having an AOL linker moiety and a 3'-AOM capping group.

[0026] Figure 3 We show the effects of Illumina's PCR products on the post-cleavage wash step using fully functionalized nucleotides (ffN) with 3'-AOM capping groups with and without palladium scavengers. Comparison of phasing values ​​on instruments.

[0027] Figure 4 The results show that when using fully functionalized nucleotides (ffN) with 3'-AOM capping groups and AOL linker moieties, Illumina's Key sequencing metrics on the instrument including phasing, prephasing, and error rate, compared to the same sequencing metrics when using a standard ffN with a 3'-O-azidomethyl capping group.

[0028] Figure 5 The results show that when using fully functionalized nucleotides (ffN) with 3'-AOM capping groups and AOL linker moieties, Illumina's Comparison of key sequencing metrics across instruments, including phasing and prephasing.

[0029] Figure 6 The results show that when using fully functionalized nucleotides (ffN) with 3'-AOM capping groups and AOL linker moieties, Illumina's Key sequencing metrics on the instrument including phasing, prephasing, and error rate, compared to the same sequencing metrics when using a standard ffN with a 3'-O-azidomethyl capping group.

[0030] Fig. 7A and Figure 7B The results of the Illumina iSeq sequencing using fully functionalized nucleotides (ffN) with 3'-AOM capping groups and AOL linker moieties are shown in Figure 2. TM Error rate and Q30 sequencing metrics for 2 × 300 sequencing runs on the instrument, compared to the same sequencing metrics when using standard ffN with 3'-O-azidomethyl end-capping groups.

[0031] Fig. 8A and Figure 8B The results of the Illumina iSeq sequencing using fully functionalized nucleotides (ffN) with 3'-AOM capping groups and AOL linker moieties are shown in Figure 2. TM Error rate and Q30 sequencing metrics for 2 × 150 sequencing runs on the instrument, compared to the same sequencing metrics when using standard ffN with 3'-O-azidomethyl end-capping groups.

[0032] 9A to 9EThe main sequencing metrics (phasing, signal decay percentage, error rate) are shown as a function of blue laser power when the green laser power is constant. These sequencing experiments were performed using fully functionalized nucleotides (ffN) with 3'-AOM capping groups and AOL linker moieties on an Illumina NovaSeq in the presence of the palladium scavenger L-cysteine. TM The sequencing was performed on the WT 5000 instrument, compared to the same sequencing metrics when using the standard protocol and ffN with a 3'-O-azidomethyl capping group.

[0033] Fig.10 We show that the Illumina iSeq DNA sequencing technology can be used to detect the presence of a fully functionalized nucleotide (ffN) with a 3'-AOM capping group and an AOL linker moiety, using the same palladium cleavage mixture in the first step of chemical linearization by SBS. TM Key sequencing metrics (%PF, error rate, Q30, and signal decay) on the instrument (1 x 150 cycles) and compared to SBS at the same ffN using standard enzymatic linearization. DETAILED DESCRIPTION

[0034] Embodiments of the present disclosure relate to nucleosides and nucleotides with 3' acetal capping groups for sequencing applications (e.g., sequencing by synthesis (SBS)). In some embodiments, the nucleoside or nucleotide comprises a label covalently attached thereto by a cleavable linker, wherein the cleavable linker comprises an acetal portion that allows the cleavage of the 3' acetal capping group and the label in a single reaction step. The 3' acetal capping group provides improved stability during the synthesis of fully functionalized nucleotides (ffN), and provides great stability during preparation in solution, storage, and operation on a sequencing instrument. In addition, the 3' acetal capping group described herein can also achieve low prephasing, lower signal attenuation, thereby improving data quality, which enables longer readings from sequencing applications.

[0035] definition

[0036] 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" as well as other forms such as "include / includes / included" is not limiting. The use of the term "have" as well as other forms such as "have / has / had" is not limiting. As used in this specification, the terms "comprise(s)" and "comprising" are to be interpreted as having an open-ended meaning, whether in a transitional phrase or in the body of the claims. That is, the above terms are to 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 also include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the listed features or components, but may also include additional features or components.

[0037] Where a range of values ​​is provided, it is understood that the upper and lower limit of that range and every intervening value between the upper and lower limits is encompassed within the embodiments.

[0038] As used herein, common organic abbreviations are defined as follows:

[0039] ℃ Temperature value in degrees Celsius

[0040] dATP deoxyadenosine triphosphate

[0041] dCTP deoxycytidine triphosphate

[0042] dGTP deoxyguanosine triphosphate

[0043] dTTP deoxythymidine triphosphate

[0044] ddNTP dideoxynucleotide triphosphate

[0045] ffN Fully functionalized nucleotides

[0046] RT Room temperature

[0047] SBS Sequencing by Synthesis

[0048] SM Starting Material

[0049] As used herein, the term "array" refers to a colony of following different probe molecules: these probe molecules are attached 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 located at different addressable positions on a substrate. Alternatively, or in addition thereto, the array can include separate substrates carrying different probe molecules, wherein different probe molecules can be identified according to the position of the substrate on the surface to which the substrate is attached or according to the position of the substrate in the liquid. The exemplary arrays where the substrate is separated are located on the surface include but are not limited to those arrays including beads in the hole, as described in, for example, U.S. Patent No. 6,355,431B1, US 2002 / 0102578 and PCT Publication No. WO 00 / 63437. For example, in U.S. Patent No. 6,524,793, an exemplary format that can be used in the present invention to distinguish beads in a liquid array, for example, using a microfluidic device such as a fluorescence activated cell sorter (FACS), is described. Additional examples of arrays that can be used in the present invention include, but are not limited to, those described in U.S. Patent Nos. 5,429,807, 5,436,327, 5,561,071, 5,583,211, 5,658,734, 5,837,858, 5,874,219, 5,919,523, 6,136,269, 6,287,768, 6,287,776, 6,288,220, 6,297,006, 6,291,193, 6,346,413, 6,416,949, 6,482,591, 6,514,751, and 6,610,482, WO 93 / 17126, WO 95 / 11995, WO 95 / 35505, EP 742 287 and EP 799 897.

[0050] As used herein, the term "covalently attached" or "covalently bonded" 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 a functionalized surface of a substrate, as opposed to adhering to the surface via other means (e.g., adhesion or electrostatic interactions). It should be understood that a polymer covalently attached to a surface may also be bonded via means other than covalent attachment.

[0051] As used herein, any "R" group refers to a substituent that can be attached to the specified 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 these atoms, the intervening bonds and the two R groups is the ring mentioned. For example, when the following substructure is present:

[0052]

[0053] And R 1 and R 2 is defined as being selected from the group consisting of 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 may be selected from hydrogen or alkyl, or alternatively, the substructure has the structure:

[0054]

[0055] wherein A is an aromatic ring or a carbocyclic group containing a double bond as depicted.

[0056] It is understood that certain group naming conventions may include either a monoradical or a diradical, 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".

[0057] As used herein, the term "halogen" or "halo" means any of the radioactive stable atoms of column 7 of the periodic table, for example, fluorine, chlorine, bromine or iodine, with fluorine and chlorine being preferred.

[0058] As used herein, "C" wherein "a" and "b" are integers a To C b" refers 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 can contain from "a" to "b" (inclusive) carbon atoms. For example, a "C1 to C4 alkyl" group refers to all alkyl groups having 1 to 4 carbons, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH 2CH2-, CH3CH2CH(CH3)- and (CH3)3C-; C3 to C4 cycloalkyl groups refer to all cycloalkyl groups having 3 to 4 carbon atoms, i.e. cyclopropyl and cyclobutyl. Similarly, a "4 to 6 membered heterocyclyl" group refers to all heterocyclyl groups having 4 to 6 total ring atoms, such as azetidine, oxetane, oxazoline, pyrrolidine, piperidine, piperazine, morpholine, etc. If for an alkyl, alkenyl, alkynyl, cycloalkyl or aryl group Without specifying "a" and "b", the broadest range described in these definitions will be assumed. As used herein, the term "C1-C6" includes C1, C2, C3, C4, C5, and C6, as well as ranges defined by any two of these numbers. For example, C1-C6 alkyl includes C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl, C2-C6 alkyl, C1-C3 alkyl, and the like. Similarly, C2-C6 alkenyl includes C2 C3-C8 cycloalkyl groups include hydrocarbon rings containing 3, 4, 5, 6, 7 and 8 carbon atoms or ranges defined by any two numbers, such as C3-C7 cycloalkyl or C5-C6 cycloalkyl.

[0059] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., does not contain double bonds and triple bonds). An alkyl group can have 1 to 20 carbon atoms (whenever it appears in this article, a numerical range such as "1 to 20" refers to each integer in the given range; for example, "1 to 20 carbon atoms" means that the alkyl group can be composed of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, but this definition also covers the term "alkyl" in which no numerical range is specified). The alkyl group can also be a medium-sized alkyl group with 1 to 9 carbon atoms. The alkyl group can also be a low alkyl group with 1 to 6 carbon atoms. The alkyl group can be named "C 1- C4 alkyl" or similar designations. By way of example only, "C 1-"C6 alkyl" means that there are one to six carbon atoms in the alkyl chain, that is, the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. Typical alkyl groups include, but are by no means limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like.

[0060] As used herein, "alkoxy" refers to a radical of the formula --OR (wherein R is an alkyl radical as defined above), such as "C 1- "C9 alkoxy" includes, but is not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy and tert-butoxy, etc.

[0061] As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain containing one or more double bonds. An alkenyl group can have from 2 to 20 carbon atoms, but this definition also encompasses the term "alkenyl" where no numerical range is specified. An alkenyl group can also be a medium-sized alkenyl group having from 2 to 9 carbon atoms. An alkenyl group can also be a low-sized alkenyl group having from 2 to 6 carbon atoms. An alkenyl group can be designated as "C 2- C6 alkenyl" or similar designations. By way of 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 the group consisting of ethenyl, propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl, buta-1,2-dienyl and buta-1,2-dien-4-yl. Typical alkenyl groups include, but are by no means limited to, ethenyl, propenyl, butenyl, pentenyl and hexenyl, etc.

[0062] As used herein, "alkynyl" refers to a straight or branched hydrocarbon chain containing one or more triple bonds. Alkynyl groups can have from 2 to 20 carbon atoms, but this definition also covers the term "alkynyl" where no numerical range is specified. Alkynyl groups can also be medium-sized alkynyl groups having from 2 to 9 carbon atoms. Alkynyl groups can also be low-sized alkynyl groups having from 2 to 6 carbon atoms. Alkynyl groups can be designated as "C 2- C6 alkynyl" or similar designations. By way of example only, "C 2- "C6 alkynyl" means that there are two to six carbon atoms in the alkynyl chain, that is, the alkynyl chain is selected from the group consisting of ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl and 2-butynyl. Typical alkynyl groups include, but are by no means limited to, ethynyl, propynyl, butynyl, pentynyl and hexynyl, etc.

[0063] 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 backbone of the chain. A heteroalkyl group may have from 1 to 20 carbon atoms, but this definition also encompasses the term "heteroalkyl" where no numerical range is specified. A heteroalkyl group may also be a medium-sized heteroalkyl group having from 1 to 9 carbon atoms. A heteroalkyl group may also be a low-sized heteroalkyl group having from 1 to 6 carbon atoms. A heteroalkyl group may be designated as "C 1- C6 heteroalkyl" or similar designations. A 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 in addition there are one or more heteroatoms in the backbone of the chain.

[0064] The term "aromatic" refers to a ring or ring system having a conjugated π electron system, and includes both carbocyclic aromatic groups (e.g., phenyl) and heterocyclic aromatic groups (e.g., pyridine). The term includes monocyclic groups or fused-ring polycyclic (i.e., rings that share adjacent pairs of atoms) groups, provided that the entire ring system is aromatic.

[0065] 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 aryl is a ring system, each ring in the ring system is aromatic. An aryl group can have 6 to 18 carbon atoms, but this definition also encompasses the term "aryl" where no numerical range is specified. In some embodiments, an aryl group has 6 to 10 carbon atoms. An aryl group can be designated as "C 6- C 10 Aryl", "C6 or C 10 Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl.

[0066] "Aralkyl" or "arylalkyl" is an aryl group attached as a substituent through an alkylene group, such as "C 7-14 "Aralkyl" and the like, including but not limited to benzyl, 2-phenylethyl, 3-phenylpropyl and naphthylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., C 1- C6 alkylene group).

[0067] As used herein, "heteroaryl" refers to an aromatic ring or ring system (i.e., two or more condensed 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 skeleton. When heteroaryl is a ring system, each ring in the ring system is aromatic. The heteroaryl group can have 5 to 18 ring members (i.e., the number of atoms (including carbon atoms and heteroatoms) constituting the ring skeleton), but the present definition also encompasses the term "heteroaryl" in which no numerical range is specified. In some embodiments, the heteroaryl group has 5 to 10 ring members or 5 to 7 ring members. The heteroaryl group can be named "5 to 7 yuan heteroaryl", "5 to 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, quinolyl, isoquinolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothiophenyl.

[0068] "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, furanylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., C 1- C6 alkylene group).

[0069] As used herein, "carbocyclyl" means a non-aromatic cyclic ring or ring system containing only carbon atoms in the ring system skeleton. When the carbocyclyl is a ring system, two or more rings can be joined 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. Therefore, carbocyclyl includes cycloalkyl, cycloalkenyl and cycloalkynyl. The carbocyclyl group can have 3 to 20 carbon atoms, but this definition also covers the term "carbocyclyl" in which no numerical range is specified. The carbocyclyl group can also be a medium-sized carbocyclyl with 3 to 10 carbon atoms. The carbocyclyl group can also be a carbocyclyl with 3 to 6 carbon atoms. The carbocyclyl group can be named "C 3- C6 carbocyclyl" or similar designations. Examples of carbocyclyl rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicyclo[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl.

[0070] As used herein, "cycloalkyl" means a fully saturated carbocyclyl ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0071] As used herein, "heterocyclyl" refers to a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring backbone. The heterocyclyl group can be joined together in a fused, bridged or spiro 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 a non-aromatic ring or an aromatic ring in the ring system. The heterocyclyl group can have 3 to 20 ring members (i.e., the number of atoms (including carbon atoms and heteroatoms) constituting the ring backbone), but this definition also covers the term "heterocyclyl" in which no numerical range is specified. The heterocyclyl group can also be a medium-sized heterocyclyl group with 3 to 10 ring members. The heterocyclyl group can also be a heterocyclyl group with 3 to 6 ring members. The heterocyclyl group can be named "3 to 6-membered heterocyclyl" or similar names. In preferred six-membered monocyclic heterocyclyls, the heteroatoms are selected from one to a maximum of three from 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, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidinyl, pyrrolidinedionyl, 4-piperidinone, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxinyl, 1,4-dioxinyl, 1,4-dioxinyl, 1,3-oxathianyl, 1,4-oxathianyl, 1,4-oxathianyl, 2 H-1,2-oxazinyl, trioxanyl, hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithienyl, 1,3-dithianyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinone, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl, thiomorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl and tetrahydroquinoline.

[0072] As used herein, "alkoxyalkyl" or "(alkoxy)alkyl" refers to an alkoxy group attached through an alkylene group, such as C 2- C8 alkoxyalkyl, or (C1-C6 alkoxy)C1-C6 alkyl, such as –(CH2) 1-3 -OCH3.

[0073] As used herein, "-O-alkoxyalkyl" or "-O-(alkoxy)alkyl" refers to an alkoxy group attached via an -O-(alkylene) group, such as -O-(C1-C6alkoxy)C1-C6alkyl, e.g. -O-(CH2) 1-3 -OCH3.

[0074] As used herein, "(heterocyclyl)alkyl" refers to a heterocycle or heterocyclyl group as defined above attached as a substituent via an alkylene group as defined above. The alkylene group and heterocyclyl group of the (heterocyclyl)alkyl may be substituted or unsubstituted. Examples include, but are not limited to, (tetrahydro-2H-pyran-4-yl)methyl, (piperidin-4-yl)ethyl, (piperidin-4-yl)propyl, (tetrahydro-2H-thiopyran-4-yl)methyl, and (1,3-thiazin-4-yl)methyl.

[0075] As used herein, "(cycloalkyl)alkyl" or "(carbocyclyl)alkyl" refers to a cycloalkyl or carbocyclyl group (as defined herein) attached as a substituent via an alkylene group. Examples include, but are not limited to, cyclopropylmethyl, cyclobutylmethyl, cyclopentylethyl, and cyclohexylpropyl.

[0076] An "O-carboxyl" group is one in which R is selected from hydrogen, C 1- C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C 3- C7 carbocyclic group, C 6- C 10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl "-OC(=O)R" groups are as defined herein.

[0077] A "C-carboxyl" group is one in which R is selected from the group consisting of hydrogen, C 1- C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C 3- C7 carbocyclic group, C 6- C 10 The "-C(=O)OR" group of the group consisting of aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl, as defined herein. Non-limiting examples include carboxyl (ie, -C(=O)OH).

[0078] A "sulfonyl" group is a group in which R is selected from hydrogen, C 1- C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C 3- C7 carbocyclic group, C 6- C 10 The "-SO2R" group of aryl, 5- to 10-membered heteroaryl and 3- to 10-membered heterocyclyl are as defined herein.

[0079] A "sulfinyl" group refers to a "-S(=O)OH" group.

[0080] An "S-sulfonamido" group refers to a group in which R A and RB are each independently selected from hydrogen, C 1- C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C 3- C7 carbocyclic group, C 6- C 10 "-SO2NR" of aryl, 5- to 10-membered heteroaryl and 3- to 10-membered heterocyclic group A R B " group, as defined herein.

[0081] An "N-sulfonamido" group is one in which R A and R B are each independently selected from hydrogen, C 1- C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C 3- C7 carbocyclic group, C 6- C 10 "-N(R)" of aryl, 5- to 10-membered heteroaryl and 3- to 10-membered heterocyclic group A )SO2R B " group, as defined herein.

[0082] A "C-amido" group is one in which R A and R B are each independently selected from hydrogen, C 1- C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C 3- C7 carbocyclic group, C 6- C 10 "-C(=O)NR" of aryl, 5- to 10-membered heteroaryl and 3- to 10-membered heterocyclic group A R B " group, as defined herein.

[0083] An "N-amido" group is one in which R A and R B are each independently selected from hydrogen, C 1- C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C 3- C7 carbocyclic group, C 6- C 10 "-N(R)" of aryl, 5- to 10-membered heteroaryl and 3- to 10-membered heterocyclic group A )C(=O)R B " group, as defined herein.

[0084] An "amino" group is one in which R A and RB are each independently selected from hydrogen, C 1- C6 alkyl, C 2- C6 alkenyl, C 2- C6 alkynyl, C 3- C7 carbocyclic group, C 6- C 10 "-NR" of aryl, 5- to 10-membered heteroaryl and 3- to 10-membered heterocyclic group A R B " group, as defined herein. Non-limiting examples include free amino groups (ie, -NH2).

[0085] An "aminoalkyl" group refers to an amino group attached through an alkylene group.

[0086] An "(alkoxy)alkyl" group refers to an alkoxy group attached through an alkylene group, such as "(C 1- C6 alkoxy)C 1- C6 alkyl" etc.

[0087] The term "hydroxyl" as used herein refers to an -OH group.

[0088] The term "cyano" as used herein refers to a "-CN" group.

[0089] The term "azido" as used herein refers to a -N3 group.

[0090] As used herein, the term "propargylamine" refers to an amino group substituted with a propargyl group (HC≡C-CH2-). When propargylamine is used as a divalent moiety in the context, it includes -C≡C-CH2-NR A -, where R A is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclic, C6-C 10 Aryl, 5- to 10-membered heteroaryl and 3- to 10-membered heterocyclyl are as defined herein.

