Modified dye and use thereof

Abstract

A modified dye and use thereof. Specifically, provided is a class of compounds with novel structures or stereoisomers or salts thereof. The compound is structurally designed on the basis of a disulfide bond, and can be used as a cleavable linker to be linked to a fluorescent dye. The fluorescent dye linked to the aforementioned compound or isomer or salt thereof is cut off by means of the cleavable linker after photographing is completed, and then sequencing in the next cycle is performed. Compared with existing linkers, when the fluorescent dye linked to the aforementioned compound or isomer or salt thereof is used for sequencing (such as COOLMPS sequencing), the background signal can be significantly reduced.

Description

Modified dyes and applications thereof TECHNICAL FIELD

[0001] The present disclosure relates to the field of nucleic acid sequencing, and in particular, the present disclosure relates to modified dyes and applications thereof. BACKGROUND

[0002] The CoolMPS antibody sequencing technology is a nucleic acid sequencing technology developed based on the DNBSEQ sequencing platform, which utilizes fluorescently labeled antibodies to specifically recognize four bases and the blocking group at the 3' hydroxyl end of the Cold dNTPs (referred to as Cold dNTPs) blocked by a removable group. After specific binding with the Cold dNTPs, the light signal is collected by a high-resolution imaging system, and after digital processing, the nucleic acid sequence of the sequence to be sequenced is obtained. In the preparation process of the fluorescently labeled antibody, the fluorescent group needs to be coupled with the antibody through a linker structure.

[0003] Sequencing by synthesis is the soul of second-generation sequencing. In the sequencing process, the recognition of bases comes from the recognition of modified fluorescent dyes on the bases. Based on the COOLMPS sequencing technology and principle of Huada Gene, the existing linker will cause the continuous increase of the two-chain background signal. High background signal will seriously reduce the signal-to-noise ratio and cause difficulties in accurate base recognition, thereby limiting the read length of sequencing.

[0004] Therefore, it is of great significance to develop and screen a removable linker-modified dye suitable for nucleic acid sequencing scenarios.

[0005] SUMMARY

[0006] In order to solve the problem of the background signal, the present disclosure provides a kind of compound with novel structure or its stereoisomer or its salt, which is based on disulfide bond structure design, and can be connected with fluorescent dye as removable linker. The fluorescent dye connected with the aforementioned compound or its isomer or its salt is disconnected by the removable linker after the end of the photograph, and then the next cycle of sequencing is carried out. Compared with the existing linker, the fluorescent dye connected with the aforementioned compound or its isomer or its salt can significantly reduce the background signal when used for sequencing (such as COOLMPS sequencing).

[0007] Therefore, in the first aspect of the present disclosure, the present disclosure provides a compound represented by formula I or its stereoisomer or its salt,

[0008] H-[NH-CH2-C(=O)]n0-NH-(CH2-CH2-O) n1 -(CH2) n2 -(S-S) n3 -L 1-(CH2) n4 -(S-S) n5 -(CH2) n6 -L 2 -C(=O)OH

[0009] Formula I

[0010] wherein:

[0011] n0 is selected from 0, 1, 2, 3, 4, 5, 6;

[0012] Preferably, n0 is selected from 0, 1 ;

[0013] n1 is selected from 0, 1, 2, 3, 4, 5, 6;

[0014] Preferably, n1 is selected from 0, 1, 2, 3, 4;

[0015] More preferably, n1 is selected from 0, 1, 2;

[0016] Most preferably, n1 is 0;

[0017] n2 is selected from 0, 1, 2, 3, 4, 5, 6;

[0018] Preferably, n2 is selected from 0, 1, 2, 3, 4;

[0019] More preferably, n2 is selected from 0, 1, 2;

[0020] Most preferably, n2 is selected from 0, 2;

[0021] n3 is selected from 0, 1 ;

[0022] n4 is selected from 0, 1, 2, 3, 4, 5, 6;

[0023] Preferably, n4 is selected from 0, 1, 2, 3, 4;

[0024] More preferably, n4 is selected from 0, 1, 2;

[0025] Most preferably, n4 is selected from 0, 2;

[0026] n5 is selected from 0, 1 ;

[0027] n6 is selected from 1, 2, 3, 4, 5, 6;

[0028] Preferably, n6 is selected from 1, 2, 3, 4;

[0029] More preferably, n6 is selected from 1, 2;

[0030] L 1 is selected from a direct bond,

[0031] m is selected from 1, 2, 3, 4, 5;

[0032] Preferably, m is selected from 1, 2, 3;

[0033] More preferably, m is 2;

[0034] Preferably, L is selected from a direct bond, 1 L is selected from a direct bond, 2

[0035] R is selected from hydrogen, C1-C6 alkyl; 1 2 3 4 each independently selected from hydrogen, C1-C6 alkyl;

[0036] Preferably, R is selected from hydrogen, C1-C4 alkyl; 1 2 3 4 each independently selected from hydrogen, C1-C4 alkyl;

[0037] More preferably, R is hydrogen; 1 2 3 4 each is hydrogen;

[0038] Preferably, L is selected from a direct bond, 2 and the following condition is fulfilled: n3 and n5 are not simultaneously 0.

[0039] In some embodiments, the compound has the structural formula of Formula I-1,

[0040] NH2-(CH2) n2 -(S-S) n3 -L 1 -(CH2) n4 -(S-S) n5 -(CH2) n6 -L 2 -C(=O)OH

[0041] Formula I-1

[0042] wherein:

[0043] n2 is selected from 0, 1, 2, 3, 4;

[0044] Preferably, n2 is selected from 0, 1, 2;

[0045] More preferably, n2 is selected from 0, 2;

[0046] n3 is selected from 0, 1 ;​​​​​​​​​​​​

[0047] n4 is selected from 0, 1, 2, 3, 4;

[0048] Preferably, n4 is selected from 0, 1, 2;

[0049] More preferably, n4 is selected from 0, 2;

[0050] n5 is selected from 0, 1 ;

[0051] n6 is selected from 1, 2, 3, 4;

[0052] Preferably, n6 is selected from 1, 2, 3;

[0053] More preferably, n6 is 2;

[0054] L 1 is selected from a direct bond,

[0055] m is selected from 1, 2, 3;

[0056] Preferably, m is 2;

[0057] Preferably, L 1 is selected from a direct bond,

[0058] L 2 is selected from a direct bond,

[0059] R 1 , R 2 , R 3 , R 4 are each independently selected from hydrogen, Ci-C6alkyl;

[0060] Preferably, R 1 , R 2 , R 3 , R 4 are each independently selected from hydrogen, Ci-C4alkyl;

[0061] More preferably, R 1 , R 2 , R 3 , R 4 are each hydrogen;

[0062] Preferably, L 2 is selected from a direct bond,

[0063] and the following condition is fulfilled: n3 and n5 are not simultaneously 0.

[0064] In some embodiments, the compound has the structural formula of Formula I-1-1,

[0065] NH2-L 1 -(CH2) n4 -(S-S) n5 -(CH2) n6 -L 2 -C(=O)OH

[0066] Formula I-1-1

[0067] wherein:

[0068] n4 is selected from 0, 1, 2, 3, 4;

[0069] Preferably, n4 is selected from 0, 1, 2;

[0070] More preferably, n4 is selected from 0, 2;

[0071] n5 is selected from 0, 1 ;

[0072] n6 is selected from 1, 2, 3, 4;

[0073] Preferably, n6 is selected from 1, 2, 3;

[0074] More preferably, n6 is 2;

[0075] L 1 is selected from a direct bond,

[0076] m is selected from 1, 2, 3;

[0077] Preferably, m is 2;

[0078] Preferably, L 1 is selected from a direct bond,

[0079] L 2 is selected from a direct bond,

[0080] R 1 , R 2 , R 3 , R 4 are each independently selected from hydrogen, Ci-C6-alkyl;

[0081] Preferably, R 1 , R 2 , R 3 , R 4 are each independently selected from hydrogen, Ci-C4-alkyl;

[0082] More preferably, R 1 , R 2 , R 3 , R 4 are all hydrogen;

[0083] Preferably, L 2 selected from a direct bond,

[0084] In some embodiments, the compound is selected from:

[0085] In a second aspect of the present disclosure, the present disclosure provides a modified dye comprising:

[0086] a fluorescent group; and a fluorescent modifying group attached to the fluorescent group, the fluorescent modifying group being a compound of any of the first aspect or a stereoisomer thereof or a salt thereof;

[0087] Optionally, the modified dye further comprises a protecting group attached to the fluorescent modifying group.

[0088] In some embodiments, the modified dye is a compound of Formula II or a stereoisomer thereof or a salt thereof,

[0089] Dye-L-P

[0090] Formula II

[0091] wherein:

[0092] Dye is selected from

[0093] Preferably, Dye is

[0094] L is -[NH-CH2-C(=O)]n0-NH-(CH2-CH2-O) n1 -(CH2) n2 -(S-S) n3 -L 1 -(CH2) n4 -(S-S) n5 -(CH2) n6 -L 2 -C(=O)-;

[0095] n0 is selected from 0, 1, 2, 3, 4, 5, 6;

[0096] Preferably, n0 is selected from 0, 1;

[0097] n1 is selected from 0, 1, 2, 3, 4, 5, 6;

[0098] Preferably, n1 is selected from 0, 1, 2, 3, 4;

[0099] More preferably, n1 is selected from 0, 1, 2;

[0100] Most preferably, n1 is 0;

[0101] n2 is selected from 0, 1, 2, 3, 4, 5, 6;

[0102] Preferably, n2 is selected from 0, 1, 2, 3, 4;

[0103] More preferably, n2 is selected from 0, 1, 2;

[0104] Most preferably, n2 is selected from 0, 2;

[0105] n3 is selected from 0, 1 ;

[0106] n4 is selected from 0, 1, 2, 3, 4, 5, 6;

[0107] Preferably, n4 is selected from 0, 1, 2, 3, 4;

[0108] More preferably, n4 is selected from 0, 1, 2;

[0109] Most preferably, n4 is selected from 0, 2;

[0110] n5 is selected from 0, 1 ;

[0111] n6 is selected from 1, 2, 3, 4, 5, 6;

[0112] Preferably, n6 is selected from 1, 2, 3, 4;

[0113] More preferably, n6 is selected from 1, 2;

[0114] L 1 is selected from a direct bond,

[0115] m is selected from 1, 2, 3, 4, 5;

[0116] Preferably, m is selected from 1, 2, 3;

[0117] More preferably, m is 2;

[0118] Preferably, L 1 is selected from a direct bond,

[0119] L 2 is selected from a direct bond,

[0120] R 1 , R 2 , R 3 , R 4 are each independently selected from hydrogen, C1-C6 alkyl;

[0121] Preferably, R 1 R 2 R 3 R 4 Each is independently selected from hydrogen and C1-C4 alkyl groups;

[0122] More preferably, R 1 R 2 R 3 R 4 All are hydrogen;

[0123] Preferably, L 2 Selected from direct keys,

[0124] And it satisfies the following condition: n3 and n5 are not both 0 at the same time;

[0125] P is selected from hydroxyl group,

[0126] Preferably, P is selected from hydroxyl groups,

[0127] More preferably, P is

[0128] In some embodiments, in the compound represented by Formula II, L is -NH-(CH2). n2 -(SS) n3 -L 1 -(CH2) n4 -(S- S) n5 -(CH2) n6 -L 2 -C(=O)-,

[0129] n2 is selected from 0, 1, 2, 3, and 4;

[0130] Preferably, n2 is selected from 0, 1, and 2;

[0131] More preferably, n2 is selected from 0 and 2;

[0132] n3 is selected from 0 and 1;

[0133] n4 is selected from 0, 1, 2, 3, and 4;

[0134] Preferably, n4 is selected from 0, 1, and 2;

[0135] More preferably, n4 is selected from 0 or 2;

[0136] n5 is selected from 0 and 1;

[0137] n6 is selected from 1, 2, 3, and 4;

[0138] Preferably, n6 is selected from 1, 2, and 3;

[0139] More preferably, n6 is 2;

[0140] L 1 is selected from a direct bond,

[0141] m is selected from 1, 2, 3;

[0142] Preferably, m is 2;

[0143] Preferably, L 1 is selected from a direct bond,

[0144] L 2 is selected from a direct bond,

[0145] R 1 , R 2 , R 3 , R 4 are each independently selected from hydrogen, Ci-C6alkyl;

[0146] Preferably, R 1 , R 2 , R 3 , R 4 are each independently selected from hydrogen, Ci-C4alkyl;

[0147] More preferably, R 1 , R 2 , R 3 , R 4 are each hydrogen;

[0148] Preferably, L 2 is selected from a direct bond,

[0149] and the following condition is met: n3 and n5 are not simultaneously 0.