[0091] As used herein, the term "propargylamide" refers to a C-amido or N-amido group substituted with a propargyl group (HC≡C-CH2-). When propargylamide is used as a divalent moiety in the context, it includes -C≡C-CH2-NR A -C(=O)- or -C≡C-CH2-C(=O)-NR A -, where R A is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclic, C6-C 10 Aryl, 5- to 10-membered heteroaryl and 3- to 10-membered heterocyclyl are as defined herein.

[0092] As used herein, the term "allylamine" refers to an amino group substituted with an allyl group (CH2=CH-CH2-). When allylamine is used as a divalent moiety in the context, it includes -CH=CH-CH2-NR A -, where R A is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclic, C6-C 10 Aryl, 5- to 10-membered heteroaryl and 3- to 10-membered heterocyclyl are as defined herein.

[0093] As used herein, the term "allylamide" refers to a C-amido or N-amido group substituted with an allyl group (CH2=CH-CH2-). When allylamide is used as a divalent moiety in the context, it includes -CH=CH-CH2-NR A -C(=O)- or -CH=CH-CH2-C(=O)-NR A -, where R A is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclic, C6-C 10 Aryl, 5- to 10-membered heteroaryl and 3- to 10-membered heterocyclyl are as defined herein.

[0094] When a group is described as "optionally substituted", it can be unsubstituted or substituted. Similarly, when a group is described as "substituted", the substituent can be selected from one or more of the substituents indicated. As used herein, the substituted group is derived from an unsubstituted parent group in which there is an exchange of one or more hydrogen atoms 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, which are independently selected from: C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 heteroalkyl, C3-C7 carbocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7 carbocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3- to 10-membered heterocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3- to 10-membered heterocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C1-C6 alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), (5- to 10-membered heteroaryl)C1-C6 alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5- to 10-membered heteroaryl(C1-C6)alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), halo, -CN, hydroxy, C1-C6 alkoxy, (C1-C6 alkoxy)C1-C6 alkyl, -O(C1-C6 alkoxy)C1-C6 alkyl; (C1-C6 haloalkoxy)C1-C6 alkyl; -O(C1-C6 haloalkoxy)C1-C6 alkyl; aryloxy, mercapto (sulfhydryl), halo (C1-C6) alkyl (e.g.,

[0095] -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-acylamino, N-acylamino, S-sulfonylamino, N-sulfonylamino, C-carboxyl, O-carboxyl, acyl, cyanate, isocyanate, thiocyanate, isothiocyanate, sulfinyl, sulfonyl, -SO3H, sulfinyl, -OSO2C 1-4 Alkyl, monophosphate, diphosphate, triphosphate and oxo (=0). Wherever a group is described as "optionally substituted", the group may be substituted with the above substituents.

[0096] When a substituent is described as a diradical (i.e., having two points of attachment to the rest of the molecule), it is understood that the substituent may be attached in any orientation unless otherwise indicated. Thus, for example, a substituent depicted as -AE- or include substituents oriented such that A is attached at the leftmost point of attachment of the molecule, as well as substituents in which A is attached at the rightmost point of attachment of the molecule. In addition, if a group or substituent is depicted as and L is defined as an optionally present linker moiety; then when L is absent (or missing), such group or substituent is equal to

[0097] 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, and in DNA it is deoxyribose, i.e., a sugar lacking the hydroxyl groups present in ribose. The nitrogenous heterocyclic bases may be purine or pyrimidine bases. Purine bases include adenine (A) and guanine (G) and their modified derivatives or analogs. Pyrimidine bases include cytosine (C), thymine (T) and uracil (U) and their modified derivatives or analogs. The C-1 atom of deoxyribose is bonded to the N-1 of pyrimidine or the N-9 of purine.

[0098] As used herein, "nucleoside" is structurally similar to a nucleotide, but lacks a phosphate moiety. An example of a nucleoside analog is a nucleoside analog in which a label is attached to a base and no phosphate group is attached to a sugar molecule. The term "nucleoside" is used herein in the conventional sense as understood by those skilled in the art. 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 may have a substituted base and / or sugar moiety. In addition, nucleosides may be incorporated into larger DNA and / or RNA polymers and oligomers.

[0099] The term "purine base" is used herein with the common meaning understood by those skilled in the art, and includes its tautomers. Similarly, the term "pyrimidine base" is used herein 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, deazapurine, adenine, 7-deazaadenine, guanine, 7-deazaguanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g., 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 (e.g., 5-methylcytosine).

[0100] As used herein, when oligonucleotide or polynucleotide are described as " comprising " nucleoside as described herein or nucleotide or with nucleoside as described herein or nucleotide labeling, this means that nucleoside as described herein or nucleotide and oligonucleotide or polynucleotide form covalent bond.Similarly, when nucleoside or nucleotide are described as a part of oligonucleotide or polynucleotide, such as " being incorporated into " oligonucleotide or polynucleotide, this means that nucleoside as described herein or nucleotide and oligonucleotide or polynucleotide form covalent bond.In some such embodiments, covalent bond is formed between 5' phosphate groups of the nucleotide of phosphodiester bond between 3' carbon atom of oligonucleotide or polynucleotide and 5' carbon atom of nucleotide described in this article.

[0101] As used herein, the term "cleavable linker" is not intended to imply that the entire linker needs to be removed. The cleavage site can be located at a position on the linker that ensures that a portion of the linker remains attached to the detectable label and / or nucleoside or nucleotide moiety after cleavage.

[0102] As used herein, "derivatives" or "analogs" means synthetic nucleotides or nucleoside derivatives with modified base moieties and / or modified sugar moieties. Such derivatives and analogs are discussed in, for example, Scheit, NucleotideAnalogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90: 543-584, 1990. Nucleotide analogs can also include modified phosphodiester bonds, including phosphorothioate bonds, phosphorodithioate bonds, alkylphosphonate bonds, aniline phosphate bonds and phosphoramidate bonds. As used herein, "derivatives", "analogs" and "modified" can be used interchangeably, and are covered by the terms "nucleotides" and "nucleosides" defined herein.

[0103] As used herein, the term "phosphate" is used with the ordinary meaning understood by those skilled in the art and includes its protonated form (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.

[0104] As used herein, the term "protecting group" refers to any atom or group of atoms added to a molecule in order to prevent existing groups in the molecule from undergoing undesired chemical reactions. Sometimes, "protecting group" and "capping group" can be used interchangeably.

[0105] As used herein, the term "phasing" refers to a phenomenon in SBS, which is caused by the incomplete removal of 3' terminators and fluorophores and the failure to complete the incorporation of a portion of the DNA chain within the cluster by the polymerase in a given sequencing cycle. Prephasing is caused by the incorporation of nucleotides that do not have an effective 3' terminator, wherein the incorporation event occurs 1 cycle in advance due to termination failure. Phasing and prephasing cause the measured signal intensity of a specific cycle to be composed of the signal from the current cycle and the noise from the previous and next cycles. As the number of cycles increases, the sequence score of each cluster affected by phasing and prephasing increases, hindering the identification of the correct base. Prephasing can be caused by the presence of trace amounts of unprotected or uncapped 3'-OH nucleotides during sequencing by synthesis (SBS). Unprotected 3'-OH nucleotides may be generated during the manufacturing process, or they may be generated during the storage process and the reagent handling process. Therefore, there are nucleotide analogs that reduce the incidence of prephasing, which is surprising and provides a huge advantage over existing nucleotide analogs in SBS applications. For example, the provided nucleotide analogs can result in shorter SBS cycle times, lower phasing and prephasing values, and longer sequencing read lengths.

[0106] Nucleosides or nucleotides having a 3' acetal end capping group

[0107] Some embodiments of the present disclosure relate to a nucleotide or nucleoside molecule comprising a nucleobase attached to a detectable label via a cleavable linker, and a ribose or deoxyribose moiety, wherein the cleavable linker comprises a moiety having the structure:

[0108]

[0109] wherein X and Y are each independently O or S; and R 1a , R 1b , R 2 , R 3a and R 3bEach is independently H, halogen, unsubstituted or substituted C1-C6 alkyl, or C1-C6 haloalkyl. In some embodiments, the ribose or deoxyribose moiety comprises a 3′-OH protecting group as described herein. In some embodiments, the cleavable linker may also include L 1 or L 2 , or both, where L 1 and L 2 This is described in detail below.

[0110] In some embodiments, a nucleoside or nucleotide described herein comprises or has the structure of Formula (I):

[0111]

[0112] Wherein B is a nucleobase;

[0113] R 4 is H or OH;

[0114] R 5 is H, 3′-OH end-capping group or phosphoramidite;

[0115] R 6 is H, a monophosphate, a diphosphate, a triphosphate, a thiophosphate, a phosphate analog, a reactive phosphorus-containing group, or a hydroxyl protecting group;

[0116] L is and

[0117] L 1 and L 2 Each is independently an optionally present linker moiety.

[0118] In some embodiments of the cleavable linker moieties described herein, X and Y are each O. In some other embodiments, X is S and Y is O, or X is O and Y is S. In some embodiments, R 1a , R 1b , R 2 , R 3a and R 3b Each is H. In other embodiments, R 1a , R 1b , R 2 , R 3a and R 3b At least one of is halogen (e.g., fluorine, chlorine) or unsubstituted C1-C6 alkyl (e.g., methyl, ethyl, isopropyl, isobutyl or tert-butyl). In some such cases, R 1a and R 1b Each is H, and R 2 , R 3a and R 3bAt least one of them is unsubstituted C1-C6 alkyl or halogen (e.g., R 2 is an unsubstituted C1-C6 alkyl group, and R 3a and R 3b Each is H; or R 2 is H, and R 3a and R 3b One or both of are halogen or unsubstituted C1-C6 alkyl). In one embodiment, the cleavable linker or L comprises (“AOL” connector portion).

[0119] In some embodiments of nucleosides or nucleotides described herein, the nucleobase ("B" in formula (I)) is a purine (adenine or guanine), a deazapurine, or a pyrimidine (e.g., cytosine, thymine, or uracil). In other embodiments, the deazapurine is a 7-deazapurine (e.g., 7-deazaadenine or 7-deazaguanine). Non-limiting examples of B include Or their optionally substituted derivatives and analogs. In some other embodiments, the labeled nucleobase comprises the structure

[0120] In some embodiments of the nucleosides or nucleotides described herein, the ribose or deoxyribose moiety comprises a 3′-OH capping group (ie, R 5 In some embodiments, the 3′-OH end-capping group or R 5 for And where R a , R b , R c , R d and R e are each independently H, halogen, unsubstituted or substituted C1-C6 alkyl, or C1-C6 haloalkyl. a and R b Each is H, and R c , R d and R e At least one of is independently halogen (e.g., fluorine, chlorine) or unsubstituted C1-C6 alkyl (e.g., methyl, ethyl, isopropyl, isobutyl or tert-butyl). c is an unsubstituted C1-C6 alkyl group, and R d and R e Each is H. In another example, R c is H, and R d and R e One or both of R is halogen or unsubstituted C1-C6 alkyl.5 Other non-limiting embodiments include In one embodiment, R 5 for Together with the 3′ oxygen group, it forms ("AOM") group, which is attached to the 3' carbon atom of the ribose or deoxyribose moiety. In other embodiments, the 3'-OH capping group or R 5 Can include an azido moiety (e.g., -CH2N3 or azidomethyl). Additional embodiments of 3'-OH end-capping groups are described in U.S. Patent Publication No. 2020 / 0216891A1, which is incorporated by reference in its entirety, including additional examples of 3' acetal end-capping groups such as These capping groups are attached to the 3' carbon atom of the ribose or deoxyribose moiety.

[0121] In some other embodiments of the nucleosides or nucleotides described herein, R in formula (I) 5 is a phosphoramidite. In such embodiments, R 6 is an acid-cleavable hydroxyl protecting group that allows subsequent monomer coupling under automated synthesis conditions.

[0122] In some embodiments of the nucleosides or nucleotides described herein, L 1 exists, and L 1 In some other embodiments, L 1 Including or for In other embodiments, the asterisk * indicates that L 1 The point of attachment to the nucleobase (eg, C5 for a pyrimidine base, or C7 for a 7-deazapurine base).

[0123] In some embodiments, the nucleotides described herein are fully functionalized nucleotides (ffN) comprising a 3′-OH blocking group described herein and a dye compound covalently attached to a nucleobase via a cleavable linker described herein, wherein the cleavable linker comprises a nucleotide having the structure L 1 , and * indicates L 1With the attachment point of nucleobase (for example, C5 position of cytosine, thymine or uracil base, or C7 position of 7-deazaadenine or 7-deazaguanine). In some cases, the ffN with allylamine or allylamide linker part described herein is also referred to as ffN-DB or ffN-(DB), wherein "DB" refers to the double bond in the linker part. In some cases, the sequencing run performed using the ffN set (including ffA, ffT, ffC and ffG) in which one or more ffNs are ffN-DB provides a higher ffN incorporation rate compared with the ffN set (also referred to as ffN-PA or ffN-(PA)) with propargylamine or propargylamide linker part described herein. For example, the ffN-DB collection described herein having an allylamine or allylamide linker moiety and a 3′-AOM capping group can improve the incorporation rate by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or 500% when treated under the same conditions for the same period of time compared to the ffN-PA collection having a 3′-O-azidomethyl capping group, thereby improving the phasing value. In other embodiments, the incorporation rate / speed is measured by the surface kinetics Vmax on the surface of a substrate (e.g., a flow cell or cBot system). For example, the ffN-DB collection having a 3′-AOM capping group can improve the Vmax value (ms) when treated under the same conditions for the same period of time compared to the ffN-PA collection having a 3′-O-azidomethyl capping group. -1) at least increase by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or 500%. In some embodiments, the incorporation rate / speed is measured at ambient temperature or a temperature (such as 4 to 10°C) lower than ambient temperature. In other embodiments, the incorporation rate / speed 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 incorporation rate / speed is measured in a solution of an alkaline pH environment (e.g., pH 9.0, 9.2, 9.4, 9.6, 9.8 or 10.0). In some such embodiments, the incorporation rate / speed is measured in the presence of an enzyme such as a polymerase (e.g., DNA polymerase), a terminal deoxynucleotidyl transferase or a reverse transcriptase. In some embodiments, ffN-DB is ffT-DB, ffC-DB or ffA-DB. In one embodiment, the ffN-DB set with improved phasing values ​​described herein includes ffT-DB, ffC, ffA and ffG. In another embodiment, the ffN-DB set with improved phasing values ​​described herein includes ffT-DB, ffC-DB, ffA and ffG. In yet another embodiment, the ffN-DB set with improved phasing values ​​described herein includes ffT-DB, ffC-DB, ffA-DB and ffG.

[0124] In other embodiments, when the nucleobase of the nucleotides described herein is thymine or its optionally substituted derivatives and analogs (ie, the nucleotide is T), L 1 Contains an allylamine moiety or an allylamide moiety, or an optionally substituted variant thereof. In a specific example, L 1 Including or for and * indicates L 1 The attachment point to the C5 position of the thymine base. In some embodiments, the T nucleotides described herein are fully functionalized T nucleotides (ffT) labeled with a dye molecule via a cleavable linker comprising Directly attached to the C5 position of thymine base (i.e., ffT-DB). In some cases, when using ffT-DB in the presence of a palladium catalyst in sequencing applications, it can significantly improve sequencing indicators, such as phasing, pre-phasing and error rate. For example, when using the ffT-DB with 3′-AOM blocking group as described herein, compared with the situation when using the standard ffT-PA with 3′-O-azidomethyl blocking group, the ffT-DB can improve one or more sequencing indicators as described herein by at least 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 2500% or 3000%.

[0125] Additional embodiments of nucleosides or nucleotides described herein include those having Formula (Ia), (Ia'), (Ib), (Ic), (Ic'), or (Id):

[0126]

[0127]

[0128] In some other embodiments of the nucleosides or nucleotides described herein, L 2 exists, and L 2 include wherein n and m are each independently an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and the phenyl moiety is optionally substituted. In some such embodiments, n is 5, and L 2 The phenyl moiety of is unsubstituted. In other embodiments, m is 4.

[0129] In any of the nucleosides or nucleotides described herein, the cleavable linker or L 1 / L 2 It may also contain a disulfide moiety or an azide moiety (such as ), or a combination thereof. L 1 or L 2 Additional non-limiting examples of linker moieties in include:

[0130] Additional linker moieties are disclosed in WO 2004 / 018493 and U.S. Patent Publication No. 2016 / 0040225, both of which are incorporated herein by reference.

[0131] In any of the nucleosides or nucleotides described herein, the nucleoside or nucleotide comprises a 2' deoxyribose moiety (ie, R in Formulas (I) and (Ia) to (Id)4 In some other aspects, the 2' deoxyribose contains one, two or three phosphate groups at the 5' position of the sugar ring. In some other aspects, the nucleotides described herein are nucleotide triphosphates (i.e., R in formulas (I) and (Ia) to (Id) 6 to form triphosphates).

[0132] In any of the nucleoside or nucleotide embodiments described herein, the detectable label may include a fluorescent dye.

[0133] Additional embodiments of the present disclosure relate to oligonucleotides or polynucleotides comprising nucleosides or nucleotides described herein. For example, an oligonucleotide or polynucleotide incorporating a nucleotide of formula (Ia′) comprises the following structure:

[0134]

[0135] In some such embodiments, the oligonucleotide or polynucleotide is hybridized to a template or target polynucleotide. In some such embodiments, the template polynucleotide is immobilized on a solid support.

[0136] Additional embodiments of the present disclosure are directed to solid supports comprising an array of a plurality of immobilized template or target polynucleotides, at least a portion of such immobilized template or target polynucleotides being hybridized to an oligonucleotide or polynucleotide comprising nucleosides or nucleotides described herein.

[0137] In any of the embodiments of nucleotides or nucleosides described herein, the 3'-OH blocking group and the cleavable linker (and attached label) can be removed under the same or substantially the same chemical reaction conditions, for example, the 3'-OH blocking group and the detectable label can be removed in a single chemical reaction. In other embodiments, the 3'-OH blocking group and the detectable label are removed in two separate steps.

[0138] In some embodiments, the 3'-blocked nucleotides or nucleosides described herein provide superior stability during storage or during treatment with reagents for sequencing applications in solution or lyophilized form compared to the same nucleotides or nucleosides protected with standard 3'-OH blocking groups disclosed in the prior art (e.g., 3'-O-azidomethyl blocking groups). For example, the acetal blocking groups disclosed herein can improve stability by 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% when treated under the same conditions for the same period of time compared to 3'-OH protected by azidomethyl, thereby reducing the pre-phase value and increasing the sequencing read length. In some embodiments, stability is measured at ambient temperature or a temperature below ambient temperature (such as 4 to 10°C). In other embodiments, stability is measured at elevated temperatures (such as 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C). In some such embodiments, stability is measured in a solution of an alkaline pH environment (e.g., pH 9.0, 9.2, 9.4, 9.6, 9.8, or 10.0). In some such embodiments, stability is measured in the presence or absence of an enzyme, wherein the enzyme is such as a polymerase (e.g., DNA polymerase), a terminal deoxynucleotidyl transferase, or a reverse transcriptase.

[0139] In some embodiments, the 3'-blocked nucleotides or nucleosides described herein provide superior deblocking rates in solution during the chemical cleavage 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 blocking groups disclosed herein can increase the deblocking rate by 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 3'-OH protected by azidomethyl when using standard deblocking reagents (such as tris(hydroxypropyl)phosphine), thereby shortening the total time of the sequencing cycle. In some embodiments, the unblocking rate is measured at ambient temperature or a temperature below ambient temperature (such as 4 to 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 of an alkaline pH environment (e.g., 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'-blocked nucleosides or nucleotides) is about 10: 1, about 5: 1, about 2: 1 or about 1: 1.

[0140] In some embodiments, a palladium deblocking agent (e.g., Pd(0)) is used to remove a 3' acetal capping group (e.g., an AOM capping group). Pd can form a chelate complex with the two oxygen atoms of the AOM group and the double bond of the allyl group, so that the deblocking agent directly adjacent to the functional group is removed and the deblocking rate can be accelerated. For example, after Pd cleaves the linker and 3' capping group of the incorporated nucleotide of Formula (Ia), (Ia'), (Ib), (Ic), (Ic') or (Id) described herein, the remaining linker construct on the copy polynucleotide can comprise the following structure:

[0141]

[0142] The wavy lines refer to the attachment of oxygen to the remaining phosphodiester bonds of the copied polynucleotide chain. For example,

[0143]

[0144] The allylamido or propargylamido moiety can be further cleaved by Pd catalyst. In addition, the remaining linker construct attached to the detectable label has the following structure:

[0145] For example

[0146] Cleavage conditions for cleavable linkers

[0147] The cleavable linkers described herein can be removed or cleaved under various chemical conditions. Non-limiting cleavage conditions include palladium catalysts such as Pd(II) complexes (e.g., Pd(OAc)2, allylpalladium(II) chloride dimer [(allyl)PdCl]2 or Na2PdCl4) in the presence of a water-soluble phosphine ligand such as tris(hydroxypropyl)phosphine (THP or THPP) or tris(hydroxymethyl)phosphine (THMP). In some embodiments, the 3' acetal capping group can be cleaved under the same or substantially the same cleavage conditions as those used for the cleavable linker.

[0148] Palladium catalyst

[0149] In some embodiments, the 3'acetal capping group and the cleavable linker described herein can be cleaved by a palladium catalyst. In some such embodiments, the Pd catalyst is water-soluble. In some such embodiments, the Pd catalyst is a Pd(0) complex (e.g., tris(3,3',3"-phosphinyltri(benzenesulfonate)palladium(0) nonahydrate). In some cases, the Pd(0) complex can be generated in situ by reducing the Pd(II) complex with a reagent such as an olefin, an alcohol, an amine, a phosphine, or a metal hydride. Suitable palladium sources include Pd(CH3CN)2Cl2, [PdCl(allyl)]2, [Pd(allyl)(THP)]Cl, [Pd(allyl)(THP)2]Cl, Pd(OAc)2, Pd(PPh3)4, Pd(dba)2, Pd(Acac)2, PdCl2(COD), and Pd(TFA)2. In one such embodiment, the Pd(0) complex is generated in situ from Na2PdCl4. In another embodiment, the palladium source is an allylpalladium(II) chloride dimer [(allyl)PdCl]2 or [PdCl(C3H5)]2. In some embodiments, the Pd(0) catalyst is generated in aqueous solution by mixing the Pd(II) complex with a phosphine. Suitable phosphines include water-soluble phosphines such as tri(hydroxypropyl)phosphine (THP), tri(hydroxymethyl)phosphine (THMP), 1,3,5-triaza-7-phosphaadamantane (PTA), bis(p-sulfonic acid phenyl)phenylphosphine dihydrate potassium salt, tri(carboxyethyl)phosphine (TCEP), and triphenylphosphine-3,3',3'-trisulfonic acid trisodium salt.

[0150] In some embodiments, the palladium catalyst is prepared by mixing [(allyl)PdCl]2 with THP in situ. The molar ratio of [(allyl)PdCl]2 and THP can be about 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 [(allyl)PdCl]2 and THP is 1:10. In some other embodiments, the palladium catalyst is prepared by mixing a water-soluble Pd reagent Na2PdCl4 with THP in situ. The molar ratio of Na2PdCl4 and THP can be about 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 Na2PdCl4 and THP is about 1:3. In another embodiment, the molar ratio of Na2PdCl4 and THP is about 1:3.5. In some other 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 lysis mixture may contain additional buffer reagents, such as primary amines, secondary amines, tertiary amines, natural amino acids, non-natural amino acids, carbonates, phosphates or borates, or combinations thereof. In other embodiments, the buffer reagents include ethanolamine (EA), tris (hydroxymethyl) aminomethane (Tris), glycine, sodium carbonate, sodium phosphate, sodium borate, dimethylethanolamine (DMEA), diethylethanolamine (DEEA), N,N,N',N'-tetramethylethylenediamine (TMEDA), N,N,N',N'-tetraethylethylenediamine (TEEDA) or 2-piperidineethanol, or combinations thereof. In one embodiment, one or more buffer reagents include DEEA. In another embodiment, one or more buffer reagents contain one or more inorganic salts, such as carbonates, phosphates or borates, or combinations thereof. In one embodiment, the inorganic salt is a sodium salt.

[0151] In other embodiments, the cleavage conditions of the cleavable linker are different from the cleavage conditions of the 3'-OH blocking group. For example, in addition, when the 3' blocking group is a 3'-O-azidomethyl group, the -CH2N3 moiety can be converted to an amino group by a phosphine. Alternatively, the azido group in -CH2N3 can be converted to an amino group by contacting these molecules with a thiol, particularly a water-soluble thiol such as dithiothreitol (DTT). In one embodiment, the phosphine is THP.

[0152] Compatible with linearization

[0153] 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 by using nucleic acid array technology. These arrays are usually composed of a high-density matrix of polynucleotides that are fixed to a solid carrier material.

[0154] WO 98 / 44151 and WO 00 / 18957 both describe nucleic acid amplification methods, which allow amplification products to be fixed on a solid support to form arrays, which are composed of clusters or "colonies", wherein clusters or "colonies" are formed by multiple identical immobilized polynucleotide chains and multiple identical immobilized complementary chains. This type of array is referred to as a "clustered array" in this article. The nucleic acid molecules present in the DNA colonies on the clustered array prepared according to these methods can provide templates for sequencing reactions, such as described in WO 98 / 44152. The products of solid phase amplification reactions (such as those described in WO 98 / 44151 and WO 00 / 18957) are so-called "bridged" structures, which are formed by annealing paired immobilized polynucleotide chains and immobilized complementary chains (both chains are attached to the solid support at 5' ends). In order to provide a more suitable template for nucleic acid sequencing, it is preferred to remove substantially all or at least a portion of one of the immobilized chains of the "bridged" structure, so as to produce a template that is at least partially single-stranded. Therefore, the single-stranded portion of the template will be available for hybridization with the sequencing primer. The process of removing all or part of an immobilized strand in a "bridging" double-stranded nucleic acid structure is called "linearization". There are many ways of linearization, including but not limited to enzymatic cleavage, photochemical cleavage, or chemical cleavage. Non-limiting examples of linearization methods are disclosed in PCT Publication No. WO 2007 / 010251, U.S. Patent Publication No. 2009 / 0088327, U.S. Patent Publication No. 2009 / 0118128, and U.S. Patent Publication No. 2019 / 0352327, which are incorporated by reference in their entirety.

[0155] In particular, amplification (e.g., bridge amplification or exclusive amplification) forms an array consisting of clusters or "colonies", wherein the clusters or "colonies" are formed by multiple identical immobilized target polynucleotide chains and multiple identical immobilized complementary chains. The target chain and the complementary chain form a polynucleotide complex that is at least partially double-stranded, wherein the two chains are fixed to a solid support at their 5' ends. The double-stranded polynucleotide is contacted with an aqueous solution of a palladium catalyst, which cleaves one chain at a cleavage site comprising an allyl-modified nucleoside (e.g., an allyl-modified T nucleoside) to remove at least a portion of one of the immobilized chains, thereby producing a template that is at least partially single-stranded. Therefore, the single-stranded portion of the template will be available for hybridization with a sequencing primer to trigger the first round of SBS (read segment 1). In some embodiments, the allyl-modified nucleoside is located in the P5 primer sequence. This method is referred to as the first chemical linearization, compared to standard enzymatic linearization, in which the removal or cleavage is promoted by an enzymatic cleavage reaction that cleaves the U position on the P5 primer using the enzyme USER.

[0156] In some embodiments, the conditions for cleaving the cleavable linker and / or deprotecting or removing the 3′-OH blocking group are also compatible with these linearization methods. In other embodiments, this cleavage condition is compatible with a chemical linearization method including the use of a Pd complex and a phosphine. In some embodiments, the Pd complex is a Pd(II) complex (e.g., Pd(OAc)2, [(allyl)PdCl]2 or Na2PdCl4), which generates Pd(0) in situ in the presence of a phosphine (e.g., THP). The chemical linearization method for cleaving the allyl-modified T nucleoside in the P5 primer sequence using a Pd catalyst is described in detail in U.S. Patent Publication No. 2019 / 0352327, which is incorporated by reference in its entirety. In other embodiments, the Pd cleavage mixture disclosed herein (e.g., a DEEA-containing buffer solution of [Pd(allyl)Cl)2] and THP) can be used directly in the first chemical linearization step. The reduced number of reagents allows for further simplification of the instrument (flow paths and cartridges).

[0157] Unless otherwise indicated, references to nucleotides are intended to apply to nucleosides as well.

[0158] Labeled nucleotides

[0159] According to one aspect of the present disclosure, the described 3'-OH blocked nucleotides also include detectable labels, which are referred to as labeled nucleotides or fully functionalized nucleotides (ffN). Labels (e.g., fluorescent dyes) are conjugated via cleavable joints in a variety of ways, including hydrophobic attraction, ion attraction, and covalent bonding. In some aspects, dyes are conjugated to nucleotides by covalent bonding achieved via cleavable joints. In some cases, the nucleotides of such labels are also referred to as "modified nucleotides". One of ordinary skill in the art understands that the label can be covalently bonded to a joint by reacting the functional group (e.g., carboxyl) of the label with the functional group (e.g., amino) of a joint.

[0160] Labeled nucleosides and nucleotides can be used to label polynucleotides formed by enzymatic synthesis, such as (by way of non-limiting example) in PCR amplification, isothermal amplification, solid phase amplification, polynucleotide sequencing (e.g., solid phase sequencing), nick translation reactions, and the like.

[0161] In some embodiments, the dye can be covalently attached to an oligonucleotide or nucleotide via a nucleotide base. For example, a labeled nucleotide or oligonucleotide can have a label attached to the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base via a cleavable linker moiety.

[0162] Unless otherwise indicated, references to nucleotides are intended to apply to nucleosides as well. The application will also be further described with reference to DNA, however the description will also apply to RNA, PNA and other nucleic acids unless otherwise indicated.

[0163] Nucleoside and nucleotide can be marked at the site on sugar or nucleobase.As known in the art, "nucleotide" is composed of nitrogenous base, sugar and one or more phosphate groups.In RNA, sugar is ribose, and in DNA, sugar is deoxyribose, that is, lacks the sugar of the hydroxyl group present in ribose.Nitrogenous base is a derivative of purine or pyrimidine.Purine is adenine (A) and guanine (G), and 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, wherein esterification occurs on the hydroxyl group of C-3 or C-5 attached to sugar.Nucleotide is usually monophosphate, diphosphate or triphosphate.

[0164] A "nucleoside" is structurally similar to a nucleotide, but lacks a phosphate moiety. An example of a nucleoside analog is one in which the label is attached to the base and there is no phosphate group attached to the sugar molecule.

[0165] Although bases are commonly referred to as purines or pyrimidines, it will be appreciated by the skilled artisan that derivatives and analogs that do not change the ability of a nucleotide or nucleoside to undergo Watson-Crick base pairing are available. "Derivative" or "analog" means a compound or molecule whose core structure is identical or very similar to that of the parent compound, but which has a chemical or physical modification that allows the derived nucleotide or nucleoside to be connected to another molecule, such as a different or additional side group. For example, the base can be a deazapurine. In a particular embodiment, the derivative should be able to undergo Watson-Crick pairing. "Derivatives" and "analogs" 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 may also include modified phosphodiester bonds, including phosphorothioate bonds, phosphorodithioate bonds, alkylphosphonate bonds, aniline phosphonate bonds, phosphoramidite bonds, and the like.

[0166] In a particular embodiment, the nucleoside or nucleotide of the label can be enzymatically incorporated and enzymatically extended. Therefore, the linker portion can have enough length to connect the nucleotide to the compound so that the compound does not significantly interfere with the overall binding and recognition of the nucleic acid replicator to the nucleotide. Therefore, the linker can also include a spacer unit. The spacer distance is, for example, the distance of the nucleotide base from the cleavage site or label.

[0167] The present disclosure also encompasses polynucleotides incorporating dye compounds. Such polynucleotides can be DNA or RNA composed of deoxyribonucleotides or ribonucleotides joined with phosphodiester bonds, respectively. Polynucleotides can include naturally occurring nucleotides combined with at least one modified nucleotide (e.g., labeled with a dye compound) as shown herein, non-naturally occurring (or modified) nucleotides or any combination thereof except the nucleotides of the labeling described herein. Polynucleotides according to the present disclosure can also include non-natural backbone bonds and / or non-nucleotide chemical modifications. It is also envisioned that a chimeric structure consisting of a mixture of ribonucleotides and deoxyribonucleotides comprising at least one labeled nucleotide.

[0168] Non-limiting exemplary labeled nucleotides as described herein include:

[0169]

[0170]

[0171] wherein L represents a cleavable linker (optionally including L as described herein 2 ), R represents a ribose or deoxyribose moiety as described above, or a ribose or deoxyribose moiety substituted with one, two or three phosphates at the 5' position.

[0172] In some embodiments, non-limiting exemplary fluorescent dye conjugates are as follows:

[0173]

[0174]

[0175] wherein PG represents a 3′-OH capping group as described herein; n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and k is 0, 1, 2, 3, 4, or 5. In one embodiment, —O—PG is AOM. In another embodiment, —O—PG is —O—azidomethyl. In one embodiment, n is 5. Refers to the point of attachment of the dye to the cleavable linker as a result of the reaction between the amino group of the linker moiety and the carboxyl group of the dye.

[0176] Sequencing methods

[0177] According to the labeled nucleotides or nucleosides of the present disclosure, they can be used in any analytical method (such as a method including detecting a fluorescent label attached to a nucleotide or nucleoside), whether used by itself, incorporated into a larger molecular structure or conjugate or associated with a larger molecular structure or conjugate. In this context, the term "incorporated into a polynucleotide" can mean that the 5' phosphate is joined to the 3'-OH group of the second (modified or unmodified) nucleotide with a phosphodiester bond, and the second nucleotide itself can form a part of a longer polynucleotide chain. The 3' end of the nucleotides described herein can be joined to the 5' phosphate of another (modified or unmodified) nucleotide with a phosphodiester bond or can not be joined to the 5' phosphate of another (modified or unmodified) nucleotide with a phosphodiester bond. Therefore, in a non-limiting embodiment, the present disclosure provides a method for detecting 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 a fluorescent signal from a detectable label (e.g., a fluorescent compound) attached to the nucleotide. The method may comprise: a synthesis step (a), wherein one or more nucleotides according to the present disclosure are incorporated into a polynucleotide; and a detection step (b), wherein the one or more nucleotides incorporated into the polynucleotide are detected by detecting or quantitatively measuring the fluorescence of the one or more nucleotides.

[0178] Additional aspects of the present disclosure include a method for preparing a growing polynucleotide that is complementary to a target single-stranded polynucleotide in a sequencing reaction, the method comprising incorporating a nucleotide described herein into the growing complementary polynucleotide, wherein incorporation of the nucleotide prevents the introduction of any subsequent nucleotide into the growing complementary polynucleotide.

[0179] Some embodiments of the present disclosure relate to a method for determining the sequence of a target single-stranded polynucleotide, the method comprising:

[0180] (a) comprising a 3′-OH end-capping group as described herein (attached to the 3′ oxygen group) and a detectably labeled nucleotide as described herein (e.g., dATP, dCTP, dGTP, dTTP, or dUTP) are incorporated into a copy polynucleotide strand that is complementary to at least a portion of a target polynucleotide strand;

[0181] (b) detecting the identity of the nucleotide incorporated into the copied polynucleotide chain; and

[0182] (c) chemically removing the label and 3'-OH blocking group from the nucleotides incorporated into the copy polynucleotide chain.

[0183] In some embodiments, the sequencing method further comprises (d) washing the chemically removed label and 3′-OH blocking group from the copy polynucleotide chain using a post-cleavage washing solution. In some such embodiments, the 3′-OH blocking group and the detectable label are removed before the next complementary nucleotide is introduced. In some embodiments, the washing step (d) also removes unincorporated nucleotides. In other embodiments, the method may include a separate washing step for washing unincorporated nucleotides from the copy polynucleotide chain before step (b).

[0184] In some embodiments, steps (a) to (d) are repeated until the sequence of the portion of the target 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.

[0185] Add to mixture

[0186] In some embodiments of the methods described herein, step (a) (also referred to as an incorporation step) includes contacting a mixture containing one or more nucleotides (e.g., dATP, dCTP, dGTP, dTTP, or dUTP) with a copy polynucleotide / target polynucleotide complex in an incorporation solution comprising a polymerase and one or more buffers. In some such embodiments, the polymerase is a DNA polymerase, such as Pol 812, Pol 1901, Pol 1558, or Pol 963. The amino acid sequences of Pol 812, Pol 1901, Pol 1558, or Pol 963 DNA polymerases are described in, for example, U.S. Patent Publication Nos. 2020 / 0131484A1 and 2020 / 0181587A1, both of which are incorporated herein by reference. In some embodiments, one or more buffers include primary amines, secondary amines, tertiary amines, natural amino acids, or non-natural amino acids, or combinations thereof. In additional embodiments, the buffer includes ethanolamine or glycine, or combinations thereof. In one embodiment, the buffer comprises glycine, or is glycine. In some embodiments, the use of glycine in the spiked mixture can improve the phasing value compared to a standard buffer (such as ethanolamine (EA)) under the same conditions. For example, the use of glycine reduces or decreases the phasing value by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% compared to the use of ethanolamine under the same conditions. In some cases, the use of glycine provides a % phasing value of less than about 0.15%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% in an SBS sequencing run with at least 50 cycles. In additional embodiments, use of glycine provides a %Phasing value of less than about 0.08% in Read 1 of an SBS sequencing run having at least 150 cycles.

[0187] Lysis mixture

[0188] In some embodiments of the methods described herein, step (c) (also referred to as the cleavage step) comprises contacting the incorporated nucleotides and the copy polynucleotide chain with a cleavage solution comprising a palladium catalyst as described herein. In some such embodiments, the 3'-OH blocking group and the detectable label are removed in a single step of the reaction. In one such embodiment, the 3' blocking group is AOM and the cleavable linker includes an AOL moiety, both of which are removed or cleaved in a single step of the chemical reaction. In other embodiments, the cleavage solution (also referred to as the cleavage mixture) comprises a Pd catalyst as described herein.

[0189] In some other embodiments, the Pd catalyst is a Pd(0) catalyst. In some such embodiments, Pd(0) is prepared by mixing a Pd(II) reagent in situ with one or more phosphine ligands. In some such embodiments, the palladium catalyst can be prepared by mixing [(allyl)PdCl]2 with THP in situ. The molar ratio of [(allyl)PdCl]2 and THP can be about 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 [(allyl)PdCl]2 and THP is 1:10 (i.e., the molar ratio of Pd:THP is 1:5). In some other embodiments, the palladium catalyst can be prepared by mixing a water-soluble Pd(II) reagent Na2PdCl4 with THP in situ. The molar ratio of Na2PdCl4 to THP can be about 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 Na2PdCl4 to THP is about 1:3. In another embodiment, the molar ratio of Na2PdCl4 to THP is about 1:3.5. Other non-limiting examples of Pd catalysts include Pd(CH3CN)2Cl2.