[0150] In some embodiments, in the compound of formula II, L is -NH-L 1 -(CH2) n4 -(S-S) n5 -(CH2) n6 -L 2 -C(=O)-,

[0151] wherein:

[0152] n4 is selected from 0, 1, 2, 3, 4;

[0153] Preferably, n4 is selected from 0, 1, 2;

[0154] More preferably, n4 is selected from 0, 2;

[0155] n5 is selected from 0, 1 ;

[0156] n6 is selected from 1, 2, 3, 4;

[0157] Preferably, n6 is selected from 1, 2, 3;

[0158] More preferably, n6 is 2;

[0159] L 1 is selected from a direct bond,

[0160] m is selected from 1, 2, 3;

[0161] Preferably, m is 2;

[0162] Preferably, L 1 is selected from a direct bond,

[0163] L 2 is selected from a direct bond,

[0164] R 1 , R 2 , R 3 , R 4 are each independently selected from hydrogen, Ci-C6alkyl;

[0165] Preferably, R 1 , R 2 , R 3 , R 4 are each independently selected from hydrogen, Ci-C4alkyl;

[0166] More preferably, R 1 , R 2 , R 3 , R 4 are all hydrogen;

[0167] Preferably, L 2 is selected from a direct bond,

[0168] In some embodiments, the compound of Formula II, L is selected from:

[0169] In some embodiments, the compound of Formula II is selected from:

[0170] In a third aspect of the disclosure, the disclosure provides an antibody conjugated dye comprising:

[0171] an antibody; and

[0172] a dye, said dye being linked to said antibody, said dye being a modified dye according to any of the technical solutions of the second aspect.

[0173] In a fourth aspect of the present disclosure, the present disclosure provides a modified nucleotide, comprising:

[0174] a nucleotide, and

[0175] a modified dye, said modified dye being bound to said nucleotide via an affinity reagent, said modified dye being linked to said affinity reagent, said modified dye being as defined in any of the technical solutions of the second aspect;

[0176] Preferably, said affinity reagent is capable of specifically recognizing and binding to an epitope of said nucleotide molecule;

[0177] More preferably, said affinity reagent is selected from the group consisting of an antibody, an aptamer, an Affimer, a Knottin;

[0178] Most preferably, said affinity reagent is an antibody.

[0179] In some embodiments, said nucleotide comprises:

[0180] a ribose or deoxyribose;

[0181] a phosphate, said phosphate being linked to said ribose or deoxyribose;

[0182] a base, said base being linked to said ribose or deoxyribose; and

[0183] a reversible blocking group, said reversible blocking group being linked to said ribose or deoxyribose, or said reversible blocking group being linked to said base.

[0184] In a fifth aspect of the present disclosure, the present disclosure provides a method of controlling nucleic acid synthesis, comprising: incorporating a modified nucleotide according to any of the technical solutions of the fourth aspect into a nucleic acid molecule to be synthesized;

[0185] Preferably, said incorporation of said modified nucleotide is achieved by a polymerase;

[0186] Preferably, said method comprises: using a polymerase to incorporate said modified nucleotide into a nucleic acid molecule to be synthesized;

[0187] Preferably, said method comprises: using a polymerase to perform a nucleotide polymerization reaction under conditions allowing a polymerase to perform a nucleotide polymerization reaction, thereby incorporating said modified nucleotide into a 3' end of a nucleic acid molecule to be synthesized;

[0188] Preferably, the method further comprises: removing the reversible blocking group and the affinity reagent carried by the incorporated modified nucleotide, and the modified dye connected with the affinity reagent, and performing the next round of nucleotide polymerization reaction.

[0189] In the fifth aspect of the present disclosure, the present disclosure further provides a method for controlling nucleic acid synthesis, comprising: incorporating a nucleotide with a reversible blocking group and the antibody-coupled dye of any of the technical solutions in the third aspect into a nucleic acid molecule to be synthesized in sequence respectively;

[0190] Preferably, the incorporation of the nucleotide with a reversible blocking group is achieved by a polymerase.

[0191] Preferably, the method comprises: using a polymerase to incorporate the nucleotide with a reversible blocking group into the nucleic acid molecule to be synthesized; and then incorporating the antibody-coupled dye of any of the technical solutions in the third aspect into the nucleic acid molecule to be synthesized.

[0192] Preferably, the method comprises: using a polymerase to perform a nucleotide polymerization reaction under conditions allowing the polymerase to perform the nucleotide polymerization reaction, so as to incorporate the nucleotide with a reversible blocking group into the 3' end of the nucleic acid molecule to be synthesized; and then incorporating the antibody-coupled dye of any of the technical solutions in the third aspect into the nucleic acid molecule to be synthesized.

[0193] Preferably, the method further comprises: removing the reversible blocking group on the incorporated nucleotide with a reversible blocking group and the incorporated antibody-coupled dye, and performing the next round of nucleotide polymerization reaction.

[0194] In the sixth aspect of the present disclosure, the present disclosure provides a method for preparing a growing polynucleotide complementary to a target polynucleotide in a sequencing reaction, comprising a nucleotide polymerization reaction, and incorporating the modified nucleotide of any of the technical solutions in the fourth aspect into the growing complementary polynucleotide, wherein the incorporation of the modified nucleotide ensures that only one nucleotide molecule is polymerized in one round of polymerization reaction.

[0195] Preferably, the incorporation of the modified nucleotide is achieved by a polymerase.

[0196] Preferably, the method comprises: using a polymerase to incorporate the modified nucleotide into the growing complementary polynucleotide.

[0197] Preferably, the method comprises: using a polymerase to perform a nucleotide polymerization reaction under conditions allowing the polymerase to perform the nucleotide polymerization reaction, so as to incorporate the modified nucleotide into the 3' end of the growing complementary polynucleotide.

[0198] In the sixth aspect of the present disclosure, the present disclosure further provides a method for preparing a growing polynucleotide complementary to a target polynucleotide in a sequencing reaction, which comprises a nucleotide polymerization reaction, and sequentially incorporating a nucleotide with a reversible blocking group and the antibody-conjugated dye of any of the technical solutions of the third aspect into the growing complementary polynucleotide, respectively, wherein the incorporation of the nucleotide with a reversible blocking group ensures that only one nucleotide molecule is polymerized in one round of polymerization reaction;

[0199] Preferably, the incorporation of the nucleotide with a reversible blocking group is achieved by a polymerase.

[0200] Preferably, the method comprises: using a polymerase to incorporate the nucleotide with a reversible blocking group into the growing complementary polynucleotide; and then, incorporating the antibody-conjugated dye of any of the technical solutions of the third aspect into the growing complementary polynucleotide.

[0201] Preferably, the method comprises: using a polymerase to perform a nucleotide polymerization reaction under conditions allowing the polymerase to perform the nucleotide polymerization reaction, so as to incorporate the nucleotide with a reversible blocking group into the 3' end of the growing complementary polynucleotide; and then, incorporating the antibody-conjugated dye of any of the technical solutions of the third aspect into the growing complementary polynucleotide.

[0202] In the seventh aspect of the present disclosure, the present disclosure provides a nucleic acid intermediate formed in determining the sequence of a target polynucleotide, wherein,

[0203] The nucleic acid intermediate is formed by the following steps:

[0204] incorporating one nucleotide complementary to the target polynucleotide into a growing nucleic acid chain to form the nucleic acid intermediate, wherein the incorporated one complementary nucleotide is the modified nucleotide of any of the technical solutions of the fourth aspect;

[0205] Alternatively, the nucleic acid intermediate is formed by the following steps:

[0206] incorporating one nucleotide complementary to the target polynucleotide into a growing nucleic acid chain to form the nucleic acid intermediate, wherein the incorporated one complementary nucleotide is the modified nucleotide of any of the technical solutions of the fourth aspect, and the growing nucleic acid chain has been previously incorporated with at least one nucleotide complementary to the target polynucleotide, and the previously incorporated at least one nucleotide complementary to the target polynucleotide is the modified nucleotide of any of the technical solutions of the fourth aspect, which has been removed of the reversible blocking group and the affinity reagent and the modified dye connected with the affinity reagent.

[0207] In the seventh aspect of the present disclosure, the present disclosure further provides a nucleic acid intermediate formed in determining the sequence of a target polynucleotide, wherein,

[0208] The nucleic acid intermediate is formed by the steps of:

[0209] incorporating into a growing nucleic acid chain, in sequence, a nucleotide complementary to the target polynucleotide and the antibody-conjugated dye of any of the third aspect, to form the nucleic acid intermediate, wherein the nucleotide carries a reversible blocking group;

[0210] Alternatively, the nucleic acid intermediate is formed by the steps of:

[0211] incorporating into a growing nucleic acid chain, in sequence, a nucleotide complementary to the target polynucleotide and the antibody-conjugated dye of any of the third aspect, to form the nucleic acid intermediate, wherein the incorporated complementary nucleotide carries a reversible blocking group, and the growing nucleic acid chain has previously incorporated at least one nucleotide complementary to the target polynucleotide, the previously incorporated at least one nucleotide complementary to the target polynucleotide being a nucleotide that has had the reversible blocking group and the antibody-conjugated dye removed.

[0212] In an eighth aspect of the disclosure, the disclosure provides a method of determining the sequence of a target polynucleotide, comprising:

[0213] 1) detecting incorporation of a nucleotide complementary to the target polynucleotide into a growing nucleic acid chain, wherein the incorporated at least one complementary nucleotide is the modified nucleotide of any of the fourth aspect, and,

[0214] 2) determining the identity of the incorporated nucleotide;

[0215] Preferably, the incorporation of the nucleotide complementary to the target polynucleotide is effected by a polymerase;

[0216] Preferably, the reversible blocking group carried by the incorporated complementary nucleotide and the affinity reagent and modified dye attached to the affinity reagent are removed prior to the introduction of the next complementary nucleotide;

[0217] Preferably, the reversible blocking group and the affinity reagent and modified dye attached to the affinity reagent are removed simultaneously.

[0218] In some embodiments, the method comprises the steps of:

[0219] (a) providing a plurality of different nucleotides, wherein at least one nucleotide is the modified nucleotide of any of the fourth aspect;

[0220] (b) incorporating the plurality of different nucleotides into a complementary sequence of the target polynucleotide,

[0221] (c) detecting the nucleotides obtained in step (b) to determine the type of nucleotide incorporated;

[0222] (d) removing the reversible blocking group and the affinity reagent carried by the nucleotides obtained in step (b) and the modified dye linked to the affinity reagent; and

[0223] (e) optionally repeating steps (a)-(d) one or more times;

[0224] to determine the sequence of the target polynucleotide;

[0225] Preferably, the incorporation of the plurality of different nucleotides is achieved by a polymerase.

[0226] In some embodiments, the method comprises the following steps:

[0227] (1) providing a first nucleotide, a second nucleotide, a third nucleotide and a fourth nucleotide, at least one of the four nucleotides is the modified nucleotide of any of the technical solutions of the fourth aspect;

[0228] (2) contacting the four nucleotides with a target polynucleotide; removing the nucleotides that are not incorporated into the growing nucleic acid strand; detecting the nucleotides incorporated into the growing nucleic acid strand; removing the reversible blocking group and the affinity reagent carried by the nucleotides incorporated into the growing nucleic acid strand and the modified dye linked to the affinity reagent;

[0229] Optionally, further comprising (3): repeating (1)-(2) one or more times;

[0230] Preferably, the contacting the four nucleotides with a target polynucleotide is performed in the presence of a polymerase.