[0190] In some other 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 lysis solution may contain one or more buffer reagents, such as primary amines, secondary amines, tertiary amines, carbonates, phosphates or borates, or combinations thereof. In other embodiments, the buffer reagents include ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, sodium carbonate, sodium phosphate, sodium borate, dimethylethanolamine (DMEA), diethylethanolamine (DEEA), N,N,N′,N′-tetramethylethylenediamine (TEMED), N,N,N′,N′-tetraethylethylenediamine (TEEDA) or 2-piperidineethanol, or combinations thereof. In one embodiment, the buffer reagent includes DEEA, or is DEEA. In another embodiment, the buffer contains one or more inorganic salts, such as carbonates, phosphates or borates, or combinations thereof. In one embodiment, the inorganic salt is a sodium salt. In additional embodiments, the cleavage solution contains a palladium (Pd) catalyst (e.g., [(allyl)PdCl]2 / THP or Na2PdCl4 / THP) and one or more buffer reagents described herein (e.g., a tertiary amine such as DEEA), and has a pH of about 9.0 to about 10.0 (e.g., 9.6 or 9.8).

[0191] In other embodiments, the mark and 3' blocking group are removed in two separate chemical reactions. In some cases, removing the mark from the nucleotides incorporated into the copy polynucleotide chain includes contacting the copy chain containing the incorporated nucleotides with a first cleavage solution containing a Pd catalyst described herein. In some cases, removing the 3'-OH blocking group from the nucleotides incorporated into the copy polynucleotide chain includes contacting the copy chain containing the incorporated nucleotides with a second cleavage solution. In some such embodiments, the second cleavage solution contains one or more phosphines, such as trialkylphosphines. 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 3'-OH blocking group is 3'-O-azidomethyl, and the second cleavage solution contains THP.

[0192] In some embodiments, the cleavage solution described herein can also be used in the prior chemical linearization step described herein. In particular, one or more first chains of double-stranded polynucleotides fixed on a solid support are cleaved by palladium catalysis to produce a single-stranded (or at least partially single-stranded) template, which can be used for hybridization with a sequencing primer and subsequent sequencing applications (e.g., the first round of sequencing while synthesis (read segment 1)), thereby achieving chemical linearization of clustered polynucleotides prepared for sequencing. In some embodiments, each double-stranded polynucleotide comprises a first chain and a second chain. The first chain is generated by extending the first extension primer fixed to the solid support. In some embodiments, the first chain comprises a cleavage site that can be cleaved by a palladium complex (e.g., a Pd(0) complex). In a specific embodiment, the cleavage site is located in the first extension primer portion of the first chain. In an additional embodiment, the cleavage site comprises a thymidine or nucleotide analog with an allyl functionality. In some embodiments of the methods described herein, the target single-stranded polynucleotide is formed by chemically cleaving a complementary chain from a double-stranded polynucleotide. In other embodiments, the complementary strand and the target polynucleotide in the duplex are both immobilized at their 5' ends on a solid support. In other embodiments, chemically cleaving the complementary strand is performed under the same reaction conditions as chemically removing the detectable label and 3'-OH blocking group from the nucleotides incorporated into the copy polynucleotide strand (i.e., step (c) of the method described herein). In one embodiment, the first chemical linearization utilizes the same cleavage mixture described herein.

[0193] palladium( Pd ) Remover

[0194] Pd has the ability to adhere to DNA primarily in its inactive Pd(II) form, which may interfere with the binding between DNA and polymerase, resulting in increased phasing. After the unblocking step, a post-cleavage washing composition comprising a Pd scavenger compound can be used. For example, PCT Publication No. WO2020 / 126593 discloses that Pd scavengers (such as 3,3'-dithiodipropionic acid (DDPA) and lipoic acid (LA)) can be included in the scanning composition and / or the post-cleavage washing composition. The purpose of using these scavengers in the post-cleavage washing solution is to remove Pd(0) and convert Pd(0) into an inactive Pd(II) form, thereby improving pre-phasing values ​​and sequencing indicators, reducing signal degradation, and extending sequencing read lengths.

[0195] In some embodiments of the methods described herein, step (a) of the method comprises contacting the nucleotides with the copy polynucleotide chain in a spiking solution comprising a polymerase, at least one palladium scavenger, and one or more buffers. In some embodiments, the Pd scavenger spiked into the solution is a Pd(0) scavenger. In some such embodiments, the Pd scavenger comprises one or more allyl moieties independently selected from the group consisting of: -O-allyl, -S-allyl, -NR-allyl, and -N-allyl. + RR′-allyl, wherein R is H, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C2-C6 alkenyl, unsubstituted or substituted C2-C6 alkynyl, unsubstituted or substituted C6-C 10 aryl, unsubstituted or substituted 5- to 10-membered heteroaryl, unsubstituted or substituted C3-C 10 carbocyclyl, or unsubstituted or substituted 5- to 10-membered heterocyclyl; and R′ is H, unsubstituted C1-C6 alkyl or substituted C1-C6 alkyl.

[0196] In some such embodiments, the Pd(0) scavenger incorporated into the solution comprises one or more -O-allyl moieties. In other embodiments, the Pd(0) scavenger comprises or is or a combination thereof. Alternative Pd(0) scavengers are disclosed in US Serial No. 63 / 190983, which is incorporated by reference in its entirety.

[0197] In some embodiments, the concentration of the Pd(0) scavenger comprising one or more allyl moieties in the doping solution is about 0.1 mM to about 100 mM, 0.2 mM to about 75 mM, about 0.5 mM to about 50 mM, about 1 mM to about 20 mM, or about 2 mM to about 10 mM. In other embodiments, the concentration of the Pd(0) scavenger is about 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM, 10 mM, 12.5 mM, 15 mM, 17.5 mM, or 20 mM. In other embodiments, the pH of the doping solution is about 9 to 10.

[0198] In some embodiments, the molar ratio of palladium catalyst (in the starting solution) to palladium scavenger comprising one or more allylic moieties is about 1:100, 1:50, 1:20, 1:10, or 1:5.

[0199] In some other embodiments of the methods described herein, a Pd(0) scavenger comprising one or more allylic moieties as described herein is used in the scanning solution of step (b) when one or more fluorescence measurements are performed to detect the identity of the incorporated nucleotides in the copied polynucleotide. In still other embodiments, a Pd(0) scavenger comprising one or more allylic moieties may be present in both the spiking solution and the scanning solution.

[0200] In some other embodiments of the methods described herein, the post-cleavage washing step is used after removing the label and 3' blocking group. In some such embodiments, one or more palladium scavengers are also used in the washing step after the label and 3' blocking group are cleaved. In some other embodiments, the one or more Pd scavengers in the post-cleavage washing solution include a Pd (II) scavenger. In some such embodiments, the palladium scavenger includes isocyanoacetate (ICNA) salt, cysteine ​​or a salt thereof, or a combination thereof. In one embodiment, the palladium scavenger includes or is potassium isocyanoacetate or sodium isocyanoacetate. In another embodiment, the palladium scavenger includes or is cysteine ​​or a salt thereof (e.g., L-cysteine ​​or L-cysteine ​​HCl salt). Other non-limiting examples of palladium scavengers in the post-lysis wash solution can include: ethyl isocyanoacetate, methyl isocyanoacetate, N-acetyl-L-cysteine, potassium ethyl xanthate (PEX or KS-C(=S)-OEt), potassium isopropyl xanthate, glutathione, lipoic acid, ethylenediaminetetraacetic acid (EDTA), iminodiacetic acid, nitrilodiacetic acid, trimercapto-S-triazine, dimethyldithiocarbamate, dithiothreitol, mercaptoethanol, allyl alcohol, propargyl alcohol, thiols, tertiary amines and / or tertiary phosphines, or combinations thereof.

[0201] In other embodiments, the concentration of the Pd (II) scavenger (such as L-cysteine) in the post-cleavage wash solution is about 0.1 mM to about 100 mM, 0.2 mM to about 75 mM, about 0.5 mM to about 50 mM, about 1 mM to about 20 mM, or about 2 mM to about 10 mM. In other embodiments, the concentration of the Pd (II) scavenger (such as L-cysteine) is about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 6.5 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12.5 mM, 15 mM, 17.5 mM or 20 mM. In one embodiment, the concentration of the Pd scavenger (such as L-cysteine ​​or a salt thereof) in the post-cleavage wash solution is about 10 mM.

[0202] In some other embodiments of the methods described herein, all Pd scavengers (e.g., both Pd(0) scavengers and Pd(II) scavengers) are in the dosing solution and / or scanning solution, and the method does not employ a specific post-cleavage washing step to remove any trace amounts of remaining Pd species.

[0203] In some embodiments of the methods described herein, the use of a Pd scavenger (e.g., a Pd(0) scavenger having one or more allyl moieties) can reduce the prephase value by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% compared to the same sequencing run under the same conditions and without the use of a palladium scavenger. In some such embodiments, the Pd(0) scavenger can reduce the prephase value of the sequencing run to less than about 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% in an SBS sequencing run having at least 50 cycles. In some embodiments, the prephase value refers to a value measured after 50 cycles, 75 cycles, 100 cycles, 125 cycles, 150 cycles, 200 cycles, 250 cycles, or 300 cycles.

[0204] In other embodiments, a palladium scavenger (e.g., a Pd(II) scavenger such as L-cysteine ​​or a salt thereof) can reduce the prephasing value or phasing value by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% compared to the same sequencing run under the same conditions and without the palladium scavenger. In some such embodiments, the use of a Pd scavenger provides a % phasing value of less than about 0.2%, 0.15%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% in an SBS sequencing run having at least 50 cycles. In some embodiments, the phasing value refers to the value measured after 50 cycles, 75 cycles, 100 cycles, 125 cycles, 150 cycles, 200 cycles, 250 cycles, or 300 cycles. In other embodiments, the use of one or more Pd scavengers provides a % phasing value of less than about 0.05% in read 1 of an SBS sequencing run with at least 150 cycles.

[0205] In some embodiments, the post-cleavage wash solutions described herein may also be used in a separate wash step prior to the detection step (ie, step (b) in the methods described herein) to wash away any unincorporated nucleotides from step (a).

[0206] In other embodiments, the nucleotides used for incorporation into step (a) are fully functionalized A, C, T, and G nucleotide triphosphates, each of which contains a 3' blocking group (e.g., 3'-AOM) and a cleavable linker (e.g., a cleavable linker containing an AOL linker moiety) as described herein. In some such embodiments, the nucleotides herein provide superior stability in solution during a sequencing run compared to the same nucleotides protected with a standard 3'-O-azidomethyl blocking group. For example, the 3'acetal capping groups disclosed herein can improve stability by 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% compared to 3'-OH protected by azidomethyl when treated under the same conditions for the same period of time, thereby reducing the pre-phase value and making the sequencing read length longer. In some embodiments, stability is measured at ambient temperature or a temperature below ambient temperature (such as 4 to 10°C). In other embodiments, stability is measured at elevated temperatures (such as 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C). In some such embodiments, stability is measured in a solution at an alkaline pH environment (e.g., pH 9.0, 9.2, 9.4, 9.6, 9.8, or 10.0). In other embodiments, the predetermined phase value using the 3' blocking nucleotides 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 more than 50, 100, or 150 SBS cycles. In other embodiments, after more than 50, 100 or 150 SBS cycles, the phasing value using 3'-capped nucleotides 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. In one embodiment, each ffN contains a 3'-AOM group.

[0207] In some embodiments, the 3'-blocked nucleotides described herein provide superior unblocking rates in solution during the chemical cleavage step of a sequencing run compared to the same nucleotides protected with a standard 3'-O-azidomethyl blocking group. For example, the 3' acetal (e.g., AOM) blocking groups disclosed herein can increase the unblocking rate by 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 when using a standard unblocking reagent (such as tris(hydroxypropyl)phosphine), thereby shortening the total time of the sequencing cycle. In some embodiments, the unblocking time of each nucleotide is shortened by about 5%, 10%, 20%, 30%, 40%, 50% or 60%. For example, under specific chemical reaction conditions, the unblocking time of 3'-AOM and 3'-O-azidomethyl is about 4 to 5 seconds and about 9 to 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 end-capping group. In some such embodiments, the t of AOM is 1 / 2 is about 1 minute, while the t 1 / 2 In some embodiments, the unblocking rate is measured at ambient temperature or a temperature (such as 4 to 10 ° C) below ambient temperature. 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 of an alkaline pH environment (e.g., 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'-blocked nucleosides or nucleotides) 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 contains 3'-AOM blocking groups and AOL linker moieties.

[0208] In any of the embodiments of the methods described herein, the labeled nucleotide is a nucleotide triphosphate having a 2' deoxyribose sugar.In any of the embodiments of the methods described herein, the target polynucleotide strand is attached to a solid support, such as a flow cell.

[0209] In one embodiment, at least one nucleotide is incorporated into the polynucleotide by the action of a polymerase during the synthesis step. In some such embodiments, the polymerase may be DNA polymerase Pol 812 or Pol 1901. However, other methods of joining nucleotides to polynucleotides may be used, such as chemically synthesizing oligonucleotides, or connecting labeled oligonucleotides to unlabeled oligonucleotides. Therefore, when the term "incorporation" is used with respect to nucleotides and polynucleotides, polynucleotide synthesis by chemical methods as well as enzymatic methods may be encompassed.

[0210] In a specific embodiment, a synthesis step is performed, which may optionally include incubating the template polynucleotide chain with a reaction mixture containing a labeled 3'-capped nucleotide of the present disclosure. A polymerase may also be provided under conditions that allow the formation of a phosphodiester bond between a free 3'-OH group on the polynucleotide chain annealed to the template polynucleotide chain and a 5' phosphate group on the nucleotide. Thus, the synthesis step may include directing the formation of the polynucleotide chain by complementary base pairing of the nucleotide with the template chain.

[0211] In all embodiments of these methods, the detection step can be performed when the polynucleotide chain into which the labeled nucleotide is incorporated is annealed to the template chain or the target chain, or after the denaturation step in which the two chains are separated. Additional steps, such as chemical reaction steps or enzymatic reaction steps, or purification steps may be included between the synthesis step and the detection step. Specifically, the target chain into which the labeled nucleotide is incorporated can be separated or purified, and then further processed or used for subsequent analysis. By way of example, the target polynucleotide labeled with nucleotides as described herein in the synthesis step can be used as a labeled probe or primer subsequently. In other embodiments, the product of the synthesis step set forth herein can be subjected to further reaction steps, and if necessary, the products of these subsequent steps are purified or separated.

[0212] Suitable conditions for the synthesis step will be well known to those familiar with standard molecular biology techniques. In one embodiment, the synthesis step can be similar to a standard primer extension reaction, which uses nucleotide precursors (including nucleotides as described herein) to form an extended target strand complementary to the template strand in the presence of a suitable polymerase. In other embodiments, the synthesis step itself can form a part of the amplification reaction of the double-stranded amplification product producing a mark, and the double-stranded amplification product of the mark is composed of the annealed complementary strand derived from the replication of the target polynucleotide chain and the template polynucleotide chain. Other exemplary synthesis steps include nick translation, strand displacement polymerization, random DNA labeling, etc. The particularly useful polymerase for the synthesis step is a polymerase that can catalyze the incorporation of nucleotides as described herein. A variety of naturally occurring or modified polymerases can be used. By way of example, a thermostable polymerase can be used for the synthesis reaction performed using thermal cycling conditions, and a thermostable polymerase may not be desired by 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 conducted at lower temperatures such as 37° C., the polymerase need not be a thermostable polymerase, and thus the choice of polymerase will depend on many factors, such as reaction temperature, pH, strand displacement activity, etc.

[0213] In specific non-limiting embodiments, the present disclosure encompasses methods for nucleic acid sequencing, resequencing, whole genome sequencing, single nucleotide polymorphism scoring, any other application involving the detection of labeled nucleotides or nucleosides as described herein when incorporated into polynucleotides. Any of a variety of other applications that benefit from the use of polynucleotides labeled with nucleotides comprising fluorescent dyes can use nucleotides or nucleosides labeled with dyes as described herein.

[0214] In a specific embodiment, the present disclosure provides the purposes of the labeled nucleotides according to the present disclosure in the sequencing by synthesis (SBS) reaction of polynucleotides.Synthesis sequencing generally involves the use of a polymerase or a ligase to sequentially add one or more nucleotides or oligonucleotides to a growing polynucleotide chain in a 5' to 3' direction to form an extended polynucleotide chain complementary to a template nucleic acid to be sequenced. The identity of the bases present in one or more added nucleotides can be determined in a detection or "imaging" step. The identity of the added bases 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. It may be useful to use the labeled nucleotides set forth herein to determine the identity of a single base, for example, in a single nucleotide polymorphism score, and such single base extension reactions are within the scope of the present disclosure.

[0215] In one embodiment of the present disclosure, the sequence of the template polynucleotide is determined by detecting the incorporation of one or more 3'-capping nucleotides described herein into a nascent chain complementary to the template polynucleotide to be sequenced, the detection process being accomplished 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 one-by-one manner by adding nucleotides to the 3' end of the primer in a polymerase-catalyzed reaction.

[0216] In a specific embodiment, each of the different nucleotide triphosphates (A, T, G and C) can be labeled with a unique fluorophore, and a blocking group is also included at the 3' position to prevent uncontrolled polymerization. Alternatively, one of the four nucleotides can be unlabeled (dark). Polymerase incorporates nucleotides into a nascent chain complementary to the template polynucleotide, and the blocking group prevents further incorporation of nucleotides. Any unincorporated nucleotides can be washed away, and the fluorescent signal from each incorporated nucleotide can be optically "read" by a suitable device (such as a charge-coupled device using laser excitation and a suitable emission filter). Then the 3'-blocking group and the fluorescent dye compound can be removed (deprotected) simultaneously or sequentially to expose the nascent chain for further incorporation of nucleotides. Typically, the identity of the incorporated nucleotides will be determined after each incorporation step, but this is not strictly necessary. Similarly, U.S. Patent No. 5,302,509 (incorporated herein by reference) discloses a method for sequencing a polynucleotide fixed on a solid support.

[0217] As illustrated above, the method utilizes the presence of DNA polymerase to incorporate fluorescently labeled 3'-enclosed nucleotides A, G, C and T into a growing chain complementary to a fixed polynucleotide. Polymerase incorporates bases complementary to the target polynucleotide, but is prevented from further addition by 3'-enclosed groups. The label of the nucleotides incorporated can then be determined, and the enclosed groups are removed by chemical cleavage to allow further polymerization to occur. The nucleic acid template to be sequenced in the synthesis sequencing reaction can be any polynucleotide that is desired to be sequenced. The nucleic acid template for sequencing reaction will generally include a double-stranded region with a free 3'-OH group, which serves as a primer or starting point for adding additional nucleotides in the sequencing reaction. The region of the template to be sequenced will dangle the free 3'-OH group on the complementary chain. The overhanging region of the template to be sequenced can be single-stranded, but can also be double-stranded, provided that there is a "nick" on the chain complementary to the template chain to be sequenced, to provide a free 3'-OH group for initiating a sequencing reaction. In such embodiments, sequencing can be performed by strand displacement. In certain embodiments, primers with free 3'-OH groups can be added as separate components (e.g., short oligonucleotides) hybridized with the single-stranded region of template to be sequenced. Alternatively, primers to be sequenced and template strands can each form a part of a partial self-complementary nucleic acid chain that can form an intramolecular duplex (such as a hairpin loop structure). Hairpin polynucleotides and methods that can be attached to solid supports are disclosed in PCT Publication No. WO 01 / 57248 and WO 2005 / 047301, and these two PCT disclosures are each incorporated herein by reference. Nucleotide can be continuously added to the growth primer, resulting in a synthetic polynucleotide chain in the 5' to 3' direction. The properties of the bases added can be determined, particularly but not necessarily after each addition of nucleotides, thereby providing the sequence information of nucleic acid templates. Therefore, the mode in which nucleotides are incorporated into nucleic acid chains (or polynucleotides) is to form a phosphodiester bond with the 5' phosphate group of the nucleotide and the nucleotide is joined to the free 3'-OH group of nucleic acid chains.

[0218] 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 bonds, provided that these do not prevent replication of the template in the sequencing reaction.

[0219] In certain embodiments, the nucleic acid template to be sequenced can be attached to a solid support via any suitable connection method known in the art (e.g., via covalent attachment). In certain 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 way to allow direct covalent attachment of the template polynucleotide, or to fix the template polynucleotide by a hydrogel or polyelectrolyte multilayer, which itself can be non-covalently attached to the solid support.

[0220] Sequencing by Synthesis Implementation and Alternatives

[0221] Some embodiments include pyrophosphate sequencing technology. Pyrophosphate sequencing 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. Pat. Nos. 6,210,891, 6,258,568 and 6,274,320, the disclosures of which are incorporated herein by reference in their entirety). In pyrophosphate sequencing, the released PPi can be detected by being immediately converted into ATP by adenosine triphosphate (ATP) sulfurylase, and the level of ATP generated is detected by photons generated by luciferase. The nucleic acid to be sequenced can be attached to a feature in an array, and the array can be imaged to capture the chemiluminescent signal generated by the incorporation of nucleotides at the feature of the array. The image can be obtained after the array is treated with a specific nucleotide type (e.g., A, T, C or G). The images obtained after adding each nucleotide type will differ in which features in the array are detected. 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 images can be stored, processed and analyzed using the methods described herein. For example, images obtained after treating the array with each different nucleotide type can be processed in the same manner as exemplified herein for images obtained from different detection channels for a reversible terminator-based sequencing method.

[0222] In another exemplary type of SBS, cycle sequencing is accomplished by stepwise addition of reversible terminator nucleotides comprising, for example, cleavable or photobleachable dye labels, as described, for example, in WO 04 / 018497 and U.S. Pat. No. 7,057,026, the disclosures of which are incorporated herein by reference. This 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, in which not only termination can be reversed but also the fluorescent label can be cleaved, facilitates efficient cyclic reversible termination (CRT) sequencing. Polymerases can also be co-engineered to efficiently incorporate and extend from these modified nucleotides.

[0223] Preferably, in the sequencing embodiment based on reversible terminator, the label does not substantially inhibit extension under SBS reaction conditions. However, the detection label can be removable, such as removed by cleavage or degradation. Images can be captured after the label is incorporated into the arrayed nucleic acid feature portion. In a specific embodiment, each cycle involves four different nucleotide types being delivered to the array simultaneously, and each nucleotide type has a label that is spectrally different. Four images can then be obtained, each image using a detection channel that is selective to one label in the four different labels. Alternatively, different nucleotide types can be added sequentially, and the image of the array can be obtained between each addition step. In such embodiments, each image will show the nucleic acid features in which a specific type of nucleotide has been incorporated. Due to the different sequence content of each feature portion, different feature portions will be present or not present in different images. However, the relative position of the feature portion will remain unchanged in the image. The image obtained by such reversible terminator-SBS method can be stored, processed and analyzed as described herein. After the image capture step, the label can be removed, and the reversible terminator portion can be removed to perform subsequent cycles of nucleotide addition and detection. Removing labels after they have been detected in a particular cycle and before subsequent cycles can provide the advantage of reducing background signal and crosstalk between cycles. Examples of useful labeling and removal methods are set forth below.

[0224] Some embodiments can utilize less than four different marks to detect four different nucleotides.For example, the method and system described in the material of the combined U.S. Patent Publication No. 2013 / 0079232 can be utilized to perform SBS.As a first example, a pair of nucleotide types can be detected at the same wavelength, but based on the intensity difference of a member in the pair relative to another member, or based on a member in the pair causing a change (for example, by chemical modification, photochemical modification or physical modification) that a signal that is obvious compared with the signal of another member of the pair detected appears or disappears.As a second example, three of the four different nucleotide types can be detected under specific conditions, and the fourth nucleotide type lacks a mark (for example, the minimum detection caused by background fluorescence, etc.) that can be detected under those conditions or detected minimally under those conditions.The first three nucleotide types can be determined to be incorporated into nucleic acid based on the existence of its corresponding signal, and the fourth nucleotide type can be determined to be incorporated into nucleic acid based on the absence of any signal or the minimum detection of any signal.As a third example, a nucleotide type can include a 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 channel and the second channel (e.g., dTTP with at least one label detected in both channels when excited by the first excitation wavelength and / or the second excitation wavelength), and a fourth nucleotide type that lacks a label detected or minimally detected in either channel (e.g., dGTP without a label).

[0225] In addition, as described in the materials of the incorporated U.S. publication 2013 / 0079232, a single channel can be used to obtain sequencing data. In such so-called single dye sequencing methods, a first nucleotide type is marked, but the mark is removed after the first image is generated, and a second nucleotide type is marked only after the first image is generated. A third nucleotide type retains its mark in both the first and second images, and a fourth nucleotide type remains unmarked in both images.

[0226] Some embodiments can utilize sequencing while connecting. Such technology utilizes DNA ligase to incorporate oligonucleotides and determine the incorporation of such oligonucleotides. Oligonucleotides generally have different labels related to the identity of specific nucleotides in the sequence of oligonucleotide hybridization. As with other SBS methods, images can be obtained after the array of nucleic acid feature parts is treated with labeled sequencing reagents. Each image will show the nucleic acid feature parts of the labeling that have been incorporated into a specific type. Due to the different sequence content of each feature part, different feature parts will be present or absent in different images, but the relative position of the feature parts will remain unchanged in the image. The images obtained by the sequencing method based on connection can be stored, processed and analyzed as described herein. Exemplary SBS systems and methods that can be used with the methods and systems described herein are described in U.S. Patent Nos. 6,969,488, 6,172,218 and 6,306,597, and the disclosures of these patents are incorporated herein by reference in their entirety.

[0227] Some embodiments can utilize nanopore sequencing (Deamer, DW and 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 entirety). 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. When the target nucleic acid passes through the nanopore, each base pair can be identified by measuring the fluctuation of the conductivity of the pore. (U.S. Patent No. 7,001,792; Soni, GV and 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. and 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 entirety). The data obtained from nanopore sequencing can be stored, processed and analyzed as described herein. In particular, according to the exemplary processing of optical images and other images described herein, the data can be processed like an image.

[0228] Some other embodiments of the sequencing method relate to the use of 3'-terminated 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. A large number of discrete DNA nanoballs can be produced by a rolling circle amplification (RCA) process. The nanoball mixture is then distributed on a patterned glass slide surface containing features that allow a single nanoball to be associated with each position. During the DNA nanoball production process, the DNA is fragmented and then connected to the first of the four adapter sequences. The template is amplified, circularized, and then cleaved with a type II endonuclease. A second set of adapters is added, followed by amplification, circularization, and cleavage. 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 produce a large number of concatemers called DNA nanoballs, which are then deposited on a flow cell. Goodwin et al., “Coming of age: ten years of next-generation sequencing technologies,” Nat Rev Genet. 2016;17(6):333-51.

[0229] Some embodiments may utilize methods involving real-time monitoring of DNA polymerase activity. Nucleotide incorporation may be detected by fluorescence resonance energy transfer (FRET) interactions between a polymerase carrying a fluorophore and a γ-phosphate labeled nucleotide (as described, for example, in U.S. Pat. Nos. 7,329,492 and 7,211,414, both of which are incorporated herein by reference), or may be detected with a zero-mode waveguide (as described, for example, in U.S. Pat. No. 7,315,019, which is incorporated herein by reference), and may be detected using fluorescent nucleotide analogs and engineered polymerases (as described, for example, in U.S. Pat. No. 7,405,281 and U.S. Patent Publication No. 2008 / 0108082, both of which are incorporated herein by reference). Illumination can be limited to the z-scale volume around the surface-tethered polymerase, allowing the incorporation of fluorescently labeled nucleotides to be observed 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 nano structures.", Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference in their entirety). Images obtained by such methods can be stored, processed, and analyzed as described herein.

[0230] Some SBS embodiments include detecting the proton released when nucleotides are incorporated into extension products. For example, sequencing based on the detection of releasing protons can use commercially available electrical detectors and related technologies from Ion Torrent (Guilford, CT, Life Technologies subsidiary), or sequencing methods and systems described in U.S. Patent Publication Nos. 2009 / 0026082, 2009 / 0127589, 2010 / 0137143 and 2010 / 0282617 (all of these patent publications are incorporated herein by reference). The method for amplifying target nucleic acids using kinetic exclusion set forth herein can be easily applied to substrates for detecting protons. More specifically, the method set forth herein can be used to produce amplicon clone colonies for detecting protons.

[0231] The above-mentioned SBS method can be advantageously carried out in a variety of formats so that a plurality of different target nucleic acids are manipulated simultaneously. In a specific embodiment, different target nucleic acids can be processed in a common reaction vessel or on the surface of a specific substrate. This allows to conveniently deliver sequencing reagents, remove unreacted reagents and detect incorporation events in a variety of ways. In the embodiment using surface-bound target nucleic acids, the target nucleic acid can be an array format. In the array format, the target nucleic acid can be usually attached to the surface in a spatially distinguishable manner. The target nucleic acid can be combined by directly covalently connecting, being attached to beads or other particles or being attached to polymerase or other molecules attached to the surface. The array can include a single copy of the target nucleic acid at each site (also referred to as feature), or multiple copies with the same sequence can be present at each site or feature. Multiple copies can be produced by amplification methods (such as bridge amplification or emulsion PCR as described in further detail below).

[0232] The methods described herein can use arrays having features at any of a variety of densities, including, 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 density.

[0233] The advantage of the method described herein is that they provide rapid and effective detection of multiple target nucleic acids in parallel. Therefore, the present disclosure provides an integrated system that can use technology known in the art (such as those illustrated above) to prepare and detect nucleic acids. Therefore, the integrated system of the present disclosure can include amplification reagents and / or sequencing reagents that can be delivered to one or more fixed DNA fragments. The system includes components such as pumps, valves, reservoirs, fluid pipelines, etc. The circulation cell can be configured for and / or for detecting target nucleic acids in the integrated system. Exemplary circulation cells are described in, for example, U.S. Patent Publication No. 2010 / 0111768 and U.S. Patent Application No. 13 / 273,666, and these two patents are each incorporated herein by reference. As illustrated for the circulation cell, one or more fluid components of the integrated system can be used for amplification method and detection method. Taking nucleic acid sequencing embodiment as an example, one or more fluid components of the integrated system can be used for the amplification method described herein and for delivering sequencing reagents in sequencing method (such as those illustrated above). Alternatively, the integrated system may include a separate fluid system to perform amplification method and perform detection method. Examples of integrated sequencing systems that can generate amplified nucleic acids and also determine nucleic acid sequences include, but are not limited to, MiSeq TM The ELISA platform (Illumina, Inc., San Diego, CA), and the apparatus described in U.S. Patent Application No. 13 / 273,666, which is incorporated herein by reference.

[0234] The array that wherein polynucleotide has been directly attached to the carrier based on silica is for example disclosed in WO 00 / 06770 (incorporated herein by reference), wherein polynucleotide is fixed on the glass carrier by the reaction between the epoxy side group on glass and the internal amino group on polynucleotide.In addition, polynucleotide can be attached to the solid carrier by the reaction of sulfenyl nucleophilic substance and solid carrier, for example, as described in WO 2005 / 047301 (incorporated herein by reference).Another example of the template polynucleotide of solid support is, wherein template polynucleotide is attached to the hydrogel carried on the solid support based on silica or other solid support, for example, as described in WO 00 / 31148, WO 01 / 01143, WO02 / 12566, WO 03 / 014392, U.S. Patent number 6,465,178 and WO 00 / 53812, these patents are separately incorporated herein by reference.

[0235] 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 WO 2005 / 065814, which is incorporated herein by reference. Specific hydrogels that can be used include those described in WO 2005 / 065814 and U.S. Patent Publication No. 2014 / 0079923. In one embodiment, the hydrogel is PAZAM (poly (N- (5-azidoacetamidopentyl) acrylamide-co-acrylamide)).

[0236] The DNA template molecules can be attached to beads or microparticles, for example, as described in U.S. Pat. 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 their generation are described in the following documents: Nature, 437, 376-380 (2005); Science, 309, 5741, 1728-1732 (2005), each of which is incorporated herein by reference. It is within the scope of the present disclosure to sequence an array of such beads using the nucleotides shown herein.

[0237] 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. Therefore, the method of the present disclosure is applicable to all types of high-density arrays, including single-molecule arrays, clustered arrays, and bead arrays. The labeled nucleotides disclosed herein can be used to sequence templates on substantially any type of array (including but not limited to those formed by fixing nucleic acid molecules on a solid support).

[0238] However, the labeled nucleotides of the present disclosure are particularly advantageous in the context of sequencing the clustered array. In the clustered array, different regions (commonly referred to as sites or features) on the array include multiple polynucleotide template molecules. Generally speaking, the multiple polynucleotide molecules can not be distinguished individually by optical means, but are detected as a whole. Depending on the formation mode of the array, each site on the array can include multiple copies of a single polynucleotide molecule (for example, the site is homogeneous for a specific single-stranded nucleic acid species or double-stranded nucleic acid species) or even multiple copies of a small amount of different polynucleotide molecules (for example, multiple copies of two different nucleic acid species). The clustered array of nucleic acid molecules can be produced using technology known in the art. By way of example, WO 98 / 44151 and WO 00 / 18957 (each incorporated herein) describe a method for amplifying nucleic acids, wherein template and amplification product are both kept fixed on a solid support, so as to form an array composed of a cluster or "colony" of immobilized nucleic acid molecules. The nucleic acid molecules present on the clustered array prepared according to these methods are suitable templates for sequencing using nucleotides labeled with dye compounds of the present disclosure.

[0239] Labeled nucleotides disclosed herein can also be used for sequencing the template on the single molecule array. As used herein, the term "single molecule array" or "SMA" refers to a polynucleotide molecule group distributed (or arranged) on a solid support, wherein the spacing between any single polynucleotide and all other polynucleotides of the molecule group makes it possible to distinguish the single polynucleotide molecule individually. Therefore, in some embodiments, the target nucleic acid molecule fixed to the surface of the solid support can be distinguished by optical means. This means that one or more different signals (each signal represents a polynucleotide) will appear in the distinguishable region of the specific imaging device used.

[0240] Single molecule detection can be achieved, wherein the spacing between adjacent polynucleotide molecules on the array is at least 100 nm, more specifically at least 250 nm, more specifically at least 300 nm, and even more specifically at least 350 nm. Therefore, each molecule can be individually resolved and detected as a single molecule fluorescent spot, and the fluorescence from the single molecule fluorescent spot also exhibits single-step photobleaching.

[0241] The terms "individually resolved" and "individually resolved" are used herein to define that, when visualized, it is possible to distinguish a molecule on an array from its adjacent molecules. The spacing between the 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 published patent applications WO 00 / 06770 and WO 01 / 57248, which are each incorporated herein by reference. Although one use of the nucleotides disclosed herein is for sequencing while synthesizing, the effectiveness of these nucleotides is not limited to this type of method. In fact, these nucleotides can be advantageously used in any sequencing method that requires detection of fluorescent labels attached to the nucleotides incorporated into the polynucleotides.

[0242] In particular, the labeled nucleotides disclosed herein can be used in automated fluorescent sequencing protocols, especially fluorescent dye-terminator cycle sequencing based on the chain termination sequencing method of Sanger and colleagues. Such methods typically use enzymes and cycle sequencing to incorporate fluorescently labeled dideoxynucleotides into primer extension sequencing reactions. The so-called Sanger sequencing method and related protocols (Sanger-type) utilize randomized chain termination of labeled dideoxynucleotides.

[0243] Thus, the present disclosure also encompasses labeled nucleotides that are dideoxynucleotides lacking hydroxyl groups at both the 3' and 2' positions, such dideoxynucleotides being suitable for use in Sanger-type sequencing methods and the like.

[0244] It will be appreciated that the labeled nucleotides of the present disclosure incorporating a 3' blocking group may also be used in Sanger methods and related protocols, since the same effect as that achieved by using dideoxy nucleotides may be achieved by using nucleotides having a 3'-OH blocking group: both prevent incorporation of subsequent nucleotides. In cases where nucleotides according to the present disclosure and having a 3' blocking group are to be used in Sanger-type sequencing methods, it will be appreciated that the dye compound or detectable label attached to the nucleotide need not be attached via a cleavable linker, since in each case of incorporation of a labeled nucleotide of the present disclosure; no subsequent incorporation of a nucleotide is required, and therefore no removal of the label from the nucleotide is required.

[0245] In any embodiment of the SBS method described herein, the nucleotide used in the sequencing application is a 3' blocking nucleotide described herein, for example, a nucleotide of formula (I) and (la) to (ld). In any embodiment, the 3' blocking nucleotide is a nucleotide triphosphate.

[0246] In some sequencing methods, the nucleotides incorporated are unlabeled. After incorporation, one or more fluorescent labels can be introduced by using an affinity reagent containing a label of one or more fluorescent dyes. For example, one, two, three or each of the four different types of nucleotides (e.g., dATP, dCTP, dGTP and dTTP or dUTP) incorporated into the buffer of step (a) can be unlabeled. Each of these four types of nucleotides (e.g., dNTP) has a 3'-OH blocking group (e.g., 3'-AOM) as described herein to ensure that only a single base can be added to the 3' end of the copy polynucleotide by a polymerase. After incorporation of unlabeled nucleotides, an affinity reagent specifically bound to the dNTP incorporated is introduced to provide an extension product containing the label of the dNTP incorporated. U.S. Patent Publication No. 2013 / 0079232 discloses the use of unlabeled nucleotides and affinity reagents in sequencing by synthesis. The improved sequencing method using unlabeled nucleotides disclosed in the present invention may include the following steps:

[0247] (a'-1) will contain the 3'-OH end-capping group described herein incorporating an unlabeled nucleotide (e.g., dATP, dCTP, dGTP, dTTP, or dUTP) (attached to the 3′ oxygen group) into a copy polynucleotide chain complementary to at least a portion of the target polynucleotide chain to produce an extended copy polynucleotide;

[0248] (a'-2) contacting the extended copy polynucleotide with a set of affinity reagents under conditions where one of the affinity reagents specifically binds to the incorporated unlabeled nucleotide to provide a labeled extended copy polynucleotide;

[0249] (b') detecting the identity of the nucleotide incorporated into the copy polynucleotide chain by performing one or more fluorescence measurements of the labeled extended copy polynucleotide; and

[0250] (c') chemically removing the detectable label from the extended copy polynucleotide and removing the 3'-OH blocking group from the nucleotides incorporated into the copy polynucleotide chain.

[0251] Affinity agents may include: small molecules or protein tags (such as streptavidin-biotin, anti-DIG and DIG, anti-DNP and DNP), antibodies (including but not limited to binding fragments of antibodies, single-chain antibodies, bispecific antibodies, etc.), aptamers, knottins, Affimer proteins, or any other known agent that binds to the incorporated nucleotide with suitable specificity and affinity. In other embodiments, an affinity agent can be labeled with multiple copies of the same fluorescent dye. In some embodiments, the Pd catalyst also removes the labeled affinity agent. For example, the hapten portion of the unlabeled nucleotide can be removed by a cleavable linker as described herein. (e.g., an AOL linker) is attached to a nucleobase, and the cleavable linker can be cleaved by a Pd catalyst. In some embodiments, the method further comprises a post-cleavage washing step (d) as described herein. In some embodiments, the method further comprises repeating steps (a'-1) to (c') or (a'-1) to (d) until the sequence of at least a portion of the target polynucleotide chain is determined. In some embodiments, the cycle is repeated at least 50 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, or at least 300 times.