[0231] In some embodiments, the method comprises the following steps:

[0232] (a) providing a mixture comprising a duplex, at least one modified nucleotide as described in any of the technical solutions of the fourth aspect, a polymerase and a cleavage reagent; the duplex comprises a growing nucleic acid strand and a nucleic acid strand to be sequenced;

[0233] (b) performing a reaction comprising the following steps (i), (ii) and (iii), optionally, repeating one or more times:

[0234] Step (i): using the polymerase, incorporating the modified nucleotide into the growing nucleic acid strand to form a nucleic acid intermediate comprising a reversible blocking group and an affinity reagent and a modified dye linked to the affinity reagent:

[0235] Step (ii): detecting the nucleic acid intermediate;

[0236] Step (iii): cleaving the reversible blocking group and the affinity reagent and the modified dye attached to the affinity reagent comprised in the nucleic acid intermediate using a cleaving reagent;

[0237] Preferably, the cleaving reagent used for cleaving the reversible blocking group and the affinity reagent and the modified dye attached to the affinity reagent is the same reagent.

[0238] In some embodiments, the duplex is immobilized on a support.

[0239] In some embodiments, the growing nucleic acid strand is a primer.

[0240] In some embodiments, the primer forms the duplex by annealing to the nucleic acid strand to be sequenced.

[0241] In some embodiments, the duplex, the modified nucleotide, and the polymerase together form a reaction system.

[0242] In some embodiments, the modified nucleotide is incorporated into the growing nucleic acid strand using a polymerase under conditions that allow the polymerase to perform nucleotide polymerization reaction, forming a nucleic acid intermediate comprising a reversible blocking group and an affinity reagent and a modified dye attached to the affinity reagent.

[0243] In some embodiments, the solution phase of the reaction system of the previous step is removed before any one of the steps of detecting the nucleic acid intermediate.

[0244] In some embodiments, the cleaving reagent is contacted with the duplex or the growing nucleic acid strand in a reaction system.

[0245] In some embodiments, the cleaving reagent is capable of cleaving the reversible blocking group and the affinity reagent and the modified dye attached to the affinity reagent carried by the modified nucleotide incorporated into the growing nucleic acid strand without affecting the phosphodiester bond on the backbone of the duplex.

[0246] In some embodiments, the solution phase of the reaction system of this step is removed after any one of the steps of cleaving the reversible blocking group and the affinity reagent and the modified dye attached to the affinity reagent comprised in the nucleic acid intermediate.

[0247] In some embodiments, after step (ii), further comprising: determining the type of the modified nucleotide incorporated into the growing nucleic acid strand in step (i) based on the signal detected in step (ii), and determining the type of the nucleotide at the corresponding position in the nucleic acid strand to be sequenced based on the principle of base complementary pairing.

[0248] In an eighth aspect of the present disclosure, the present disclosure also provides a method for determining the sequence of a target polynucleotide, comprising:

[0249] 1) detecting incorporation of nucleotides complementary to the target polynucleotide in the growing nucleic acid strand, wherein at least one of the incorporated complementary nucleotides is a nucleotide carrying a reversible blocking group, and the nucleotide carrying a reversible blocking group is bound to an antibody-conjugated dye as described in any of the technical solutions of the third aspect, and

[0250] 2) determining the type of the incorporated nucleotides;

[0251] Preferably, the incorporation of the nucleotides complementary to the target polynucleotide is achieved by a polymerase.

[0252] Preferably, the reversible blocking group carried by the incorporated complementary nucleotide and the bound antibody-conjugated dye are removed before the next complementary nucleotide is introduced.

[0253] Preferably, the reversible blocking group and the antibody-conjugated dye are removed simultaneously.

[0254] In some embodiments, the method comprises the following steps:

[0255] (a) providing a plurality of different nucleotides and antibody-conjugated dyes, wherein the nucleotides are nucleotides carrying a reversible blocking group, and the antibody-conjugated dyes are as described in any of the technical solutions of the third aspect;

[0256] (b) incorporating the plurality of different nucleotides and the antibody-conjugated dyes into the complementary sequence of the target polynucleotide, respectively and sequentially,

[0257] (c) detecting the nucleotides obtained in step (b), thereby determining the type of the incorporated nucleotides;

[0258] (d) removing the reversible blocking group carried by the nucleotides obtained in step (b) and the bound antibody-conjugated dye; and

[0259] (e) optionally repeating steps (a)-(d) one or more times;

[0260] thereby determining the sequence of the target polynucleotide.

[0261] Preferably, the incorporation of the plurality of different nucleotides is achieved by a polymerase.

[0262] In some embodiments, the method comprises the following steps:

[0263] (1) providing first, second, third and fourth nucleotides, each of which carries a reversible blocking group, and an antibody-conjugated dye as described in any of the technical solutions of the third aspect;

[0264] (2) contacting the four nucleotides with a target polynucleotide; removing the nucleotides that are not incorporated into the growing nucleic acid chain; contacting the antibody-conjugated dye with the target polynucleotide; removing the unbound antibody-conjugated dye; detecting the nucleotides incorporated into the growing nucleic acid chain; removing the reversible blocking group carried by the nucleotides incorporated into the growing nucleic acid chain and the bound antibody-conjugated dye;

[0265] Optionally, further comprising (3) repeating (1)-(2) one or more times.

[0266] Preferably, the contacting the four nucleotides with a target polynucleotide is performed in the presence of a polymerase.

[0267] In some embodiments, the method comprises the following steps:

[0268] (a) providing a mixture comprising a duplex, a nucleotide carrying a reversible blocking group, an antibody-conjugated dye, a polymerase and an excision reagent; the duplex comprises a growing nucleic acid chain and a nucleic acid chain to be sequenced; the antibody-conjugated dye is as described in any of the technical solutions of the third aspect;

[0269] (b) performing a reaction comprising the following steps (i), (ii) and (iii), optionally, repeating one or more times:

[0270] Step (i): using the polymerase, incorporating the nucleotide carrying a reversible blocking group into the growing nucleic acid chain, and then incorporating the antibody-conjugated dye into the growing nucleic acid chain, to finally form a nucleic acid intermediate comprising a reversible blocking group and an antibody-conjugated dye:

[0271] Step (ii): detecting the nucleic acid intermediate;

[0272] Step (iii): using the excision reagent, excising the reversible blocking group and the antibody-conjugated dye comprised by the nucleic acid intermediate;

[0273] Preferably, the excision reagent used for excising the reversible blocking group and the antibody-conjugated dye is the same reagent.

[0274] In some embodiments, the duplex is immobilized on a support.

[0275] In some embodiments, the growing nucleic acid chain is a primer.

[0276] In some embodiments, the primer forms the duplex by annealing to the nucleic acid strand to be sequenced.

[0277] In some embodiments, the duplex, the nucleotide with reversible blocking group, the antibody-conjugated dye, and the polymerase together form a reaction system.

[0278] In some embodiments, the nucleotide with reversible blocking group is incorporated into the growing nucleic acid strand using a polymerase under conditions that allow the polymerase to perform nucleotide polymerization reaction, followed by incorporation of the antibody-conjugated dye into the growing nucleic acid strand, to finally form a nucleic acid intermediate comprising the reversible blocking group and the antibody-conjugated dye.

[0279] In some embodiments, the solution phase of the reaction system of the previous step is removed before any one of the steps of detecting the nucleic acid intermediate.

[0280] In some embodiments, the excision reagent is contacted with the duplex or the growing nucleic acid strand in a reaction system.

[0281] In some embodiments, the excision reagent is capable of excising the reversible blocking group and the bound antibody-modified dye carried by the nucleotide incorporated into the growing nucleic acid strand, and does not affect the phosphodiester bond on the duplex backbone.

[0282] In some embodiments, the solution phase of the reaction system of the previous step is removed after any one of the steps of excising the reversible blocking group and the antibody-conjugated dye comprised in the nucleic acid intermediate.

[0283] In some embodiments, after step (ii), further comprising: determining the type of the nucleotide with reversible blocking group incorporated into the growing nucleic acid strand in step (i) according to the signal detected in step (ii), and determining the type of the nucleotide at the corresponding position in the nucleic acid strand to be sequenced based on the principle of base complementary pairing.

[0284] In a ninth aspect of the present disclosure, the present disclosure provides a kit comprising the compound of any one of the first aspect or a stereoisomer thereof or a salt thereof, or comprising the modified dye of any one of the second aspect, or comprising the antibody-conjugated dye of any one of the third aspect, or comprising the modified nucleotide of any one of the fourth aspect.

[0285] In some embodiments, the kit further comprises: reagents for processing nucleic acid molecules; primers for initiating nucleotide polymerization reaction; polymerases for performing nucleotide polymerization reaction; one or more buffer solutions; one or more washing solutions; or any combination thereof.

[0286] In a tenth aspect of the present disclosure, the present disclosure provides use of the compound of any one of the first aspect or a stereoisomer thereof or a salt thereof, or the modified dye of any one of the second aspect, or the antibody-conjugated dye of any one of the third aspect, or the modified nucleotide of any one of the fourth aspect, or the kit of any one of the ninth aspect for determining the sequence of a target polynucleotide. Advantages

[0287] The fluorescent dye connected with the compound of the present disclosure or its isomer or its salt has novel structure, and when used for sequencing (such as COOLMPS sequencing), the background signal is significantly reduced, the signal-to-noise ratio is improved, the sequencing quality is greatly improved, and the read length of sequencing is significantly prolonged. BRIEF DESCRIPTION OF DRAWINGS

[0288] Figure 1: Hydrogen spectrum and carbon spectrum spectrum of AF532 S-S Linker4 NHS.

[0289] Figure 2: Hydrogen spectrum and carbon spectrum spectrum of AF532 S-S Linker5 NHS.

[0290] Figure 3: Hydrogen spectrum and carbon spectrum spectrum of AF532 S-S Linker6 NHS.

[0291] Figure 4: Hydrogen spectrum and carbon spectrum spectrum of AF532 S-S Linker1 NHS.

[0292] Figure 5: A schematic diagram of the steps of a sequencing experiment.

[0293] Figure 6: Background signal detection result graph when AF532 S-S Linker4 NHS (i.e. SS4-AF532), AF532 S-S Linker5 NHS (i.e. SS5-AF532), AF532 S-S Linker6 NHS (i.e. SS6-AF532) are used for C base detection in PE50 sequencing.

[0294] Figure 7: Background signal detection result graph when AF532 S-S Linker4 NHS (i.e. SS4-AF532), AF532 S-S Linker5 NHS (i.e. SS5-AF532), AF532 S-S Linker6 NHS (i.e. SS6-AF532) are used for G base detection in PE50 sequencing.

[0295] Figure 8: Background signal detection results of AF532 S-S Linker4 NHS (i.e. SS4-AF532), AF532 S-S Linker5 NHS (i.e. SS5-AF532), AF532 S-S Linker6 NHS (i.e. SS6-AF532) for T base detection in PE50 sequencing.

[0296] Figure 9: Background signal detection results of AF532 S-S Linker3 NHS (i.e. SS3-AF532), AF532 S-S Linker4 NHS (i.e. SS4-AF532), AF532 S-S Linker5 NHS (i.e. SS5-AF532), AF532 S-S Linker6 NHS (i.e. SS6-AF532) for A base detection in PE50 sequencing. DETAILED DESCRIPTION

[0297] The embodiments of the present disclosure will be described in detail below with examples, but those skilled in the art will understand that the following examples are only for illustration of the present disclosure and should not be regarded as limiting the scope of the present disclosure. The specific conditions not noted in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not noted by the manufacturer, which are all conventional products that can be obtained by purchase.

[0298] Unless otherwise specified, the groups and substituents of the present disclosure have the ordinary meaning in the chemical art.

[0299] In various parts of the specification, the substituents of the compounds of the present disclosure are disclosed according to the group categories or ranges. It is specifically pointed out that the present disclosure includes each independent secondary combination of the individual members of these group categories and ranges. For example, the term "C1-C6 alkyl" specifically refers to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0300] In addition, it should be noted that, unless otherwise explicitly stated, the description "each of… is / are independently selected from" and "… is / are each independently selected from" used throughout this document can be interchangeable, and should be interpreted broadly, which can mean that the specific options expressed between the same or different symbols in different groups do not affect each other, or that the specific options expressed between the same or different symbols in the same group do not affect each other.