[0252] Reagent test kit

[0253] The present disclosure also provides a kit comprising one or more 3'-capped nucleosides and / or nucleotides described herein, for example, 3'-capped nucleotides of formula (I) and (Ia) to (Id). Such a kit will typically include at least one 3'-capped nucleotide or nucleoside with a detectable label (e.g., a fluorescent dye), and at least one additional component. The additional component may be one or more of the components identified in 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 shown below. In some other embodiments, the kit may include four types of labeled nucleotides, i.e., fully functionalized nucleotides (A, C, T, and G) described herein, wherein each type of nucleotide comprises a 3'-AOM capping group and an AOL linker portion described herein. In other embodiments, G is unlabeled and does not comprise an AOL linker. In further embodiments, one or more of the remaining three nucleotides (i.e., A, C, and T) comprise L. 1 , it is allylamine or allylamide linker moiety.In one embodiment, this test kit includes unlabeled WG, the WA of mark, the WA of mark and the WT-DB of mark as described herein.In another embodiment, this test kit includes unlabeled WG, the WA of mark, the WA of mark, the WT-DB of mark and the WT-DB of mark as described herein.

[0254] In a specific embodiment, the kit can include at least one labeled 3' blocked nucleotide or nucleoside, and labeled or unlabeled nucleotides or nucleosides. 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. Combinations of nucleotides can be provided as independent individual components (e.g., one nucleotide type per container or tube) or as a nucleotide mixture (e.g., two or more nucleotides mixed in the same container or tube).

[0255] In the case where the kit includes a plurality of, particularly two or three, or more particularly four 3'-terminated nucleotides labeled with dye compounds, different nucleotides can be labeled with different dye compounds, or one can be dark, not labeled with dye compounds. In the case where different nucleotides are labeled with different dye compounds, a feature of the kit is 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 commercial capillary-based DNA sequencing platform) when two or more such dyes are present in a sample. When two nucleotides labeled with fluorescent dye compounds are provided in the form of a kit, some embodiments are characterized in that the spectrally distinguishable fluorescent dyes can be excited at the same wavelength, such as, excited by the same laser. When four 3'-terminated nucleotides (A, C, T and G) labeled with fluorescent dye compounds are provided in the form of a kit, some embodiments are characterized in that two fluorescent dyes in the spectrally distinguishable fluorescent dyes can be excited at one wavelength, and the other two spectrally distinguishable dyes can be excited at another wavelength. The specific excitation wavelengths are 488nm and 532nm.

[0256] In one embodiment, the kit comprises a first 3'-terminated nucleotide labeled with a first dye, and a second nucleotide labeled with a second dye, wherein the dyes have a maximum absorbance difference of at least 10 nm, particularly 20 nm to 50 nm. More specifically, the two dye compounds have a Stokes shift between 15 nm and 40 nm, wherein "Stokes shift" is the distance between the peak absorption wavelength and the peak emission wavelength.

[0257] In an alternative embodiment, the kit of the present disclosure may include 3'-terminated nucleotides in which the same base is labeled with two or more different dyes. The first nucleotide (e.g., 3'-terminated T nucleotide triphosphate or 3'-terminated G nucleotide triphosphate) can be labeled with a first dye. The second nucleotide (e.g., 3'-terminated C nucleotide triphosphate) can be labeled with a second dye spectrally different from the first dye, such as a "green" dye that absorbs light at a wavelength less than 600nm, and a "blue" dye that absorbs light at a wavelength less than 500nm, such as 400nm to 500nm, particularly 450nm to 460nm. The third nucleotide (e.g., 3'-terminated A nucleotide triphosphate) can be labeled as a mixture of the first dye and the second dye, or a mixture of the first dye, the second dye and the third dye, and the fourth nucleotide (e.g., 3'-terminated G nucleotide triphosphate or 3'-terminated T nucleotide triphosphate) can be "dark", and does not include a label. In one example, nucleotides 1 to 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 to 4 can be labeled "blue 1", "blue 2", "blue 1 / blue 2", and dark.

[0258] In a specific embodiment, the kit can include four labeled 3'-capped nucleotides (e.g., A, C, T, G), wherein each type of nucleotide includes the same 3'-capping group and fluorescent label, and wherein each fluorescent label has a different maximum fluorescence value, and each fluorescent label can be distinguished from the other three labels. The kit can be such that two or more of the fluorescent labels have similar maximum absorbances but different Stokes shifts. In some other embodiments, one type of nucleotide is unlabeled.

[0259] Although the kit is described herein with a configuration having different nucleotides labeled with different dye compounds as an example, it should be understood that the kit may include 2, 3, 4 or more different nucleotides having the same dye compound. In some embodiments, the kit also includes an enzyme and a buffer suitable for the enzyme to function. In some such embodiments, the enzyme is a polymerase, a terminal deoxynucleotidyl transferase or a reverse transcriptase. In a specific embodiment, the enzyme is a DNA polymerase, such as DNA polymerase 812 (Pol 812) or DNA polymerase 1901 (Pol 1901). In some other embodiments, the kit may include an incorporation mixture as described herein. In other embodiments, the kit containing the incorporation mixture as described herein also includes at least one Pd scavenger (e.g., a Pd(0) scavenger comprising one or more allylic moieties as described herein). The Pd(0) scavenger includes one or more allylic moieties each independently selected from the group consisting of: -O-allyl, -S-allyl, -NR-allyl and -N-allyl. + RR′-allyl, wherein R is H, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C2-C6 alkenyl, unsubstituted or substituted C2-C6 alkynyl, unsubstituted or substituted C6-C 10 aryl, unsubstituted or substituted 5- to 10-membered heteroaryl, unsubstituted or substituted C3-C 10 carbocyclic group, or unsubstituted or substituted 5- to 10-membered heterocyclic group; and R' is H, unsubstituted C1-C6 alkyl or substituted C1-C6 alkyl. In some such embodiments, the Pd(0) scavenger incorporated into the solution comprises one or more -O-allyl moieties. In other embodiments, the Pd(0) scavenger comprises or is or a combination thereof. Alternative Pd(0) scavengers are disclosed in U.S. Serial No. 63 / 190983, which is incorporated by reference in its entirety. In one embodiment, the Pd(0) scavenger incorporated into the mixture comprises or is In another embodiment, the Pd(0) scavenger incorporated into the mixture comprises or is

[0260] Other components to be included in such kits may include buffers, etc. Nucleotides of the present disclosure and any other nucleotide components including mixtures of different nucleotides may be provided in a concentrated form in a kit and diluted before use. In such embodiments, a suitable dilution buffer may also be included. For example, the mixture kit may include one or more buffers selected from the following: primary amines, secondary amines, tertiary amines, natural amino acids or non-natural amino acids, or a combination thereof. In other embodiments, the buffer incorporated into the mixture includes ethanolamine or glycine, or a combination thereof.

[0261] Similarly, one or more components of the components identified in the methods shown herein may be included in the kit of the present disclosure. In other embodiments, the kit may include a palladium catalyst as described herein. In some embodiments, the Pd catalyst is produced by mixing a Pd (II) complex (i.e., a Pd precatalyst) with one or more water-soluble phosphines as described herein. In some such embodiments, the kit comprising the Pd catalyst is a cracking mixture kit. In other embodiments, the cracking mixture kit may include [Pd (allyl) Cl] 2 or Na2PdCl4 and a water-soluble phosphine THP for producing active Pd (0) material. The molar ratio of the Pd (II) complex (e.g., [Pd (allyl) Cl] 2 or Na2PdCl4) to a water-soluble phosphine (e.g., THP) may be about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. In other embodiments, the lysis mixture kit may also include one or more buffer reagents selected from the group consisting of the following items: primary amines, secondary amines, tertiary amines, carbonates, phosphates, borates, and combinations thereof. Non-limiting examples of buffer reagents in the lysis mixture kit are selected from the group consisting of the following items: ethanolamine (EA), tris (hydroxymethyl) aminomethane (Tris), glycine, carbonates, phosphates, borates, dimethylethanolamine (DMEA), diethylethanolamine (DEEA), N, N, N', N'-tetramethylethylenediamine (TEMED), N, N, N', N'-tetraethylethylenediamine (TEEDA), 2-piperidineethanol, and combinations thereof. In one embodiment, the lysis mixture kit includes DEEA. In other embodiments, the lysis mixture kit includes 2-piperidineethanol.

[0262] In some other embodiments, the kit may include one or more palladium scavengers (e.g., Pd (II) scavengers described herein). In some such embodiments, the kit is a post-lysis wash buffer kit. Non-limiting examples of Pd scavengers in the post-lysis wash buffer kit include isocyanoacetate (ICNA) salts, ethyl isocyanoacetate, methyl isocyanoacetate, cysteine ​​or its salts, L-cysteine ​​or its salts, N-acetyl-L-cysteine, potassium ethyl xanthate, potassium isopropyl xanthate, glutathione, lipoic acid, ethylenediaminetetraacetic acid (EDTA), iminodiacetic acid, nitrilodiacetic acid, trimercapto-S-triazine, dimethyldithiocarbamate, dithiothreitol, mercaptoethanol, allyl alcohol, propargyl alcohol, thiol, tertiary amine and / or tertiary phosphine, or a combination thereof. In one embodiment, the post-lysis wash buffer kit includes L-cysteine ​​or its salt.

[0263] In any of the embodiments of the kits described herein, the Pd scavenger (eg, a Pd(0) or Pd(II) scavenger described herein) is in a separate container / compartment from the Pd catalyst.

[0264] Example

[0265] Additional embodiments are disclosed in more detail in the following examples, which are not intended to limit the scope of the claims in any way.

[0266] Example 1. Synthesis of fully functionalized nucleotides with 3' AOM and AOL linker moieties

[0267] Scheme 1. Synthesis of AOL linker

[0268]

[0269] Synthetic intermediate AOL LN2 :

[0270] The acetal compound LN1 (2.43 g, 9.6 mmol) was dissolved in anhydrous CH2Cl2 (100 mL) under N2, and the solution was cooled to 0°C with an ice bath. 2,4,6-Trimethylpyridine (7.6 mL, 57.5 mmol) was added, followed by the dropwise addition of trimethylsilyl trifluoromethanesulfonate (7.0 mL, 38.7 mmol). The mixture was stirred at 0°C for 2 hours, then allyl alcohol (13 L, 191.1 mmol) was added, and the reactants were refluxed overnight. The reaction was quenched with a 98:2 mixture of MeOH / H2O, and the resulting solution was stirred for another 3 hours at RT. The mixture was diluted with CH2Cl2 (100 mL) and water (200 mL), and the aqueous layer was acidified to pH 2 to 3 with 2N HCl. The aqueous layer was separated, and the organic layer was further extracted with acidic water. The organic layer was dried over MgSO4, filtered, and the volatiles were evaporated under reduced pressure. The crude product was purified by flash chromatography on silica gel to afford AOL LN2 as a colorless oil (2.56 g, 86%).

[0271] Synthetic intermediate AOL LN3 :

[0272] To a solution of AOL LN2 (2.17 g, 7.0 mmol) in ethanol (17.5 mL) was added 4 M NaOH (17.5 mL, 70 mmol) in water and the mixture was stirred at RT for 3 hours. Thereafter, all volatiles were removed under reduced pressure and the residue was dissolved in 75 mL of water. The solution was acidified to pH 2-3 with 2N HCl and then extracted with dichloromethane (DCM). The combined organic fractions were dried over MgSO4, filtered and the volatiles were evaporated under reduced pressure. AOL LN3 was obtained without further purification as a colorless oil that solidified upon storage at -20°C (1.65 g, 84%). LC-MS (ES): (negative ion) m / z 281 (MH + ); (positive ion) m / z 305 (M+Na + ).

[0273] Synthetic intermediate AOL LN4 :

[0274] A solution of AOL LN3 (1.62 g, 5.74 mmol) in anhydrous DMF (20 mL) was stirred under vacuum for 5 min and then cooled to 0°C with an ice bath. N,N-diisopropylethylamine (1.2 mL, 6.89 mmol) was added dropwise under N2, followed by PyBOP (3.30 g, 6.34 mmol). The reaction was stirred at 0°C for 30 min, then a solution of N-(5-aminopentyl)-2,2,2-trifluoroacetamide hydrochloride (1.62 g, 6.90 mmol) in anhydrous DMF (3.0 mL) was added, followed immediately by additional N,N-diisopropylethylamine (1.4 mL, 8.04 mmol). The reaction was removed from the ice bath and stirred at RT for 4 h. The volatiles were removed under reduced pressure and the residue was dissolved in EtOAc (150 mL). The solution was extracted with 20 mM aqueous KHSO4, water, and saturated aqueous NaHCO3. The organic layer was dried over MgSO4, filtered and the volatiles were evaporated under reduced pressure. The crude product was purified by silica gel flash chromatography to obtain AOL LN4 as a colorless oil (2.16 g, 82%). LC-MS (ES): (negative ion) m / z 461 (MH + ), 497(M-Cl - ).

[0275] Synthetic AOL linker

[0276] To a CH3CN solution (13 mL) of AOL LN4 (350 mg, 0.76 mmol) was added TEMPO (48 mg, 0.31 mmol), followed by an aqueous solution (6.5 mL) of NaH2PO4·2H2O (762 mg, 4.88 mmol) and NaClO2 (275 mg, 3.04 mmol). An aqueous solution of NaClO (14% available chlorine, 0.83 mL, 1.94 mmol) was added, and the solution immediately turned dark brown. The reaction was stirred for 6 hours at RT and then quenched with a 100 mM Na2S2O3 aqueous solution until the mixture became colorless. The acetonitrile was removed under reduced pressure, and the residue was diluted with water and then basified with triethylamine. The aqueous phase was extracted with EtOAc (10 mL) and then concentrated under reduced pressure. The crude product was purified by reverse phase flash chromatography on a C18 column to give AOL as a colorless oil (triethylammonium salt, 310 mg, 71%). LC-MS(ES):(negative ion)m / z 475(MH + ); (positive ion) m / z499 (M+Na + ), 578(M+Et3NH + ).

[0277] Synthesis of AOL-NH2 linker

[0278] To a solution of AOL (446 mg, 0.94 mmol) in methanol (10 mL) was added aqueous NH3 (35%, 40 mL) and the mixture was stirred at RT for 5.5 hours. After that, all volatiles were removed under reduced pressure and the crude product was purified by reverse phase flash chromatography on a C18 column to afford AOL NH2 as a white solid (quantitative). 1 H NMR (400MHz, DMSO-d6): δ (ppm) 8.86 (t, J = 5.5Hz, 1H, CONH), 8.28 (s, 3H, NH3 + ),7.85(s,1H,Ar-H),7.41(d,J=7.6Hz,1H,Ar-H),7.31(t,J=7.9Hz,1H,Ar-H),7.03(ddd,J=8.1,2.5,1.1 Hz,1H,Ar-H),5.87(ddt,J=17.2,10.5,5.3Hz,1H,OCH2CHCH2),5.24(dq,J=17.2,1.7Hz,1H,OCH2CHCH2,H a ),5.09(dq,J=10.5,1.5Hz,1H,OCH2CHCH2,H b ),5.02(dd,J=6.7,2.4Hz,1H,OCHO),4.41(dd,J=12.2,2.5Hz,1H,OCH2,H a ),4.18–3.99(m,3H,OCH2CHCH2 and OCH2,H b ),3.94–3.81(m,2H,OCH2COOH),3.49–3.39(m,1H,CH 2, H a ),3.21–3.10(m,1H,CH2,H b ),2.86–2.70(m,2H,CH2),1.81–1.39(m,6H,CH2). 13 C NMR (101 MHz, DMSO-d6): δ (ppm) 172.9, 166.1, 158.0, 136.2, 135.2, 129.3, 120.4, 119.3, 116.0, 111.3, 99.0, 68.8, 67.7, 66.8, 38.7, 38.4, 27.8, 26.3, 23.0. LC-MS (ESI): (negative ion) 379 (MH); (positive ion) m / z 381 (M+H + ).

[0279] General procedure for dye-AOL linker conjugation :

[0280] Dye carboxylate (0.15mmol) is dissolved in 6mL anhydrous N, N'-dimethylformamide (DMF). Add N, N-diisopropylethylamine (136μL, 0.78mmol), then add 0.5M anhydrous DMF solution (TSTU, 300μL, 0.15mmol) of N, N, N', N'-tetramethyl-O-(N-succinimide) urea tetrafluoroborate. At RT, the reactant is stirred for 1 hour under nitrogen. The aqueous solution (400μL) of AOL NH2 (0.10mmol) is added to the activated dye solution, and the reactant is stirred for 3 hours at RT. Take the crude product and purify by preparative RP-HPLC.

[0281]

[0282] Characterization of AOL-SO7181: Yield 54% (54 μmol). LC-MS (ES): (negative ion) m / z 1002 (MH + ); (positive ion) m / z 1004 (M+H + ).

[0283]

[0284] Characterization of AOL-AF550POPOS0: Yield 88% (88 μmol). LC-MS (ES): (negative ion) m / z 1034 (MH + ), 516(M-2H + ); (positive ion) m / z 1036 (M+H + ), 1137(M+Et3NH + ).

[0285]

[0286] Characterization of AOL-NR7180A: Yield 24% (23.9 μmol). LC-MS (ES): (positive ion) m / z=880 (M+H) + .

[0287]

[0288] Characterization of AOL-NR550S0: Yield 22% (21.9 μmol). LC-MS (ES): (positive ion) m / z = 963 (M+H) + . (Negative ion) m / z = 961 (MH + ).

[0289] Scheme 2. Synthesis of 5'-triphosphate-3'-AOM-A(DB) nucleotide

[0290]

[0291] Synthetic intermediate A2 :

[0292] Compound A1 (319 mg, 0.419 mmol) was dissolved in 0.8 mL of anhydrous DCM under N2 atmosphere, and pentamethylcyclopentadienyl tris(acetonitrile)ruthenium hexafluorophosphate (II) ([RuCp*(MeCN)3]PF6, 42 mg, 0.08 mmol) was then added, followed by triethoxysilane (231 μL, 1.25 mmol). The reactants were stirred under N2 for 1 hour at RT. The solution was then diluted with DCM, filtered on a silica gel plug, and the silica gel plug was washed with ethyl acetate. The solution was evaporated under reduced pressure, dried under vacuum for 10 minutes, and the residue was then dissolved in 2 mL of anhydrous THF. Cuprous iodide (15 mg, 0.08 mmol) and a THF solution of 1M TBAF (920 μL, 0.919 mmol) were added. The reactants were stirred for 2.5 hours at RT, diluted with EtOAc, and extracted with saturated NH4Cl. The aqueous phase was extracted with EtOAc. The combined organic phases were dried over MgSO4, filtered and evaporated to dryness. The residue was purified by silica gel flash chromatography. Yield: 125 mg (0.237 mmol). LC-MS (ES and CI): (positive ion) m / z 527 (M+H + ).

[0293] Synthetic intermediate A3 :

[0294] Nucleoside A2 (155 mg, 0.294 mmol) was dried over P2O5 under reduced pressure for 18 h. Anhydrous triethyl phosphate (1 mL) and some freshly activated The reaction flask was then cooled to 0°C in an ice bath. Freshly distilled POCl3 (33 μL, 0.353 mmol) was added dropwise, followed by Proton (113mg, 0.53mmol). After addition, the reactant was further stirred for 15 minutes at 0°C. Then, an anhydrous DMF solution of 0.5M di-tri-n-butylammonium pyrophosphate (2.94mL, 1.47mmol) was quickly added, followed by immediate addition of tri-n-butylamine (294μL, 1.32mmol). The reactant was kept in an ice-water bath for another 10 minutes, then poured into a 1M triethylammonium bicarbonate aqueous solution (TEAB, 10mL) to quench, and stirred for 4 hours at RT. All solvents were evaporated under reduced pressure. 35% ammonia solution (10mL) was added to the above residue, and the mixture was stirred for at least 5 hours at RT. The solvent was then evaporated under reduced pressure. The crude product was first purified by ion exchange chromatography on DEAE-Sephadex A25 (50g). The chromatographic column was eluted with triethylammonium bicarbonate (TEAB) aqueous solution. The fractions containing triphosphoric acid were combined, and the solvent was evaporated under reduced pressure to dryness. The crude material was further purified by preparative HPLC. Compound A3 was obtained as triethylammonium salt. Yield: 134 μmol (46%). LC-MS (ESI): (negative ion) m / z 614 (MH + ).

[0295] In addition, a structure The 5'-triphosphate-3'-AOM-A nucleotide and the corresponding ffA. The detailed synthesis steps are described in US Patent Application No. 16 / 724,088.

[0296] General Synthesis Process of Nucleotide Triphosphate-AOL Linkers :

[0297] Compound AOL (0.120 mmol) was co-evaporated with 2×2 mL of anhydrous N,N'-dimethylformamide (DMF) and then dissolved in 3 mL of anhydrous N,N'-dimethylacetamide (DMA). N,N-diisopropylethylamine (70 μL, 0.4 mmol) was added, followed by N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU, 36 mg, 0.120 mmol). The reaction was stirred under nitrogen for 1 hour at RT. At the same time, an aqueous solution of nucleotide triphosphates (0.08 mmol) was evaporated to dryness under reduced pressure and then resuspended in 300 μL of 0.1 M triethylammonium bicarbonate (TEAB) aqueous solution. The activated linker solution was added to the nucleotide triphosphates and the reaction was stirred for 18 hours at RT and then monitored by RP-HPLC. The solution was concentrated and 10 mL of concentrated NH4OH aqueous solution was added. The reaction was stirred for 24 hours at RT and then evaporated under reduced pressure. The crude product was first purified by ion exchange chromatography on DEAE-Sephadex A25 (50 g), eluting with aqueous triethylammonium bicarbonate (TEAB). The fractions containing triphosphoric acid were combined and the solvent was evaporated to dryness under reduced pressure. The crude material was further purified by preparative HPLC.

[0298]

[0299] Characterization of pppA(DB)-(3'AOM)-AOL: Yield 60 μmol (75%). LC-MS (ES): (negative ion) m / z 976 (MH + ), 488(M-2H + ).

[0300]

[0301] Characterization of pppA(DB)-(3'AOM)-AOL: Yield 68 μmol (72%). 1H NMR (400MHz, D2O): δ (ppm) 7.94 (d, J=1.7Hz, 1H, H-2), 7.32 (d, J=1.6Hz, 1H, H-8), 7.10–6.96 (m ,2H,Ar),6.95–6.87(m,2H,Ar),6.41–6.31(m,1H,1'-CH),6.01–5.81(m,2H,CH allyl),5.29(ddq, J=17.2,6.1,1.4 Hz,2H,CHHallyl), 5.20 (ddt, J=10.5,3.8,1.1 Hz,2H,CHHallyl), 5.02 (td, J=4.4,2.4 Hz,1H,O-CH2-O linker), 4.92–4.81 (m,2H,3'-O-CH2-O), 4.53 (dd, J=4.9,2.4 Hz,1H,3'-CH), 4.3 9 (dd, J = 16.1, 4.0 Hz, 1H, O-CHH linker), 4.32–4.19 (m, 2H, O-CHH linker, 4'-CH), 4.17–4.01 (m, 8H, 5'-CH2, CH2O linker, CH2-O allyl), 3.25–3.11 (m, 2H, CH2-NHCO), 3.04 (q, J = 7.3 Hz, 18H, Et3N), 2.92–2.82 ( m, 2H, CH2-N connector), 2.55–2.41 (m, 2H, 2'-CH2), 1.59 (p, J = 7.6 Hz, 2H, CH2-CH2-N connector), 1.47 (p, J = 7.1 Hz, 2H, CH2-CH2-NHCO), 1.31 (tt, J = 8.3, 4.4 Hz, 2H, CH2-CH2-CH2-N connector), 1.15 (t, J = 7.3 Hz, 26H). 31 P NMR (162MHz, D2O): δ (ppm)-6.18 (d, J = 20.6Hz, γ P),-11.32(d,J=19.3Hz, α P),-22.20(t,J=19.9Hz, β P).

[0302] Scheme 3. Synthesis of 5'-triphosphate-3'-AOM-C(DB) nucleotide

[0303]

[0304] Synthetic intermediate C1

[0305] 5-iodo-5'-O-(tert-butyldiphenylsilyl)-2'-deoxycytidine (3g, 5.07mmol) was dissolved in 30mL anhydrous pyridine, and trimethylsilyl chloride (1.29mL, 10.1mmol) was then added dropwise. The reactant was stirred for 1 hour at RT, then placed in an ice bath, and benzoyl chloride (648μL, 5.6mmol) was slowly added dropwise. The reactant was taken out of the ice bath and then stirred for 1 hour at RT. After completion, the solution was placed in an ice bath and quenched with 50mL cold water, then 50mL methanol and 20mL pyridine were added, and the suspension was stirred overnight at RT. The solvent was evaporated under reduced pressure, and the residue was dissolved in 200mL EtOAc and extracted with 2×200mL saturated NaHCO3 and 100mL brine. The organic phase was dried with MgSO4, filtered and evaporated to dryness. The crude product was purified by silica gel flash chromatography to obtain C1. Yield: 2.535 g (3.64 mmol, 73%). LC-MS (ESI): (positive ion) m / z 696 (M+H + ), 797(M+Et3NH + ).

[0306] Synthetic intermediate C2

[0307] N-Benzoyl-5-iodo-5'-O-(tert-butyldiphenylsilyl)-2'-deoxycytidine (C1) (695 mg, 1 mmol) and palladium (II) acetate (190 mg, 0.85 mmol) were dissolved in dry degassed DMF (10 mL), and N-allyl trifluoroacetamide (7.65 mL, 5 mmol) was then added. The solution was placed under vacuum and purged with nitrogen three times, and then degassed triethylamine (278 μL, 2 mmol) was added. The solution was heated to about 80°C and protected from light for 1 hour. The resulting black mixture was cooled to room temperature, then diluted with 50 mL EtOAc and extracted with 100 mL water. The aqueous phase was then extracted with EtOAc. The organic phases were combined, dried over MgSO4, filtered and evaporated to dryness. The crude product was purified by flash chromatography on silica gel to give C2. Yield: 305 mg (0.42 mmol, 42%). LC-MS (ES and CI): (positive ion) m / z 721 (M+H + ), 797(M+Et3NH + ).

[0308] Synthetic intermediate C3

[0309] N-Benzoyl-5-[3-(2,2,2-trifluoroacetylamino)-allyl]-5'-O-(tert-butyldiphenylsilyl) 2'-deoxycytidine (C2) (350 mg, 0.486 mmol) was dissolved in 1.1 mL of anhydrous DMSO (14.5 mmol), followed by the addition of glacial acetic acid (1.7 mL, 29.1 mmol) and acetic anhydride (1.7 mL, 17 mmol). The reactants were heated to 60 °C for 6 hours and then quenched with 50 mL of saturated aqueous NaHCO3. After the solution stopped bubbling, it was extracted with EtOAc. The organic phases were combined and washed with saturated aqueous NaHCO3, water and brine. The organic phase was dried over MgSO4, filtered and evaporated to dryness. The crude product was purified by flash chromatography on silica gel to give C3. Yield: 226 mg (0.289 mmol, 60%). LC-MS (ESI): (positive ion) m / z 781 (M+H + ), 882(M+Et3NH + ).

[0310] Synthetic intermediate C4

[0311] N-Benzoyl-5-[3-(2,2,2-trifluoroacetylamino)-allyl]-5'-O-(tert-butyldiphenylsilyl)-3'-O-methylthiomethyl-2'-deoxycytidine (C3) (210 mg, 0.27 mmol) was dissolved in 5 mL of anhydrous DCM under N2 atmosphere, cyclohexene (136 μL, 1.35 mmol) was added, and the solution was cooled to about -10°C. Freshly distilled 1M sulfuryl chloride in anhydrous DCM (320 μL, 0.32 mmol) was added dropwise and the reaction was stirred for 20 minutes. After all the starting material had been consumed, an additional portion of cyclohexene (136 μL, 1.35 mmol) was added and the reaction was evaporated to dryness under reduced pressure. The residue was quickly purged with nitrogen and then dissolved in 2.5 mL of ice-cold anhydrous DCM and ice-cold allyl alcohol (2.5 mL) was added while stirring at 0°C. The reaction was stirred at 0°C for 3 hours, then quenched with saturated aqueous NaHCO3 solution, and then further diluted with saturated aqueous NaHCO3 solution. The mixture was extracted with EtOAc. The combined organic phases were dried over MgSO4, filtered and evaporated to dryness. The residue was purified by flash chromatography on silica gel to give C4. Yield: 58% (124 mg, 0.157 mmol). LC-MS (ESI): (positive ion) m / z 791 (M+H + ).

[0312] Synthetic intermediate C5

[0313] N-Benzoyl-5-[3-(2,2,2-trifluoroacetylamino)-allyl]-5'-O-(tert-butyldiphenylsilyl)-3'-O-allyloxymethyl-2'-deoxycytidine (C4) (120 mg, 0.162 mmol) was dissolved in anhydrous THF (5 mL) under N2 atmosphere and then placed at 0°C. Glacial acetic acid (29 μL, 0.486 mmol) was added, followed by a 1.0 M TBAF solution in THF (486 μL, 0.486 mmol). The solution was stirred at 0°C for 3 hours. The solution was diluted with EtOAc and then extracted with 0.025N HCl and brine. The organic phase was dried over MgSO4, filtered and evaporated to dryness. The residue was purified by flash chromatography on silica gel to give C5. Yield: 50 mg (0.090 mmol, 55%). LC-MS (ESI): (positive ion) m / z 553 (M+H + ). (Negative ion) m / z551(MH + ), 587(M+Cl - ).

[0314] Synthetic intermediate C6

[0315] N-Benzoyl-3'-O-allyloxymethyl-5-[3-(2,2,2-trifluoroacetylamino)-allyl]-2'-deoxycytidine (C5) (50 mg, 0.0.09 mmol) was dried over P2O5 for 18 hours under reduced pressure. Anhydrous triethyl phosphate (1 mL) and some freshly activated The reaction flask was then cooled to 0°C in an ice bath. Freshly distilled POCl3 (10 μL, 0.108 mmol) was added dropwise, followed by Proton (29mg, 0.135mmol). After addition, the reactant was further stirred for 15 minutes at 0°C. Then, an anhydrous DMF solution of 0.5M di-tri-n-butylammonium pyrophosphate (1mL, 0.45mmol) was quickly added, followed by immediate addition of tri-n-butylamine (100μL, 0.4mmol). The reactant was kept in an ice-water bath for another 10 minutes, then poured into 1M triethylammonium bicarbonate aqueous solution (TEAB, 5mL) for quenching, and stirred for 4 hours at RT. All solvents were evaporated under reduced pressure. 35% ammonia solution (5mL) was added to the above residue, and the mixture was stirred for 18 hours at RT. The solvent was evaporated under reduced pressure, and the residue was resuspended in 10mL 0.1M TEAB, then filtered. The filtrate was first purified by ion exchange chromatography on DEAE-Sephadex A25 (50g). The chromatographic column was eluted with triethylammonium bicarbonate aqueous solution. The fractions containing triphosphoric acid were combined, and the solvent was evaporated under reduced pressure to dryness. The crude material was further purified by preparative HPLC. Compound C6 was obtained as a triethylammonium salt. Yield: 40.6 μmol (45%), based on ε 290 =5041M -1 cm -1 . 1 H NMR (400MHz, D2O): δ (ppm) 8.23 ​​(s, 1H, H-6), 6.53 (dd, J=15.5, 0.9Hz, 1H, Ar-CH =),6.42–6.24(m,2H,Ar-CH=CH-,1'-CH),5.98(ddt,J=17.3,10.4,5.9Hz,1H,O -CH2-CH=),5.37(dq,J=17.3,1.6Hz,1H,CHH=),5.29(ddt,J=10.4,1.6,1.1Hz, 1H,CHH=),4.89(s,2H,O-CH2-O),4.60(dt,J=6.2,3.1Hz,1H,3'-CH),4.39(t,J= 2.7Hz,1H,4'-CH),4.35(dq,J=12.0,3.8Hz,1H,5'-CHH),4.28–4.21(m,1H,5'- CHH), 4.20 (ddt, J=6.0, 2.7, 1.4Hz, 1H, = CH-CH2-O), 3.73 (dt, J=7.2, 1.4Hz, 2H, CH2-NH2),3.18(q,J=7.3Hz,20H,Et3N),2.59(ddd,J=14.1,6.1,3.3Hz,1H,2'- CHH), 2.37 (ddd, J=14.2, 7.2, 6.1Hz, 1H, 2'-CHH), 1.27 (t, J=7.3Hz, 31H, Et3N). 31P NMR (162MHz, D2O): δ (ppm)-6.06 (d, J = 20.7Hz, γ P),-11.24(d,J=19.1Hz, α P),-21.95(t,J=19.7Hz, β P). LC-MS (ESI): (negative ion) m / z 591 (MH + ).

[0316] In addition, 5'-triphosphate-3'-AOM-C nucleotide and 5'-triphosphate-3'-AOM-T(DB) nucleotide having the following structures were prepared:

[0317] Finally, 5'-triphosphate-3'-AOM-G (also called ffG-(3'-AOM)) was prepared. The detailed synthesis steps are described in U.S. Patent Publication No. 2020 / 0216891.

[0318] General synthetic procedure for fully functionalized nucleotides with AOL linker moieties

[0319] Dye-COOH (0.02 mmol) or dye-AOL (0.02 mmol) was co-evaporated with 2×2 mL of anhydrous N,N'-dimethylformamide (DMF) and then dissolved in 2 mL of anhydrous N,N'-dimethylacetamide (DMA). N,N-diisopropylethylamine (17 μL, 0.1 mmol) was added, followed by N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU, 6 mg, 0.02 mmol). The reaction was stirred under nitrogen for 1 hour at RT. At the same time, an aqueous solution of nucleotide triphosphates (0.01 mmol) was evaporated to dryness under reduced pressure and then resuspended in 200 μL of 0.1 M triethylammonium bicarbonate (TEAB) aqueous solution. The activated dye solution was added to the nucleotide triphosphates and the reaction was stirred for 18 hours at RT and then monitored by RP-HPLC. The crude product was first purified by ion exchange chromatography on DEAE-Sephadex A25 (25 g) using a linear gradient elution with triethylammonium bicarbonate (TEAB, 0.1 M to 1 M) aqueous solution. The fractions containing triphosphoric acid were combined and the solvent was evaporated to dryness under reduced pressure. The crude material was further purified by preparative HPLC.

[0320]

[0321] Characterization of ffA-(3'AOM)-AOL-BL-NR650C5: Yield 66% (6.6 μmol). LC-MS (ES): (negative ion) m / z 1931 (MH + ), 965(M-2H + ).

[0322]

[0323] Characterization of ffA-(3'AOM)-AOL-BL-NR550s0: Yield 65% (6.5 μmol). LC-MS (ES): (negative ion) m / z 1784 (MH + ), 891(M-2H + ).

[0324]

[0325] Characterization of ffA(DB)-(3'AOM)-AOL-BL-NR650C5: Yield 31% (3.1 μmol). LC-MS (ES): (negative ion) m / z 1933 (MH + ), 965(M-2H + ), 644(M-3H + ).

[0326]

[0327] Characterization of ffA(DB)-(3'AOM)-AOL-NR7181A: Yield 21% (2.12 μmol). LC-MS (ES): (negative ion) m / z=1475 (MH + ).

[0328]

[0329] Characterization of ffA-(3'-AOM)-AOL-NR7180A: Yield 41% (4.1 μmol). LC-MS (ES): (negative ion) m / z 1472 (MH + ), 736(M-2H + ).

[0330]

[0331] Characterization of ffA(DB)-(3'-AOM)-AOL-BL-NR550S0: Yield 21% (2.1 μmol). LC-MS (ES): (negative ion) m / z 1786 (MH + ), 892(M-2H +), 594(M-3H + ).

[0332]

[0333] Characterization of ffC(DB)-(3'AOM)-AOL-SO7181: Yield 48% (4.87 μmol). LC-MS (ES): (negative ion) m / z 1577 (MH + ), 788(M-2H + ), 525(M-3H + ).

[0334]

[0335] Characterization of ffC-(3'-AOM)-AOL-SO7181: Yield 56% (5.6 μmol). LC-MS (ES): (negative ion) m / z 1575 (MH + ), 787(M-2H + ).

[0336]

[0337] Characterization of ffT(DB)-3'AOM-AOL-AF550POPOS0: Yield 46% (4.6 μmol). LC-MS (ES): (negative ion) m / z 1609 (MH + ), 804(M-2H + ), 536(M-3H + ).

[0338]

[0339] Characterization of ffT(DB)-(3'AOM)-AOL-NR550s0: Yield 38% (3.8 μmol). LC-MS (ES): (negative ion) m / z=1535 (MH + ).

[0340] Example 2. Solution cleavage efficiency of different palladium reagent formulations

[0341] Figure 1A comparison of the cleavage efficiency of solutions of three different palladium reagent formulations is presented: 1) 10 mM [(allyl)PdCl]2, 100 mM THP, 100 mM ethanolamine buffer, 10 mM sodium ascorbate; 2) 20 mM Na2PdCl4, 60 mM THP, 100 mM N,N'-diethylethanolamine, 10 mM sodium ascorbate, 1 M NaCl, 0.1% Tween 20; 3) 20 mM Na2PdCl4, 70 mM THP, 100 mM N,N'-diethylethanolamine, 10 mM sodium ascorbate, 1 M NaCl, 0.1% Tween 20. The cleavage efficiency was determined by measuring the relative cleavage rate of 3'-AOM-nucleotide substrates. Briefly, a stock solution of the palladium reagent was added to a 100 mM buffer solution of 0.1 mM 3'-AOM-nucleotide substrate to give a final concentration of 1 mM Pd species. The solution was incubated at RT and the reaction kinetics were monitored by removing aliquots from the reaction at set time points, quenching them with a 1:1 EDTA / H2O2 (0.1:0.1 M) solution, and analyzing these aliquots by HPLC for the formation of 3'-OH nucleotides and the disappearance of 3'-AOM nucleotide substrate. Figure 1 As shown, the cleavage efficiency of Na2PdCl4 when only 3 or 3.5 equivalents of THP were used was comparable to that of [(allyl)PdCl]2, compared to [(allyl)PdCl]2 when 10 equivalents of THP were used.

[0342] Example 3: AOM-AOL Study on the stability of ffN in solution and its performance in sequencing

[0343] Figure 2 The fully functionalized nucleotides (ffN) with 3'-AOM capping group and AOL linker moiety (including labeled ffT-DB, labeled ffA and labeled ffC, and unlabeled ffG) are shown with the stressed standard The two sets of ffN were incubated at 45 °C for several days in a standard spike-in mix formulation excluding only DNA polymerase. For each time point, fresh polymerase was added to obtain a complete spike-in mix and then directly loaded on The sequencing conditions described previously were used. The pre-phase % is a direct indication of the percentage of 3'OH-ffN present in the mixture and is therefore directly related to the stability of the 3' capping group. The pre-phase values ​​of the two groups of ffN were recorded and plotted ( Figure 2 ). AOM-AOL-ffN did not show any increase in pre-phase compared to the standards and appeared to be much more stable than the standard ffN with 3'-O-azidomethyl end capping group and LN3 linker moiety.