[0301] The term "C1-C6alkyl" refers to any straight or branched chain saturated radical containing 1 to 6 carbon atoms, such as, for example, methyl (Me), ethyl (Et), n-propyl, isopropyl (iPr), n-butyl, isobutyl, t-butyl (t-Bu), sec-butyl, n-pentyl, t-pentyl, n-hexyl, and the like, preferably "C1-C4alkyl", specifically, for example, methyl (Me), ethyl (Et), n-propyl, isopropyl (iPr), n-butyl, isobutyl, t-butyl (t-Bu), sec-butyl.

[0302] The term "direct bond" refers to the groups on either side of the bond being directly connected, for example, the structure of the compound of Formula I would be NH2-(CH2-CH2-O) n1 -(CH2) n2 -(S-S) n3 -L 1 -(CH2) n4 -(S-S) n5 -(CH2) n6 -L 2 -C(=O)OH, if L 1 is a direct bond, then the structure of the compound of Formula I would be NH2-(CH2-CH2-O) n1 -(CH2) n2 -(S-S) n3 -(CH2) n4 -(S-S) n5 -(CH2) n6 -L 2 -C(=O)OH. The remaining analogous definitions are understood in light of the foregoing.

[0303] The stereoisomers of the compounds described herein, when specifically designated as (R)- or (S)-isomers in the chemical name, are to be understood as being predominantly in the (R)-isomer or (S)-isomer configuration, respectively. Any asymmetric carbon atom can be present in the (R)-, (S)-, or (R,S)-configuration, preferably in the (R)- or (S)-configuration.

[0304] As used herein, the term "salt of a compound" refers to (i) a salt formed between an acidic functional group present in a compound provided in the present disclosure and an appropriate inorganic or organic cation (base), and includes, but is not limited to, alkali metal salts such as sodium salts, potassium salts, lithium salts, and the like; alkaline earth metal salts such as calcium salts, magnesium salts, and the like; other metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, and the like; inorganic base salts such as ammonium salts; organic base salts such as tertiary octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylamine salts, tris(hydroxymethyl)aminomethane salts, and (ii) a salt formed between a basic functional group present in a compound provided in the present disclosure and an appropriate inorganic or organic anion (acid), and includes, but is not limited to, hydrogen halide salts such as hydrofluoride salts, hydrochloride salts, hydrobromide salts, hydroiodide salts, and the like; inorganic acid salts such as nitrate salts, perchlorate salts, sulfate salts, phosphate salts, and the like; lower alkyl sulfonic acid salts such as methanesulfonate salts, trifluoromethanesulfonate salts, ethanesulfonate salts, and the like; aryl sulfonic acid salts such as benzenesulfonate salts, p-toluenesulfonate salts, and the like; organic acid salts such as acetate salts, malate salts, fumarate salts, succinate salts, citrate salts, tartrate salts, oxalate salts, maleate salts, and the like; amino acid salts such as glycine salts, trimethylglycine salts, arginine salts, ornithine salts, glutamic acid salts, aspartic acid salts, and the like.

[0305] In the present disclosure, the dye (or fluorescent group) can be, for example, Cy5, Cy3, AF532, ATTO532, AF647, and the like. Among them, the abbreviations of each dye (or fluorescent group) and their corresponding structural formula are shown in Table A below.

[0306] Table A: Abbreviations of each dye and their corresponding structural formula

[0307] The dye (or fluorescent group) in the present disclosure also includes structural analogs of the dyes in the above table as the parent nucleus.

[0308] In addition, when the above dye is connected to the fluorescent modification group of the present disclosure (such as the compound of formula I or formula I-1 of the present disclosure or its stereoisomer or salt thereof), the connection site of the above dye connected to the above fluorescent modification group of the present disclosure is shown as a wavy line in Table B below.

[0309] Table B: Connection site of each dye connected to the fluorescent modification group of the present disclosure

[0310] In the methods of the present disclosure, a material consisting of both a growing nucleic acid strand and a nucleic acid strand to be sequenced is referred to as a "duplex" regardless of the length of the growing nucleic acid strand or the nucleic acid strand to be sequenced, which can be longer than the growing nucleic acid strand.

[0311] In the methods of the present disclosure, the nucleic acid molecule to be sequenced can be any nucleic acid molecule of interest. In certain preferred embodiments, the nucleic acid molecule to be sequenced comprises deoxyribonucleotides, ribonucleotides, modified deoxyribonucleotides, modified ribonucleotides, or any combination thereof. In the methods of the present disclosure, the nucleic acid molecule to be sequenced is not limited by its type. In certain preferred embodiments, the nucleic acid molecule to be sequenced is DNA or RNA. In certain preferred embodiments, the nucleic acid molecule to be sequenced can be genomic DNA, mitochondrial DNA, chloroplast DNA, mRNA, cDNA, miRNA, or siRNA. In certain preferred embodiments, the nucleic acid molecule to be sequenced is linear or circular. In certain preferred embodiments, the nucleic acid molecule to be sequenced is double-stranded or single-stranded. For example, the nucleic acid molecule to be sequenced can be single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), or a hybrid of DNA and RNA. In certain preferred embodiments, the nucleic acid molecule to be sequenced is single-stranded DNA. In certain preferred embodiments, the nucleic acid molecule to be sequenced is double-stranded DNA.

[0312] In the methods of the present disclosure, the nucleic acid molecule to be sequenced is not limited by its source. In certain preferred embodiments, the nucleic acid molecule to be sequenced can be obtained from any source, such as any cell, tissue, or organism (e.g., viruses, bacteria, fungi, plants, and animals). In certain preferred embodiments, the nucleic acid molecule to be sequenced is derived from a mammal (e.g., a human, a non-human primate, a rodent, or a canine), a plant, a bird, a reptile, a fish, a fungus, a bacterium, or a virus.

[0313] Methods for extracting or obtaining nucleic acid molecules from cells, tissues, or organisms are well known to those skilled in the art. Suitable methods include, but are not limited to, ethanol precipitation, chloroform extraction, and the like. Detailed descriptions of such methods can be found in, for example, J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., John Wiley & Sons, Inc., 1995. In addition, various commercial kits are available for extracting nucleic acid molecules from various sources (e.g., cells, tissues, or organisms).

[0314] In the methods of the disclosure, the nucleic acid molecules to be sequenced are not limited by their length. In certain preferred embodiments, the nucleic acid molecules to be sequenced can be at least 10 bp, at least 20 bp, at least 30 bp, at least 40 bp, at least 50 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1000 bp, or at least 2000 bp in length. In certain preferred embodiments, the nucleic acid molecules to be sequenced can be 10-20 bp, 20-30 bp, 30-40 bp, 40-50 bp, 50-100 bp, 100-200 bp, 200-300 bp, 300-400 bp, 400-500 bp, 500-1000 bp, 1000-2000 bp, or more than 2000 bp in length. In certain preferred embodiments, the nucleic acid molecules to be sequenced can have a length of 10-1000 bp to facilitate high-throughput sequencing.

[0315] In the methods of the disclosure for preparing polynucleotides or sequencing, a suitable polymerase can be used to perform the nucleotide polymerization reaction. In some exemplary embodiments, the polymerase is capable of synthesizing a new DNA strand using a DNA template (e.g., a DNA polymerase). In some exemplary embodiments, the polymerase is capable of synthesizing a new DNA strand using an RNA template (e.g., a reverse transcriptase). In some exemplary embodiments, the polymerase is capable of synthesizing a new RNA strand using a DNA or RNA template (e.g., an RNA polymerase). Thus, in certain preferred embodiments, the polymerase is selected from the group consisting of a DNA polymerase, an RNA polymerase, and a reverse transcriptase. A suitable polymerase can be selected to perform the nucleotide polymerization reaction according to the actual need. In certain preferred embodiments, the polymerization reaction is a polymerase chain reaction (PCR). In certain preferred embodiments, the polymerization reaction is a reverse transcription reaction.

[0316] In the methods of the disclosure, a KOD polymerase or a mutant thereof can be used to perform the nucleotide polymerization reaction. KOD polymerases or mutants thereof (e.g., KOD POL151, KOD POL157, KOD POL171, KOD POL174, KOD POL376, KOD POL391) have acceptable polymerization efficiency on the modified nucleosides or nucleotides of the disclosure. KOD POL391 and KOD POL171 have acceptable polymerization efficiency on the modified nucleotides of the disclosure. In certain embodiments, KOD POL391 or KOD POL171 has a polymerization efficiency on the modified nucleotides of the disclosure of 70% or more, such as 70-80%, 80-90%, or 90-100%.

[0317] In the method of producing a polynucleotide or the method of sequencing according to the present disclosure, the polymerization reaction of nucleotides is performed under suitable conditions. Suitable polymerization conditions include the composition of a solution phase and the concentration of each component, the pH of the solution phase, the polymerization temperature, and the like. The polymerization is performed under suitable conditions, which is advantageous for obtaining an acceptable, even high, polymerization efficiency.

[0318] In some embodiments according to the present disclosure, the oxygen atom at the 3' position of the deoxyribose of the modified nucleotide is protected, and thus, they are capable of terminating the polymerization of a polymerase (e.g., a DNA polymerase). For example, when the modified nucleotide is introduced to the 3' end of a growing nucleic acid chain, the polymerase will not be able to proceed to the next round of polymerization reaction due to the absence of a free hydroxyl group (-OH) at the 3' position of the deoxyribose of the modified nucleotide, and thus, the polymerization reaction will be terminated. In this case, only one base will be incorporated into the growing nucleic acid chain in each round of polymerization reaction.

[0319] Further, the protecting group of the oxygen atom at the 3' position of the deoxyribose of the modified nucleotide can be removed and converted to a free hydroxyl group (-OH). Subsequently, the next round of polymerization reaction can be performed on the growing nucleic acid chain using a polymerase and a modified nucleotide, and one base is again introduced.

[0320] Thus, the oxygen atom at the 3' position of the deoxyribose of the modified nucleotide can be reversibly blocked, i.e., the modified nucleotide comprises a reversible blocking group that is attached to the oxygen atom at the 3' position of the deoxyribose. When the modified nucleotide is incorporated to the 3' end of a growing nucleic acid chain, they will terminate the polymerase from continuing the polymerization, and the growing nucleic acid chain will not be further extended; and after the blocking group comprised by the modified nucleotide is removed, a free hydroxyl group (-OH) will be present at the 3' position, and the polymerase will be able to continue the polymerization on the growing nucleic acid chain, and the nucleic acid chain will be continued to be extended.

[0321] In other embodiments according to the present disclosure, although the hydroxyl group (-OH) at the 3' position of the deoxyribose of the modified nucleotide can not be protected, the base is protected. Thus, they are also capable of terminating the polymerization of a polymerase (e.g., a DNA polymerase). For example, when the modified nucleotide is introduced to the 3' end of a growing nucleic acid chain, the polymerase will not be able to proceed to the next round of polymerization reaction due to the steric hindrance effect or hydrogen bond interaction, and the like, at the base of the modified nucleotide, and thus, the polymerization reaction will be terminated. In this case, only one base will be incorporated into the growing nucleic acid chain in each round of polymerization reaction.

[0322] Further, the protecting group at the base of the modified nucleotide can be removed. Subsequently, the next round of polymerization reaction can be performed on the growing nucleic acid chain using a polymerase and a modified nucleotide, and one base is again introduced.

[0323] Thus, the base of the modified nucleotide can be reversibly blocked, i.e. the modified nucleotide comprises a reversible blocking group, which is attached to the base. When a modified nucleotide is incorporated at the 3’ end of a growing nucleic acid strand, it will terminate the polymerase from continuing the polymerization, terminating further elongation of the growing nucleic acid strand; and, upon removal of the blocking group comprised by the modified nucleotide, the polymerase will be able to continue the polymerization of the growing nucleic acid strand, continuing the elongation of the nucleic acid strand.