[0344] Example 4. Use of palladium scavengers in sequencing reactions

[0345] Figure 3 The results show that the Illumina PCR products can be used with and without potassium isocyanoacetate in the post-cleavage wash step when using fully functionalized nucleotides (ffN) with a 3′-AOM capping group and a standard LN3 linker moiety (including labeled ffT-DB, labeled ffA, and labeled ffC, as well as unlabeled ffG). Comparison of phasing values ​​on the instruments. Sequencing experiments on Illumina The PCR reaction was performed using a cartridge in which the standard spiking mix reagent was replaced with a freshly prepared spiking mix containing ffN with a 3'-AOM capping group and a standard LN3 linker moiety, and in which a freshly prepared palladium cleavage reagent solution (10 mM [(allyl)PdCl]2, 100 mM THP, 100 mM ethanolamine buffer, 10 mM sodium ascorbate, 1 M NaCl, 0.1% Tween 20) was added to the empty wells. Potassium isocyanoacetate was added to the standard The final concentration of 10 mM was reached in the post-lysis wash solution. The sequencing experiment was performed using a 2 × 151 cycle process that included a 5 second incubation with the palladium lysis reagent solution in addition to the standard sequencing by synthesis (SBS) protocol. Figure 3 As shown, it was observed that the % phasing decreased from 0.183 to 0.075 when 10 mM potassium isocyanoacetate was used in the post-lysis wash solution.

[0346] Figure 4 The results show that when using fully functionalized nucleotides (ffN) with 3'-AOM capping groups and AOL linker moieties (including labeled ffT-DB, labeled ffA, and labeled ffC, as well as unlabeled ffG), Illumina's Key sequencing metrics on the instrument (including phasing, prephasing, and error rate) compared to the same sequencing metrics when using a standard ffN with a 3'-O-azidomethyl end capping group and an LN3 adapter. The above was performed by running a 2×151 cycle protocol using a standard cartridge, in which the incorporation mixture reagent and the standard cleavage reagent were replaced with a freshly prepared incorporation mixture containing fully functionalized nucleotides (ffN) with a 3'-AOM blocking group and an AOL linker moiety, and a freshly prepared palladium cleavage reagent solution (10 mM [(allyl)PdCl]2, 100 mM THP, 100 mM N,N'-diethylethanolamine buffer, 10 mM sodium ascorbate, 1 M NaCl, 0.1% Tween 20), respectively. Potassium isocyanoacetate (ICNA) was added to the standard After lysis, wash solution was added to achieve a final concentration of 10 mM. Kit and workflow, a control sequencing experiment was performed with standard ffN with 3'-O-azidomethyl end-capping group. The results showed improvements in prephasing and more importantly error rate, demonstrating the full efficiency of this AOM-AOL SBS chemistry combined with a single cleavage step.

[0347] Example 5. Use of glycine in sequencing reactions

[0348] Figure 5 The results show that when using fully functionalized nucleotides (ffN) with 3'-AOM capping groups and AOL linker moieties, Illumina's Comparison of the main sequencing indicators (including phasing and pre-phasing) on ​​the instrument. In this embodiment, the entire set of ffN includes 3'-AOM-ffA-AOL (labeled), 3'-AOM-ffG-AOL (unlabeled), 3'-AOM-ffT (DB) -AOL (labeled) and 3'-AOM-ffC (DB) -AOL (labeled). Figure 5 As shown, a significant decrease in phasing values ​​was observed when glycine was used in the spiking buffer compared to the standard spiking buffer containing ethanolamine. Although there was a slight increase in prephasing values ​​when glycine was used, this was not considered a meaningful increase. The instrument was run using a cartridge, where the standard spike-in mixture reagent and the standard cleavage reagent were replaced with a freshly prepared spike-in mixture containing fully functionalized nucleotides (ffN) with 3'-AOM blocking groups and AOL linker moieties in 50 mM ethanolamine or 50 mM glycine buffer, and a freshly prepared palladium cleavage reagent solution (10 mM [(allyl) PdCl]2, 100 mM THP, 100 mM N,N'-diethylethanolamine buffer, 10 mM sodium ascorbate, 1 M NaCl, 0.1% Tween 20), respectively. Potassium isocyanoacetate (ICNA) was added to the standard MiniSeq post-cleavage wash solution to a final concentration of 10 mM. A standard MiniSeq protocol with 2×151 cycles was used.

[0349] Figure 6 The Illumina The main sequencing indicators on the instrument (including phasing, pre-phasing and error rate), and the same sequencing indicators when using a standard ffN with a 3'-O-azidomethyl end capping group and an LN3 linker portion for comparison. In this embodiment, the entire set of ffNs includes 3'-AOM-ffA-AOL (labeled), 3'-AOM-ffG-AOL (unlabeled), 3'-AOM-ffT (DB) -AOL (labeled) and 3'-AOM-ffC (DB) -AOL (labeled). The sequencing experiment was performed on a standard ffN loaded with a cartridge. The instrument was run using a 2×151 cycle protocol, in which the standard spike-in mixture reagent and the standard cleavage reagent were replaced with a freshly prepared spike-in mixture containing fully functionalized nucleotides (ffN) with 3′-AOM blocking groups in 50 mM glycine buffer and a freshly prepared palladium cleavage reagent solution (10 mM [(allyl)PdCl]2, 100 mM THP, 100 mM N,N′-diethylethanolamine buffer, 10 mM sodium ascorbate, 1 M NaCl, 0.1% Tween 20), respectively. Potassium isocyanoacetate was added to the standard The results showed that the error rate was further improved compared to the error rate generated by using standard ffN. It is believed that the phasing of the AOM-AOL series is related to Figure 5 The improvement compared to the results of the previous study was due to the use of glycine buffer and 3'-AOM-ffC(DB)-AOL.

[0350] Example 6. In iSeq TM Sequencing by Synthesis

[0351] Fig. 7A and Figure 7BThe results show that fully functionalized nucleotides (ffN) with 3'-AOM capping groups and AOL linker moieties can be used to efficiently perform PCR on Illumina iSeq TM Comparison of the main sequencing metrics (including error rate and Q30 score) for 2×300 cycles of sequencing by synthesis performed on the instrument. In this embodiment, the entire set of AOM ffNs includes 3'-AOM-ffA(DB)-AO-dye 1, 3'-AOM-ffG (unlabeled), 3'-AOM-ffT(DB)-AOL-NR550S0 and 3'-AOM-ffC(DB)-AOL-dye 2. The entire set of azidomethyl (AZM) ffNs includes the same ffN with a 3'-azidomethyl end capping group, a propargyl amido group and a LN3 linker. Dye 1 is a chromene quinoline dye disclosed in U.S. Serial No. 63 / 127061, which has a structural part when conjugated with ffA Dye 2 is a coumarin dye disclosed in U.S. Patent Publication No. 2018 / 0094140, which when conjugated with ffC has a structural moiety NR550S0 is a known green dye.

[0352] Sequencing experiments are performed in standard iSeq TM The instrument was run using a cartridge, where the standard spike-in mixture reagent and the standard cleavage reagent were replaced with a freshly prepared spike-in mixture containing fully functionalized nucleotides (ffN) with 3'-AOM capping groups and AOL linker moieties in 50 mM ethanolamine or 50 mM glycine buffer using 300% concentration of polymerase 1901 (Pol 1901) (360 μg / mL), and a freshly prepared palladium cleavage reagent solution (10 mM [(allyl)PdCl]2, 100 mM THP, 100 mM N,N'-diethylethanolamine buffer, 10 mM sodium ascorbate, 1 M NaCl, 0.1% Tween 20). L-cysteine ​​was added to the standard iSeq TM The final concentration of 10 mM was achieved in the post-lysis wash solution. iSeq was run for 2 × 301 cycles in a 2 excitation / 1 emission protocol. TM In particular, iSeq TM The instrument was set to take the first image with green excitation light (about 520 nm) and the second image with blue excitation light (about 450 nm). 2×301 SBS cycles (incorporation, then imaging, then cleavage) were performed using the standard sequencing process. The sequencing indicators are summarized in the table below.

[0353]

[0354] It was observed that the AOM ffN set provided good performance, providing superior error rates and Q30 for read 1 and read 2. The phasing values ​​when using the AOM ffN set were comparable to those produced by the AZM ffN set. However, the AOM ffN set produced significantly lower prephasing values.

[0355] Fig. 8A and Figure 8B The results show that fully functionalized nucleotides (ffN) with 3'-AOM capping groups and AOL linker moieties can be used to efficiently perform PCR on Illumina iSeq TM Comparison of key sequencing metrics (including error rate and Q30 score) for 2×150 cycles of sequencing by synthesis performed on the instrument. In this example, the full set of AOM ffNs includes 3'-AOM-ffA(DB)-AO-Dye 1, 3'-AOM-ffG (unlabeled), 3'-AOM-ffT(DB)-AOL-NR550S0, and 3'-AOM-ffC(DB)-AOL-Dye 2. The full set of AZM ffNs includes the same ffNs with a 3'-azidomethyl end capping group, propargyl amido, and LN3 linker. Sequencing experiments were performed on standard iSeq TM The instrument was run using a cartridge, where the standard spike-in mixture reagent and the standard cleavage reagent were replaced with a freshly prepared spike-in mixture containing fully functionalized nucleotides (ffN) with 3'-AOM capping groups and AOL linker moieties in 50 mM ethanolamine or 50 mM glycine buffer using 300% concentration of Pol 1901 (360 μg / mL), and a freshly prepared palladium cleavage reagent solution (10 mM [(allyl)PdCl]2, 100 mM THP, 100 mM N,N'-diethylethanolamine buffer, 10 mM sodium ascorbate, 1 M NaCl, 0.1% Tween 20), respectively. L-cysteine ​​was added to the standard iSeq TM The final concentration of 10 mM was achieved in the post-lysis wash solution. iSeq was run for 2 × 301 cycles in a 2 excitation / 1 emission protocol. TM In particular, iSeq TM The instrument was set to take the first image with green excitation light (about 520 nm) and the second image with blue excitation light (about 450 nm).2 x 301 SBS cycles (incorporation followed by imaging and then cleavage) were performed using a standard sequencing protocol.

[0356] The incorporation mixture contact time of AZM ffN is about 24.1 seconds, while the incorporation mixture contact time of AOM ffN is about 29.1 seconds. However, the longer incorporation time of AOM ffN is offset by a shorter unblocking time. The cleavage mixture contact time is about 5.8 seconds, while the cleavage mixture contact time of the AZM ffN group is about 10.2 seconds. Therefore, the total incubation time of the AZM ffN group and the AOM ffN group is about 34.4 seconds and 34.9 seconds, respectively. The sequencing indexes are summarized in the table below.

[0357]

[0358] Likewise, it was observed that the AOM ffN group provided good performance, providing superior error rate and Q30 for both read 1 and read 2. In addition, the AOM ffN group produced lower prephasing values.

[0359] Example 7. NovaSeq with blue laser power titration TM Sequencing by Synthesis

[0360] It has been observed that prolonged exposure to blue light in SBS sequencing results in high levels of signal delay and phasing due to increased light dose and power density.This example compares the performance of AOM ffN and AZM ffN in a blue laser titration sequencing experiment.

[0361] In this experiment, the AOM ffN group will be used on the improved blue / green excitation NovaSeq TM The 1×151 SBS runs on were compared with the standard ffN set with 3'-azidomethyl end groups and LN3 joints. The flow cell used was a 490nm spacing BEER2 flow cell. The power configurations for the blue laser power were: 600mW, 800mW, 1000mW, 1400mW, 1800mW, and 2400mW. The green laser power was constant at 1000mM. The following standard AZM ffNs were used: green ffT (LN3-AF550POPOS0), dark G, red ffC (LN3-SO7181), blue ffC (sPA-blue dye A), blue ffA (LN3-BL-blue dye A), green ffA (LN3-BL-NR550S0). For the AOM ffN set, the following ffNs were used: green ffT (ffT (DB) -AOL-AF550POPOS0), dark G, red ffC (ffC (DB) -AOL-SO7181), blue ffC (ffC (DB) -AOL-blue dye A), blue ffA (ffA (DB) -AOL-BL-blue dye A), green ffA (ffA (DB) -AOL-BL-NR550S0). The structures of the blue dyes labeled AOM ffC and ffA are shown below:

[0362]

[0363] For running SBS with AOM ffN group, carried out the following modification.First, 10mM L-cysteine ​​was added in the washing solution after the cracking. The cracking mixture comprises the following components: DEEA buffer and Na therein PdCl . Two 10 seconds waiting steps were added in the washing step after the cracking. In addition, 60 seconds of static mixing waiting time (contrasting with 38 seconds of AZM ffN) were used. Fig.9A The two ffN groups were shown to have similar phasing values ​​at lower blue laser powers, but the AOM ffN group was less sensitive to titration increases in blue laser power (indicated by a flatter phasing slope compared to the AZM phasing slope). In addition, much lower prephasing values ​​were observed for the AOM ffN. Fig. 9B As shown, the AOM ffN group has significantly smaller signal attenuation at higher blue laser power. Fig. 9C and Fig.9D Shows the average error rate as a function of the number of cycles. Fig.9D yes Fig. 9C The results show that while the AZM ffNs had lower error rates in the early cycles at lower blue laser power, the AOM ffN group performed much better in the later cycles at higher blue laser power. Fig.9E The average error rates for 151 cycles were summarized. Similarly, the AOM ffN group outperformed the AZM ffN group at high laser powers (e.g., at 1400 mW, 1800 mW, and 2400 mW).

[0364] Example 8. First chemical linearization using Pd cleavage mixture

[0365] In this example, the Pd cleavage mixture used in SBS was tested in the first chemical linearization step after the clustering step. The experiment compared chemical linearization with standard enzymatic linearization, in which USER caused cleavage of one of the double-stranded polynucleotides at the U position on the P5 primer. Fully functionalized nucleotides (ffN) with 3'-AOM capping groups and AOL linker moieties were used in Illumina's iSeq TM 1×150 SBS cycles were performed on the instrument. In this example, the full set of AOM ffN included 3′-AOM-ffA(DB)-AO-Dye 1, 3′-AOM-ffG (unlabeled), 3′-AOM-ffT(DB)-AOL-NR550S0, and 3′-AOM-ffC(DB)-AOL-Dye 2, as described in Example 6. The iSeq TMThe instrument was set to capture the first image with green excitation light (about 520 nm) and the second image with blue excitation light (about 450 nm) (using a 2-excitation / 1-emission scheme). TM The flow cell used on the instrument allows the first chemical linearization of the P5 primer. The chemical linearization step was performed in a Pd cleavage mixture (a solution of 10 mM [Pd(allyl)Cl]2 and 100 mM THP in a buffer containing DEEA) incubated at 63°C for 30 s. Fig.10 SBS sequencing metrics using two different linearization methods are shown. It was observed that when the Pd cleavage mixture was used in the first chemical linearization step, all major sequencing metrics fell within the standard observation range. This experiment confirms that a single reagent mixture can be used for two separate sequencing steps - the linearization step and the SBS cleavage step, which enables further simplification of the instrument (flow channel and cartridge).

Claims

1. A nucleoside or nucleotide comprising a nucleobase attached to a detectable label via a cleavable linker, wherein the nucleoside or nucleotide has the structure of formula (I): wherein B is a purine, a deazapurine or a pyrimidine; R 4 is H or OH; R 5 for And where R a , R b , R c , R d and R e Each is independently H, or unsubstituted C1-C6 alkyl; -OR 6 is monophosphate, diphosphate, or triphosphate; L is X and Y are each independently O or S; R 1a , R 1b , R 2 , R 3a and R 3b are each independently H, or unsubstituted C1-C6 alkyl; and Where L 1 for Where L 2 exists, and L 2 for wherein m and n are each independently an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; The label is a fluorescent dye.

2. The nucleoside or nucleotide according to claim 1, wherein X and Y are each O.

3. The nucleoside or nucleotide according to claim 1, wherein R 1a , R 1b , R 2 , R 3a and R 3b Each is H.

4. The nucleoside or nucleotide according to claim 1, wherein R 1a , R 1b , R 2 , R 3a and R 3b At least one of them is unsubstituted C1-C6 alkyl.

5. The nucleoside or nucleotide according to claim 1, wherein R 5 for 6. The nucleoside or nucleotide according to claim 1, comprising a structure of Formula (Ia), (Ia′), (Ib), (Ic), (Ic′) or (Id): Where R 4 , R 6 , L 2 and markings as defined in claim 1.

7. The nucleoside or nucleotide according to claim 1, wherein n is 5.

8. The nucleoside or nucleotide according to claim 1, wherein m is 4.

9. The nucleoside or nucleotide according to claim 1, wherein R 4 is H and -OR 6 It is triphosphate.

10. The nucleoside or nucleotide according to claim 7, wherein R 4 is H and -OR 6 It is triphosphate. An oligonucleotide or polynucleotide comprising the nucleotide according to claim 9.

12. The oligonucleotide or polynucleotide of claim 11, wherein the oligonucleotide or polynucleotide hybridizes to a template polynucleotide.

13. The oligonucleotide or polynucleotide according to claim 12, wherein the template polynucleotide is immobilized on a solid support.

14. The oligonucleotide or polynucleotide of claim 13, wherein the solid support comprises an array of a plurality of immobilized template polynucleotides.

15. An oligonucleotide or polynucleotide comprising the nucleotide according to claim 10.

16. A method for preparing a growing polynucleotide complementary to a target single-stranded polynucleotide in a sequencing reaction, comprising incorporating the nucleotide of claim 9 into the growing complementary polynucleotide, wherein incorporation of the nucleotide prevents any subsequent nucleotide from being introduced into the growing complementary polynucleotide.

17. A method for determining the sequence of a target single-stranded polynucleotide, comprising: (a) incorporating the nucleotide of claim 9 into a copy polynucleotide chain complementary to at least a portion of the target polynucleotide chain; (b) detecting the identity of the nucleotide incorporated into the copied polynucleotide chain; and (c) chemically removing the detectable label and the 3'-OH blocking group from the nucleotide incorporated into the copy polynucleotide chain.

18. A kit comprising one or more nucleotides of claim 9.

19. The kit of claim 18, further comprising an enzyme, at least one Pd(0) scavenger, and one or more buffers.

20. The kit according to claim 19, wherein the enzyme is DNA polymerase, terminal deoxynucleotidyl transferase or reverse transcriptase.

21. The kit of claim 19, wherein the at least one Pd(0) scavenger is:

22. The kit of claim 18, further comprising a palladium catalyst.

23. The kit of claim 22, wherein the palladium catalyst is a Pd(0) catalyst generated in situ from a Pd(II) complex and one or more water-soluble phosphines.

24. The kit of claim 23, wherein the Pd(II) complex is [Pd(allyl)Cl]2 or Na2PdCl4.

25. The kit of claim 23, further comprising one or more Pd(II) scavengers.

26. The kit of claim 25, wherein the one or more Pd(II) scavengers are selected from the group consisting of isocyanoacetate (ICNA) salts, ethyl isocyanoacetate, methyl isocyanoacetate, cysteine ​​or its salts, L-cysteine ​​or its salts, N-acetyl-L-cysteine, potassium ethyl xanthate, potassium isopropyl xanthate, glutathione, lipoic acid, ethylenediaminetetraacetic acid (EDTA), iminodiacetic acid, nitrilodiacetic acid, trimercapto-S-triazine, dimethyldithiocarbamate, dithiothreitol, mercaptoethanol, allyl alcohol, propargyl alcohol, thiol, tertiary amine, tertiary phosphine, or a combination thereof.

27. A kit comprising one or more nucleotides of claim 10.

28. The kit of claim 27, further comprising a DNA polymerase, at least one Pd(0) scavenger, and one or more buffers.

Images