[0324] The dyes (or referred to as fluorescent groups) described herein are all fluorescent dyes routinely used by those skilled in the art. For example, Welch et al. (Chem. Eur. J. 5(3): 951-960, 1999) disclose dansyl-functionalized fluorescent moieties, which can be used in the present disclosure. Zhu et al. (Cytometry 28: 206-211, 1997) describe the use of fluorescent labels Cy3 and Cy5, which can also be used in the present disclosure. Prober et al. (Science 238: 336-341, 1987), Connell et al. (BioTechniques 5(4): 342-384, 1987), Ansorge et al. (Nucl. Acids Res. 15(11): 4593-4602, 1987), and Smith et al. (Nature 321: 674, 1986) also disclose labels suitable for use. Other commercially available fluorescent labels include, but are not limited to, fluorescein, rhodamine (including TMR, Texas Red, and Rox), alexa, boron difluoride, acridine, coumarin, pyrene, benzanthracene, and cyanine.

[0325] In the present disclosure, the dye (or referred to as fluorescent group) or modified dye is carried by the nucleotide molecule by introduction of an affinity reagent (such as an antibody, an aptamer, an affinity Affimer, a Knottin), which can specifically recognize and bind to the epitope of the nucleotide molecule, the specific principle is described in WO2018129214A1. The entire relevant content in WO2018129214A1 is incorporated into the present application.

[0326] The skilled person is aware of the utility of dideoxynucleotide triphosphates in so-called Sanger sequencing methods and related protocols (Sanger-type), which rely on random chain termination at specific types of nucleotides. One example of a Sanger-type sequencing protocol is the BASS method described by Metzker.

[0327] Sanger and Sanger-type methods are typically practiced by performing an experiment in which eight types of nucleotides are provided, four of which contain a 3'-OH group; four of which lack a 3'-OH group and are labeled differently from one another. The nucleotides used that lack a 3'-OH group are dideoxynucleotides (ddNTPs). As is well known to those skilled in the art, when the ddNTPs are labeled differently, the sequence of the target oligonucleotide can be determined by assaying the position of the terminal nucleotide that is incorporated, and combining this information.

[0328] It will be appreciated that the modified nucleotides of the present application have utility in Sanger methods and related protocols, as the same effect achieved by using ddNTPs can be achieved by using the modified nucleotides described herein.

[0329] It will also be appreciated that the modified nucleotides of the present application have utility in second generation sequencing (NGS sequencing) and third generation sequencing (single molecule sequencing), as the same effect achieved by using dNTPs can be achieved by using the modified nucleotides described herein.

[0330] The technical solutions of the present disclosure are further explained and described below in connection with specific examples. Except as specifically noted, the instruments referred to in the following examples are those conventionally used in the art, and the reagents referred to are those conventionally available in the art.

[0331] The present disclosure demonstrates the structural design of disulfide bond-based linkers, synthetic routes, and applications in nucleic acid sequencing. The present disclosure designs six disulfide bond linkers as shown below, wherein the synthesis routes of 1, 3, 4, 5, and 6 and the sequencing results of 3, 4, 5, and 6 are exemplarily shown below. Those skilled in the art can understand that other compounds can be synthesized in a similar manner and obtain similar sequencing results.

[0332] Example 1: Synthesis and characterization of compounds

[0333] 1 Reagents and instruments

[0334] 1.1 Instruments:

[0335] DIONEX UltiMate 3000 liquid chromatograph-mass spectrometer (degasser: SRD-3400, binary pump: HPG-3400RS, automatic sampler: WPS-3000TRS, column oven: TCC-3000SD, detector: DAD-3000, mass spectrometer: ISQ EM-Mass spectrometer, Thermo Fisher Scientific), DIONEX UltiMate 3000 preparative liquid chromatograph (binary pump: HPG-3200BX, automatic sampler: WPS-3000TSL, detector: DAD-3000, fraction collector: Fraction Collector F, Thermo Fisher Scientific), KQ-300E ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), electronic balance (model: BCA324I-10CN, Sartorius Scientific Instruments (Beijing) Co., Ltd.), biotage automatic column chromatography machine (Biotage Trading (Shanghai) Co., Ltd.), vacuum freeze dryer (Bio-Medical Kong (Beijing) Instrument Co., Ltd.).

[0336] 1.2 Reagents:

[0337] Acetonitrile (lot number: WXBD3817V, Chromasolv, Sigma-Aldrich Reagent), ultrapure water, cystamine dihydrochloride (lot number: C13590508, 98%, Shanghai Macklin Biochemical Technology Co., Ltd.), cysteamine hydrochloride (lot number: C12728647, 98%, Shanghai Macklin Biochemical Technology Co., Ltd.), terephthalic acid monomethyl ester (lot number: C13313655, 97%, Shanghai Macklin Biochemical Technology Co., Ltd.), triethylamine (Et3N) (lot number: STBK3222, 99.5%, Sigma-Aldrich Reagent), sodium hydroxide (NaOH) (lot number: C12499732, 96%, Shanghai Macklin Biochemical Technology Co., Ltd.), methanol (lot number: 20220125, 99.5%, Shanghai Try), di-tert-butyl dicarbonate (lot number: C2111016, 98%, Shanghai Aladdin Biochemical Technology Co., Ltd.), 3,3'-dithiopropionic acid (lot number: C13551219, 99%, Shanghai Macklin Biochemical Technology Co., Ltd.), carbonyldiimidazole (CDI) (lot number: HB01BA0047, 98%, Sangon Biotech (Shanghai) Co., Ltd.), tetrahydrofuran (THF) (lot number: C12789231, 99%, Shanghai Macklin Biochemical Technology Co., Ltd.), trifluoroacetic acid (TFA) (lot number: B2210493, 99%, Shanghai Aladdin Biochemical Technology Co., Ltd.), dichloromethane (DCM) (lot number: STBK2768, 99.8%, Sigma-Aldrich Reagent), AF532 dye (Beijing Oukai Nas Technology Co., Ltd.), N’N-diisopropylethylamine (DIPEA) (lot number: C12677270, 99%, Shanghai Macklin Biochemical Technology Co., Ltd.), anhydrous N’N-dimethylacetamide (DMF) (lot number: C13087511, 99.8%, Shanghai Macklin Biochemical Technology Co., Ltd.), 4-dimethylaminopyridine (DMAP) (lot number: C12509524, 99%, Shanghai Macklin Biochemical Technology Co., Ltd.), N,N’-disuccinimidyl carbonate (DSC) (lot number: BCCB6748, 95%, Sigma-Aldrich Reagent), sodium hydroxide (NaOH) (lot number: C12499732, 96%, Shanghai Macklin Biochemical Technology Co., Ltd.), glycine ethyl ester hydrochloride (lot number: C13380325, 99%, Shanghai Macklin Biochemical Technology Co., Ltd.), sodium bicarbonate (NaHCO3) (lot number: C1266333, 99.8%, Shanghai Macklin Biochemical Technology Co., Ltd.), ethanol (lot number: 20220125, 95%, Shanghai Try).

[0338] 2 Experimental methods

[0339] 2.1 Synthetic route

[0340] (1) Synthesis of AF532 S-S Linker4 NHS

[0341] Synthetic method of compound 2:

[0342] Take a 250 mL flask, weigh compound 1 (7 g), add tetrahydrofuran (30 mL) and deionized water (30 mL) to dissolve, then add sodium hydroxide (2.46 g), adjust pH = 8-9, then add Boc20 (14.79 g), 23 °C, electromagnetic stirring for 4 hours. The reaction solution was concentrated under reduced pressure to give a crude product, which was then purified by biotage automatic column machine (silica gel, developing agent petroleum ether / ethyl acetate = 1 / 0 ~ 1 / 1) to give compound 2.

[0343] Synthetic method of compound 4:

[0344] Take a 40 mL sample bottle, weigh compound 3 (1 g), add methanol (10 mL) to dissolve, then add triethylamine (1.93 g), compound 2 (1.26 g), 21 °C, electromagnetic stirring for 15 hours. The reaction solution was concentrated under reduced pressure to give a crude product, which was then purified by biotage automatic column machine (silica gel, developing agent dichloromethane / methanol = 1 / 0 ~ 10 / 1) to give compound 4. LCMS: calcd for C 10 H 19 NO4S2[M+H] + : 282.08. Found, m / z, [M-100+H] + : 182.07.

[0345] Synthetic method of compound 5:

[0346] Take a 25 mL flask, weigh compound 4 (300 mg), add dichloromethane (5 mL) to dissolve, then add trifluoroacetic acid (2.5 mL), 22 °C, electromagnetic stirring for 1 hour. The reaction solution was concentrated under reduced pressure to give a crude product, which was then purified by biotage automatic column machine (welflash C18-I, regular C18 20-40 μm, 40 g, 0.1% formic acid / acetonitrile, acetonitrile content 0-100%) to give compound 5. 1 H NMR (600 MHz, DMSO-d6) δ 12.46 (s, 1H), 8.00 (s, 3H), 3.10 (t, J = 7.2 Hz, 2H), 2.94 - 2.88 (m, 4H), 2.64 (t, J = 7.2 Hz, 2H). 13 C NMR (150 MHz, DMSO-d6) δ 172.68, 37.78, 33.98, 33.65, 32.78. LCMS: calcd for C5H11 NO2S2[M+H] + : 182.06. Found, m / z, [M+H] + : 182.06.

[0347] Synthesis method of compound 7:

[0348] Take a 4 mL sample bottle, weigh compound 6 (30 mg), dissolve in DMF (1 mL), then add DSC (18 mg), DMAP (1 mg), 22°C, electromagnetic stirring for 4 hours. Then add compound 5 (17 mg), DIPEA (30 mg), continue electromagnetic stirring at 22°C for 15 hours. Filter the reaction solution, purify by biotage automatic column machine (welflash C18-I, regular C18 20-40 μm, 40 g, 0.1M TEAB / acetonitrile, acetonitrile content 0-100%), reverse phase liquid is concentrated under reduced pressure, freeze-dried to obtain compound 7. LCMS: calcd for C 35 H 39 N3O 10 S4[M+H] + : 790.15. Found, m / z, [M+H] + : 790.27.

[0349] Synthesis method of compound 8:

[0350] Take a 4 mL sample bottle, weigh compound 7 (15.0 mg), dissolve in DMF (1.5 mL), then add DSC (6.69 mg), DMAP (425.18 μg), 22°C, electromagnetic stirring for 3 hours. Filter the reaction solution, purify by preparative HPLC (HYPERSIL GOLD, 5 μm, 250 mm*10 mm, pre-column, 1PK, mobile phase: 0.1% formic acid aqueous solution / acetonitrile; flow rate: 5 mL / min; column equilibration time 8 min; mobile phase ratio: acetonitrile content 10-75% gradient separation for 0-23 min), obtain the separation liquid, remove the organic phase by concentrating under reduced pressure, freeze-drying to obtain compound 8 (NMR spectrum is shown in Figure 1). 1H NMR (600 MHz, DMSO-d6): δ 8.93 (t, J = 5.5 Hz, 1H), 8.79 (s, 2H), 8.14 (d, J = 8.3 Hz, 2H), 7.58 (d, J = 7.9 Hz, 2H), 6.83 (s, 2H), 3.83 (q, J = 6.6 Hz, 2H), 3.64 (q, J = 6.4 Hz, 2H), 3.17-3.12 (m, 2H), 3.09-3.04 (m, 2H), 3.00 (t, J = 6.7 Hz, 2H), 2.82 (s, 4H), 1.29-1.13 (m, 12H), 1.06-1.01 (m, 6H). 13 C NMR (151 MHz, DMSO-d6): δ 170.56, 167.99, 166.14, 155.20, 154.74, 154.58, 144.21, 135.76, 129.93, 128.24, 120.14, 114.13, 66.31, 56.49, 46.20, 42.64, 40.51, 39.17, 37.40, 37.40, 32.38, 30.92, 28.38, 28.26, 25.92, 25.69, 23.00, 22.94, 19.03, 16.18, 16.11. LCMS: calcd for C 39 H 42 N4O 12 S4[M-H] - :885.17. Found, m / z, [M-H] - :885.22.

[0351] (2) Synthesis of AF532 S-S Linker5 NHS

[0352] Synthetic method of compound 10:

[0353] Take a 500 mL flask, weigh compound 3 (5.0 g), dissolve in tetrahydrofuran (250 mL), then add CDI (4.20 g), and stir at 22°C for 5 hours. Monitor the consumption of raw materials and new points are generated, add compound 9 (9.62 g), stir at 22°C for 16 hours. The reaction solution is dried by rotary evaporation, dissolved in ethyl acetate (80 mL), then washed with 1M HCl (30 mL*3) aqueous solution, saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then concentrated under reduced pressure with dichloromethane (30 mL*3) to remove residual ethyl acetate, to obtain compound 10. LCMS: calcd for C 13 H 24 N2O5S2[M-H]- :351.11.Found, m / z, [M-H] - :351.09.

[0354] Synthesis method of compound 11:

[0355] Take a 25 mL flask, weigh compound 10 (600 mg), dissolve in dichloromethane (8 mL), then add trifluoroacetic acid (4 mL), 22°C, electromagnetic stirring for 1 hour. Pour the reaction solution into water (50 mL), extract with ethyl acetate / petroleum ether = 1 / 1 (30 mL*3), separate the aqueous phase, and concentrate under reduced pressure to obtain compound 11. 1 H NMR (600 MHz, DMSO-d6) δ 12.44 (s, 1H), 8.18 (t, J = 5.4 Hz, 1H), 7.87 (s, 3H), 3.28 (q, J = 6.6 Hz 2H), 3.13-3.10 (m, 2H), 2.92-2.86 (m, 4H), 2.62 (t, J = 7.2 Hz, 2H), 2.52-2.50 (m, 2H). 13 C NMR (150 MHz, DMSO-d6) δ 172.67, 170.92, 38.58, 36.45, 34.97, 33.62, 33.47, 32.98. LCMS: calcd for C8H 16 N2O3S2[M+H] + :253.06.Found, m / z, [M+H] + :253.08.

[0356] Synthesis method of compound 12:

[0357] Take a 4 mL sample bottle, weigh compound 6 (30 mg), dissolve in DMF (1 mL), then add DSC (18 mg), DMAP (1 mg), 22°C, electromagnetic stirring for 3 hours. Then add compound 11 (24 mg), DIPEA (30 mg), 22°C, continue electromagnetic stirring for 15 hours. Filter the reaction solution, and purify by biotage automatic column machine (welflash C18-I, regular C18 20-40 μm, 40 g, 0.1M TEAB / acetonitrile, acetonitrile content 0-100%), and reverse phase liquid is concentrated under reduced pressure to obtain compound 12. LCMS: calcd for C 38 H 44 N4O 11 S4[M+H] + :861.19.Found, m / z, [M+H] + :861.23.

[0358] Method for synthesizing compound 13:

[0359] Take a 4 mL sample bottle, weigh compound 12 (18 mg), dissolve in DMF (1 mL), then add DSC (8 mg), DMAP (1 mg), 22°C, electromagnetic stirring for 3 hours. Filter the reaction solution, purify by preparative HPLC (COSMOSIL Packed Column, 5 μm, 250 mm*20 mm, pre-column, mobile phase: 0.1% methanolic aqueous solution / acetonitrile; flow rate: 7 mL / min; column equilibration time: 8 min; mobile phase ratio: 0-25 min acetonitrile content 25-70% gradient separation, 25-30 min acetonitrile content 70% isocratic elution), prepare the solution and concentrate under reduced pressure, freeze-drying to obtain compound 13 (the nuclear magnetic resonance spectrum is shown in Figure 2). 1 H NMR (600 MHz, DMSO-d6): δ 8.95-8.67 (m, 3H), 8.18-8.13 (m, 3H), 7.58 (d, J = 8.2 Hz, 2H), 6.84 (s, 2H), 3.86-3.82 (m, 2H), 3.42-3.35 (m, 2H), 3.31 (q, J = 6.4 Hz, 2H), 3.09 (t, J = 6.8 Hz, 2H), 3.01-2.93 (m, 4H), 2.81 (s, 4H), 2.60-2.51 (m, 2H), 1.25-1.14 (m, 12H), 1.09-0.97 (m, 6H). 13 C NMR (151 MHz, DMSO-d6): δ 171.22, 170.82, 170.52, 167.94, 166.16, 155.34, 154.58, 154.36, 144.42, 136.07, 135.31, 129.86, 128.29, 122.36, 114.09, 109.34, 66.49, 46.18, 42.62, 40.51, 38.75, 35.47, 35.28, 34.49, 34.26, 32.35, 30.82, 28.44, 28.33, 25.89, 25.76, 23.01, 22.95, 16.16, 16.10, 9.05. LCMS: calcd for C 42 H 47 N5O 13 S4[M-H] - :956.21. Found, m / z, [M-H] - :956.24.

[0360] (3) Synthesis of AF532 S-S Linker6 NHS

[0361] Method for synthesizing compound 16:

[0362] Take 500 mL flask, weigh compound 15 (2.0 g), add dichloromethane (100 mL), then add CDI (2.27 g), and 22 ℃, electromagnetic stirring for 5 hours. Add compound 14 (6.19 g) and triethylamine (2.72 g). 22 ℃, electromagnetic stirring for 16 hours. Filter out the insoluble solid, spin dry the solvent, dissolve with ethyl acetate (30 mL), add 10 mL water to the reaction solution, adjust the pH to 2-3 with 1M HCl, separate the aqueous phase, wash the aqueous phase with ethyl acetate (30 mL*3), and concentrate the aqueous phase under reduced pressure to obtain compound 16. LCMS: calcd for C 13 H 18 N2O3S2[M+H] + : 315.08. Found, m / z, [M+H] + : 315.10.

[0363] Method for synthesizing compound 17:

[0364] Take 50 mL flask, weigh compound 16 (0.5 g), add methanol (5 mL) to dissolve, adjust the reaction also pH to 12-13 with 4M NaOH aqueous solution (7 mL), and stir the reaction solution at 22 ℃ for 5 hours. Concentrate the reaction solution under reduced pressure, purify by preparative liquid chromatography (HYPERSIL GOLD, 5 μm, 250 mm*10 mm, pre-column, 1PK, mobile phase: 0.1% formic acid aqueous solution / acetonitrile; flow rate: 5 mL / min; column equilibration time 3 min; mobile phase ratio: 0-30 min acetonitrile content 5-60% gradient separation), concentrate the preparative solution under reduced pressure, freeze-dry to obtain compound 17. 1 H NMR (600 MHz, DMSO-d6) δ 13.24 (s, 1H), 8.86 (t, J = 5.4 Hz, 1H), 8.03-8.01 (m, 2H), 7.94-7.92 (m, 2H), 7.84 (m, 3H) 3.58 (q, J = 6.6 Hz 2H), 3.12-3.08 (m, 2H), 2.96-2.92 (m, 4H). 13 C NMR (150 MHz, DMSO-d6) δ 166.77, 165.66, 137.97, 133.11, 129.32, 127.41, 38.74, 37.84, 36.71, 34.22. LCMS: calcd for C 12 H 16 N2O3S2[M-H] -: 299.06. Found, m / z, [M-H] - : 299.02.

[0365] Synthesis method of compound 18:

[0366] Take a 4 mL sample bottle, weigh compound 6 (21 mg), dissolve in DMF (0.5 mL), then add DSC (13 mg), DMAP (0.9 mg), 22°C, electromagnetic stirring for 3 hours. Then add compound 17 (10 mg), DIPEA (21.5 mg), and stir at 22°C for 15 hours. Filter the reaction solution, purify by preparative liquid chromatography (HYPERSIL GOLD, 5 μm, 250 mm*10 mm, pre-column, 1PK, mobile phase: 0.1M TEAB / acetonitrile; flow rate: 5 mL / min; column equilibration time 3 min; mobile phase ratio: 0~21 min acetonitrile content 5~70% gradient separation), and prepare a solution under reduced pressure, freeze-drying to obtain compound 18. LCMS: calcd for C 42 H 44 N4O 11 S4[M-H] - : 907.19. Found, m / z, [M-H] - : 907.13.

[0367] Synthesis method of compound 19:

[0368] Take a 4 mL sample bottle, weigh compound 18 (3.2 mg), dissolve in DMF (1.5 mL), then add DSC (1.4 mg), DMAP (0.8 mg), 22°C, electromagnetic stirring for 3 hours. Filter the reaction solution, purify by preparative HPLC (HYPERSIL GOLD, 5 μm, 250 mm*10 mm, pre-column, 1PK, mobile phase: 0.1% formic acid in water / acetonitrile; flow rate: 5 mL / min; column equilibration time 8 min; mobile phase ratio: 0~23 min acetonitrile content 10~75% gradient separation), and prepare a solution under reduced pressure, freeze-drying to obtain compound 19 (NMR spectrum as shown in Figure 3). 1HNMR (600 MHz, DMSO-d6): δ 9.02-8.93 (m, 2H), 8.82-8.75 (m, 2H), 8.18-8.13 (m, 4H), 8.07 (d, J = 8.6 Hz, 2H), 7.59 (d, J = 8.2 Hz, 2H), 6.87 (s, 2H), 3.85-3.82 (m, 1H), 3.66-3.63 (m, 4H), 3.13-3.10 (m, 1H), 3.00 (t, J = 6.7 Hz, 4H), 2.90 (s, 4H), 1.25-1.15 (m, 12H), 1.09-1.00 (m, 6H). 13 C NMR (151 MHz, DMSO-d6) δ 170.14, 165.05, 161.24, 140.14, 130.07, 128.15, 126.49, 113.55, 66.00, 55.95, 52.56, 45.67, 42.07, 39.95, 38.79, 37.00, 36.82, 27.87, 27.77, 25.45, 22.42, 22.36, 15.59, 15.54, 8.54. LCMS: calcd for C 46 H 47 N5O 13 S4[M-H] - : 1004.21. Found, m / z, [M-H] - : 1004.31.

[0369] (4) Synthesis of AF532 S-S Linker1 NHS

[0370] Synthesis method of compound 2:

[0371] Take a 500 mL flask, weigh compound 1 (10.83 g), dissolve in methanol (200 mL), then add triethylamine (13.91 g), then add Boc2O (10 g), 22°C, electromagnetic stirring for 3 hours. The reaction solution is concentrated under reduced pressure to obtain a crude product, then 0.2M NaH2PO4 solution (200 mL) is added, ethyl acetate / petroleum ether = 1 / 1 (100 mL) is added, a small amount of 2M HCl solution is added to adjust pH = 4, and the water phase is separated. Then re-extract with ethyl acetate / petroleum ether = 1 / 1 (100 mL), separate the water phase, adjust the pH to 9-10 with 2M NaOH aqueous solution, then extract with ethyl acetate (100 mL*3), combine the organic phase, wash with saturated sodium chloride solution (100 mL), dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain compound 2.

[0372] Method for synthesizing compound 4:

[0373] Take a 100 mL flask, weigh compound 3 (1 g), dissolve in tetrahydrofuran (10 mL), then add carbonyldiimidazole (850 mg), 22°C, electromagnetic stirring for 3 hours. Then add compound 2 (1.32 g), 22°C, continue electromagnetic stirring for 15 hours. Pour the reaction solution into water (50 mL), extract with ethyl acetate (30 mL*3), combine the organic phase, wash with saturated sodium chloride solution (50 mL), dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain a crude product. Purify by biotage automatic column machine (silica gel, developing agent dichloromethane / methanol = 1 / 0~10 / 1) to obtain compound 4. LCMS: calcd for C 15 H 28 N2O5S4[M+H] + :445.09.Found,m / z,[M+H] + :445.06, [M-100+H] + :345.06.

[0374] Method for synthesizing compound 5:

[0375] Take a 25 mL flask, weigh compound 4 (400 mg), dissolve in dichloromethane (3 mL), then add trifluoroacetic acid (1.5 mL), 22°C, electromagnetic stirring for 1 hour. Concentrate the reaction solution under reduced pressure to obtain a crude product, and purify by biotage automatic column machine (welflash C18-I, regular C18 20-40 μm, 40 g, 0.1% formic acid / acetonitrile, acetonitrile content 0-100%) to obtain compound 5. 1 H NMR (600 MHz, DMSO-d6) δ 8.96 (m, 3H) 8.14 (t, J = 6.0 Hz, 1H), 3.26 (q, J = 6.6 Hz, 2H), 3.00 (t, J = 7.2 Hz, 2H), 2.84-2.76 (m, 6H), 2.78 (t, J = 7.2 Hz, 2H) 2.58 (t, J = 6.6 Hz, 2H) 2.48 (t, J = 7.2 Hz, 2H). 13 C NMR (150 MHz, DMSO-d6) δ 172.90, 170.27, 38.00, 37.91, 36.93, 34.92, 34.21, 33.93, 33.83, 33.27. LCMS: calcd for C 10 H 20 N2O3S4[M+H] + :345.04.Found,m / z,[M+H] + :345.11.

[0376] Method for synthesizing compound 7:

[0377] Take a 4 mL sample bottle, weigh compound 6 (30 mg), dissolve in DMF (1 mL), then add DSC (18 mg), DMAP (1 mg), 22°C, electromagnetic stirring for 5 hours. Then add compound 5 (32 mg), DIPEA (30 mg), 22°C, continue electromagnetic stirring for 15 hours. Filter the reaction solution, purify by biotage automatic column machine (welflash C18-I, regular C18 20-40 μm, 40 g, 0.1 M TEAB / acetonitrile, acetonitrile content 0-100%), reverse phase liquid is reduced pressure concentrated, freeze-dried to obtain compound 7. LCMS: calcd for C 40 H 48 N4O 11 S6[M+H] + :953.16. Found, m / z, [M+H] + :953.20.

[0378] Method for synthesizing compound 8:

[0379] Take a 4 mL sample bottle, weigh compound 7 (15.0 mg), dissolve in DMF (1.5 mL), then add DSC (6.05 mg), DMAP (385.51 μg), 22°C, electromagnetic stirring for 3 hours. The reaction solution is purified by HPLC (HYPERSIL GOLD, 5 μm, 250 mm*10 mm, pre-column, 1PK, mobile phase: 0.1% formic acid aqueous solution / acetonitrile; flow rate: 5 mL / min; column equilibration time 5 min; mobile phase ratio: 0-5 min 5% acetonitrile isocratic, 5-20 min acetonitrile content 40-55% gradient separation), the separated liquid is concentrated under reduced pressure to remove the organic phase, freeze-dried to obtain compound 8 (NMR spectrum is shown in Figure 4). 1H NMR (600 MHz, DMSO-d6) δ 8.94 (t, J = 5.5 Hz, 1H), 8.79 (s, 2H), 8.20 (t, J = 5.7 Hz, 1H), 8.14 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 8.1 Hz, 2H), 6.83 (s, 2H), 3.83 (q, J = 6.4 Hz, 2H), 3.64 - 3.60 (m, 4H), 3.46 - 3.42 (m, 2H), 3.40 - 3.36 (m, 2H), 3.17 - 3.03 (m, 3H), 3.00 - 2.96 (m, 3H), 2.93 (t, J = 7.1 Hz, 1H), 2.87 - 2.75 (m, 5H), 1.23 - 1.11 (m, 12H), 1.06 - 1.00 (m, 6H). 13 C NMR (151 MHz, DMSO-d6): δ 170.62, 170.53, 167.95, 166.10, 154.70, 154.57, 144.22, 135.74, 135.65, 129.92, 128.25, 122.19, 114.12, 66.32, 56.49, 46.19, 42.64, 40.51, 39.34, 38.48, 37.68, 37.43, 35.31, 34.17, 32.32, 30.80, 28.38, 28.26, 25.90, 23.01, 22.95, 19.03, 16.18, 16.11, 9.06. LCMS: calcd for C 44 H 51 N5O 13 S6[M-H] - : 1048.18. Found, m / z, [M-H] - : 1048.49.

[0380] (5) Synthesis of AF532 S-S Linker3 NHS

[0381] Synthetic method of compound 2:

[0382] Into a 500 mL flask, compound 1 (10.83 g) was weighed, dissolved in methanol (200 mL), then triethylamine (13.91 g) was added, and di-tert-butyl dicarbonate (10 g) was added in portions, and stirred magnetically at 24 °C for 15 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product, then water (100 mL) was added, ethyl acetate / petroleum ether = 1 / 1 (200 mL) was added, and the pH was adjusted to 4-5 with 2M aqueous hydrochloric acid solution, and the aqueous phase was separated. The aqueous phase was adjusted to pH 8-9 with saturated aqueous sodium carbonate solution, and extracted with ethyl acetate (80 mL*3), and the organic phase was washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 2.

[0383] Synthesis method of compound 4:

[0384] Into a 250 mL flask, compound 3 (2 g) was weighed, dissolved in tetrahydrofuran (20 mL), then carbonyldiimidazole (1.72 g) was added, and stirred magnetically at 23 °C for 3 hours. Then compound 2 (3.36 g), triethylamine (2.25 g), and 4-dimethylaminopyridine (136 mg) were added, and stirred magnetically at 23 °C for 15 hours. The reaction solution was poured into water (100 mL), extracted with ethyl acetate (50 mL*3), the organic phase was combined, washed with 1M aqueous hydrochloric acid solution (50 mL*2), saturated aqueous sodium carbonate solution (50 mL), and saturated sodium chloride (50 mL) in sequence, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 4. LCMS: calcd for C 18 H 26 N2O5S2[M+H] + : 415.13. Found, m / z, [M-100+Na] + : 437.21, [M-100+H] + : 315.14.

[0385] Synthesis method of compound 5:

[0386] Into a 50 mL flask, compound 4 (2 g) was weighed, dissolved in dichloromethane (14 mL), then trifluoroacetic acid (7 mL) was added, and stirred magnetically at 24 °C for 1 hour. The reaction solution was concentrated under reduced pressure, then dichloromethane (20 mL*3) was added and concentrated under reduced pressure to obtain compound 5.

[0387] Synthesis method of compound 7:

[0388] Take 100 mL flask, weighed compound 6 (800 mg), added tetrahydrofuran (10 mL), then added carbonyl diimidazole (708 mg), 24 ℃, electromagnetic stirring 3 hours. Then add compound 5 (1.52 g) triethylamine (925 mg) and 4-dimethylamino pyridine (56 mg), 24 ℃, electromagnetic stirring 4 hours. The reaction liquid is poured into water (50 mL), extracted with ethyl acetate (50 mL*3), the combined organic phase is washed with saturated sodium chloride (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, to get the crude product, with ethyl acetate / petroleum ether = 1 / 1 (30 mL), beating, filtering, drying, to get compound 7. LCMS: calcd for C 20 H 29 N3O6S2[M+H] + : 472.15. Found, m / z, [M-100+H] + : 372.16.

[0389] Synthesis method of compound 8:

[0390] Take 100 mL flask, weighed compound 7 (500 mg), added methanol (10 mL) and tetrahydrofuran (10 mL), dissolved, then added sodium hydroxide (85 mg) aqueous solution (7 mL), 24 ℃, electromagnetic stirring 2h. The reaction liquid is added to 1M hydrochloric acid aqueous solution to adjust pH to 4-5, then extracted with ethyl acetate (50 mL*3), the combined organic phase is washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, to get compound 8.

[0391] Synthesis method of compound 9:

[0392] Take 50 mL flask, weighed compound 8 (400 mg), added dichloromethane (8 mL) to dissolve, then added trifluoroacetic acid (4 mL), 23 ℃, electromagnetic stirring 1 hour. The reaction liquid is concentrated under reduced pressure to get the crude product, purified by biotage automatic column machine (welflash C18-I, regular C18 20-40 μm, 80 g, 0.1% formic acid / acetonitrile, acetonitrile content 0-100%), the preparation liquid is concentrated under reduced pressure to get compound 9. 1 H NMR (600 MHz, DMSO-d6) δ 13.24 (s, 1H), 8.86 (t, J = 5.4 Hz, 1H), 8.54 (t, J = 5.4 Hz, 1H), 8.02-8.00 (m, 4H), 7.94-7.92 (m, 2H) 3.59-3.55 (m, 4H) 3.44 (q, J = 6.6 Hz, 2H) 2.92 (t, J = 7.2 Hz, 2H), 2.84 (t, J = 6.6 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) δ 166.76, 166.07, 165.63, 137.97, 133.10, 129.30, 127.42, 40.13, 38.85, 37.85, 36.96, 36.82. LCMS: calcd for C 14 H 19 N3O4S2[M+H] + :358.08.Found,m / z,[M+H] + :358.16.

[0393] Synthesis method of compound 11:

[0394] Take a 15 mL sample bottle, weigh compound 10 (50 mg), dissolve in DMF (3 mL), then add compound 9 (50 mg), triethylamine (45 mg), 23°C, electromagnetic stirring for 15 hours. The reaction solution is filtered, first purified by biotage automatic column machine (welflash C18-I, regular C18 20-40 μm, 80 g, 0.1M TEAB / acetonitrile, acetonitrile content 0-100%), then purified by preparative HPLC (HYPERSIL GOLD, 5 μm, 250 mm*10 mm, pre-column, 1PK, mobile phase: 1M TEAB / acetonitrile; flow rate: 7 mL / min; column equilibration time 5 min; mobile phase ratio: 0-25 min acetonitrile gradient 5%-100%). The separated solution is concentrated under reduced pressure to remove the organic phase, freeze-dried to obtain compound 11. LCMS: calcd for C 44 H 47 N5O 12 S4[M-H] - :964.21.Found,m / z,[M-H] - :964.27.

[0395] Synthesis method and structural data of compound 12:

[0396] Take 4 mL sample bottle, weigh compound 11 (23 mg), add DMF (2 mL) to dissolve, then add DSC (10 mg), DMAP (1 mg), 22°C, electromagnetic stirring for 3 hours. The reaction solution is filtered and purified by preparative HPLC (HYPERSIL GOLD, 5 μm, 250 mm*10 mm, pre-column, 1PK, mobile phase: 0.2% trifluoroacetic acid aqueous solution / acetonitrile; flow rate: 7 mL / min; column equilibration time 5 min; mobile phase ratio: 0-20 min acetonitrile content 30-78% gradient separation, 20-22 min acetonitrile content 78-100% gradient separation, 22-25 min 100% acetonitrile isocratic), the separated solution is concentrated under reduced pressure to remove the organic phase, freeze-dried to obtain compound 12. 1 H NMR (600 MHz, DMSO-d6): δ 9.03 (t, J = 5.9 Hz, 1H), 8.99 (t, J = 5.5 Hz, 1H), 8.79 (s, 2H), 8.24 (t, J = 5.6 Hz, 1H), 8.17 (d, J = 8.5 Hz, 4H), 8.06 (d, J = 8.6 Hz, 2H), 7.62-7.55 (m, 2H), 6.81 (s, 2H), 3.95 (d, J = 5.8 Hz, 2H), 3.83 (q, J = 6.5 Hz, 2H), 3.61 (q, J = 6.9 Hz, 2H), 3.14-3.08 (m, 2H), 2.96 (t, J = 6.9 Hz, 2H), 2.90 (s, 4H), 2.86 (t, J = 6.9 Hz, 2H), 1.20-1.14 (m, 12H), 1.05-1.01 (m, 6H). 13 C NMR (151 MHz, DMSO-d6): δ 168.56, 164.21, 163.49, 159.66, 152.58, 142.10, 138.57, 133.58, 133.43, 128.50, 127.73, 126.57, 126.31, 124.90, 120.01, 116.43, 111.99, 108.00, 64.19, 54.37, 46.94, 44.08, 41.04, 40.50, 38.38, 36.44, 35.47, 35.14, 26.24, 26.12, 23.87, 20.89, 20.83, 16.90, 14.03, 6.95. LCMS: calcd for C 48 H 50 N6O 14 S4[M-H] - : 1061.23. Found, m / z, [M-H] - : 1061.56.

[0397] Synthesis method of compound 14:

[0398] Take a 500 mL flask, weigh compound 13 (10 g), dissolve in ethanol (50 mL), then add sodium bicarbonate (18.19 g), then dispense di-tert-butyl dicarbonate (15.75 g), 23°C, electromagnetic stirring for 5 hours. Filter the reaction solution, concentrate the filtrate under reduced pressure to obtain the crude product, dissolve in ethyl acetate (200 mL), wash successively with water (100 mL), saturated sodium chloride (50 mL), dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain compound 14.

[0399] Synthesis method of compound 6:

[0400] Take a 250 mL flask, weigh compound 14 (15 g), dissolve in methanol (120 mL), then add an aqueous solution of sodium hydroxide (5.9 g) (40 mL), 24°C, electromagnetic stirring for 4 hours. Concentrate the reaction solution under reduced pressure to remove methanol, adjust the pH of the aqueous phase to 4-5 with 2M aqueous hydrochloric acid solution, then extract with ethyl acetate (100 mL*4), wash the combined organic phase with saturated sodium chloride solution (50 mL), dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain compound 6. LCMS: calcd for C7H 13 NO4[M-H] - : 174.08. Found, m / z, [M-H] - : 174.09.

[0401] Example 2: Sequencing steps and sequencing results

[0402] Experimental raw materials:

[0403] Experimental steps:

[0404] 1. Dye preparation: prepared according to the standard preparation method of NHS terminal dye. Among them, since the reactive dye has a hydrophobic molecule, it needs to be dissolved in anhydrous dimethyl sulfoxide (DMSO).

[0405] 2. Fluorescent antibody labeling: The labeling reaction of amine on the antibody with the succinimidyl ester on the dye (such as AF532 S-S Linker3 NHS, AF532 S-S Linker4 NHS, AF532 S-S Linker5 NHS or AF532 S-S Linker6 NHS prepared in Example 1, and control dye) has pH dependence. The amine acylation reaction is usually carried out at a pH higher than 7.5. The reaction is usually carried out in 0.1-0.2M sodium bicarbonate buffer (pH 8.3) at room temperature for 1 hour. Gel filtration column (such as SephadexTM G-25, BioGel TM P-30 or equivalent column) to separate the labeled antibody from free Alexa Fluor TM (Alexa Fluor TM 532 NHS ester (succinimidyl ester) dye.

[0406] 3. CoolMPS PE50 sequencing: using CoolMPS high-throughput sequencing reagent kit series (MGISEQ-2000RS FCL PE100), combined with MGISEQ-2000RS platform, using Customize, PE50 read length sequencing was performed.

[0407] The experimental steps are briefly described in FIG. 5.

[0408] Experimental results:

[0409] The experimental results are shown in FIG. 6, FIG. 7, FIG. 8 or FIG. 9. As can be seen from the figures, the background signal of the control dye continues to increase cumulatively after 50 cycles, while the background signal of the modified dye prepared by the present disclosure does not accumulate after 50 cycles, and the background signal is basically flat. As can be seen from the results, compared with the control dye, the modified dye prepared by the present disclosure (such as AF532 S-S Linker3 NHS, AF532 S-S Linker4 NHS, AF532 S-S Linker5 NHS or AF532 S-S Linker6 NHS prepared in Example 1) has the effect of significantly inhibiting the continuous increase of the double-stranded background.

[0410] Although some specific embodiments of the present disclosure have been described in detail by examples, those skilled in the art should understand that the above examples are only for illustration, and are not intended to limit the scope of the present disclosure. Those skilled in the art should understand that the above embodiments can be modified without departing from the scope and spirit of the present disclosure. The scope of the present disclosure is defined by the appended claims.

Claims

1. The compound represented by Formula I, or its stereoisomer or salt thereof, H-[NH-CH2-C(=O)]n0-NH-(CH2-CH2-O) n1 -(CH2) n2 -(SS) n3 -L 1 -(CH2) n4 -(SS) n5 -(CH2) n6 -L 2 - C(=O)OH Formula I in: n0 is selected from 0, 1, 2, 3, 4, 5, 6; Preferably, n0 is selected from 0 and 1; n1 is selected from 0, 1, 2, 3, 4, 5, 6; Preferably, n1 is selected from 0, 1, 2, 3, and 4; More preferably, n1 is selected from 0, 1, and 2; n2 is selected from 0, 1, 2, 3, 4, 5, 6; Preferably, n2 is selected from 0, 1, 2, 3, and 4; More preferably, n2 is selected from 0, 1, and 2; Preferably, n2 is selected from 0 and 2; n3 is selected from 0 and 1; n4 is selected from 0, 1, 2, 3, 4, 5, 6; Preferably, n4 is selected from 0, 1, 2, 3, and 4; More preferably, n4 is selected from 0, 1, and 2; Preferably, n4 is selected from 0 or 2; n5 is selected from 0 and 1; n6 is selected from 1, 2, 3, 4, 5, 6; Preferably, n6 is selected from 1, 2, 3, and 4; More preferably, n6 is selected from 1 and 2; L 1 Selected from direct keys, m is selected from 1, 2, 3, 4, and 5; Preferably, m is selected from 1, 2, and 3; More preferably, m is 2; Preferably, L 1 Selected from direct keys, L 2 Selected from direct keys, R 1 R 2 R 3 R 4 Each is independently selected from hydrogen and C1-C6 alkyl groups; Preferably, R 1 R 2 R 3 R 4 Each is independently selected from hydrogen and C1-C4 alkyl groups; More preferably, R 1 R 2 R 3 R 4 All are hydrogen; Preferably, L 2 Selected from direct keys, And it satisfies the following condition: n3 and n5 are not both 0.

2. The compound of claim 1, or its stereoisomer or salt thereof, wherein, The compound has the structural formula I-1, NH2-(CH2). n2 -(SS) n3 -L 1 -(CH2) n4 -(SS) n5 -(CH2) n6 -L 2 -C(=O)OH Formula I-1 in: n2 is selected from 0, 1, 2, 3, and 4; Preferably, n2 is selected from 0, 1, and 2; More preferably, n2 is selected from 0 and 2; n3 is selected from 0 and 1; n4 is selected from 0, 1, 2, 3, and 4; Preferably, n4 is selected from 0, 1, and 2; More preferably, n4 is selected from 0 or 2; n5 is selected from 0 and 1; n6 is selected from 1, 2, 3, and 4; Preferably, n6 is selected from 1, 2, and 3; More preferably, n6 is 2; L 1 Selected from direct keys, m is selected from 1, 2, and 3; Preferably, m is 2; Preferably, L 1 Selected from direct keys, L 2 Selected from direct keys, R 1 R 2 R 3 R 4 Each is independently selected from hydrogen and C1-C6 alkyl groups; Preferably, R 1 R 2 R 3 R 4 Each is independently selected from hydrogen and C1-C4 alkyl groups; More preferably, R 1 R 2 R 3 R 4 All are hydrogen; Preferably, L 2 Selected from direct keys, And it satisfies the following condition: n3 and n5 are not both 0.

3. The compound or its stereoisomer or salt according to any one of claims 1-2, wherein, The compound is selected from:

4. Modified dyes, comprising: A fluorescent group; and a fluorescent modifying group, wherein the fluorescent modifying group is connected to the fluorescent group, and the fluorescent modifying group is a compound according to any one of claims 1-3 or a stereoisomer thereof or a salt thereof; Optionally, the modified dye further comprises a protecting group connected to the fluorescent modifying group.

5. The modified dye according to claim 4, wherein, The modified dye is a compound of formula II, its stereoisomer, or a salt thereof, Dye-LP formula II in: Dye is selected from Preferably, Dye is L is -[NH-CH2-C(=O)]n0-NH-(CH2-CH2-O) n1 -(CH2) n2 -(SS) n3 -L 1 -(CH2) n4 -(SS) n5 - (CH2) n6 -L 2 -C(=O)-; n0 is selected from 0, 1, 2, 3, 4, 5, 6; Preferably, n0 is selected from 0 and 1; n1 is selected from 0, 1, 2, 3, 4, 5, 6; Preferably, n1 is selected from 0, 1, 2, 3, and 4; More preferably, n1 is selected from 0, 1, and 2; n2 is selected from 0, 1, 2, 3, 4, 5, 6; Preferably, n2 is selected from 0, 1, 2, 3, and 4; More preferably, n2 is selected from 0, 1, and 2; Preferably, n2 is selected from 0 and 2; n3 is selected from 0 and 1; n4 is selected from 0, 1, 2, 3, 4, 5, 6; Preferably, n4 is selected from 0, 1, 2, 3, and 4; More preferably, n4 is selected from 0, 1, and 2; Preferably, n4 is selected from 0 or 2; n5 is selected from 0 and 1; n6 is selected from 1, 2, 3, 4, 5, 6; Preferably, n6 is selected from 1, 2, 3, and 4; More preferably, n6 is selected from 1 and 2; L 1 Selected from direct keys, m is selected from 1, 2, 3, 4, and 5; Preferably, m is selected from 1, 2, or 3; More preferably, m is 2; Preferably, L 1 Selected from direct keys, L 2 Selected from direct keys, R 1 R 2 R 3 R 4 Each is independently selected from hydrogen and C1-C6 alkyl groups; Preferably, R 1 R 2 R 3 R 4 Each is independently selected from hydrogen and C1-C4 alkyl groups; More preferably, R 1 R 2 R 3 R 4 All are hydrogen; Preferably, L 2 Selected from direct keys, And it satisfies the following condition: n3 and n5 are not both 0; P is selected from hydroxyl group, Preferably, P is selected from hydroxyl groups, More preferably, P is 6. The modified dye according to claim 5, wherein, In the compound represented by Formula II, L is -NH-(CH2). n2 -(SS) n3 -L 1 -(CH2) n4 -(SS) n5 -(CH2) n6 -L 2 -C(=O)-, n2 is selected from 0, 1, 2, 3, and 4; Preferably, n2 is selected from 0, 1, and 2; More preferably, n2 is selected from 0 and 2; n3 is selected from 0 and 1; n4 is selected from 0, 1, 2, 3, and 4; Preferably, n4 is selected from 0, 1, and 2; More preferably, n4 is selected from 0 or 2; n5 is selected from 0 and 1; n6 is selected from 1, 2, 3, and 4; Preferably, n6 is selected from 1, 2, and 3; More preferably, n6 is 2; L 1 Selected from direct keys, m is selected from 1, 2, and 3; Preferably, m is 2; Preferably, L 1 Selected from direct keys, L 2 Selected from direct keys, R 1 R 2 R 3 R 4 Each is independently selected from hydrogen and C1-C6 alkyl groups; Preferably, R 1 R 2 R 3 R 4 Each is independently selected from hydrogen and C1-C4 alkyl groups; More preferably, R 1 R 2 R 3 R 4 All are hydrogen; Preferably, L 2 Selected from direct keys, And it satisfies the following condition: n3 and n5 are not both 0.

7. The modified dye according to any one of claims 5-6, wherein, In the compounds represented by Formula II, L is selected from:

8. The modified dye according to any one of claims 5-7, wherein, The compound represented by Formula II is selected from:

9. Antibody-conjugated dyes, comprising: Antibodies; and The dye, wherein the antibody is linked to the dye, and the dye is a modified dye as described in any one of claims 4-8.

10. Modified nucleotides, comprising: Nucleotides, and A modified dye, wherein the modified dye binds to the nucleotide via an affinity reagent, the modified dye is linked to the affinity reagent, and the modified dye is as defined in any one of claims 4-8; Preferably, the affinity reagent is capable of specifically recognizing and binding to the epitopes of the nucleotide molecule; More preferably, the affinity reagent is selected from antibodies, aptamers, affinity compounds (Affimer), and desmin (Knottin); Most preferably, the affinity reagent is an antibody.

11. The modified nucleotide of claim 10, wherein, The nucleotides comprise: Ribose or deoxyribose; Phosphoric acid, wherein the phosphate is attached to the ribose or deoxyribose; A base, wherein the base is linked to the ribose or deoxyribose; and A reversible blocking group, wherein the reversible blocking group is attached to the ribose or deoxyribose, or wherein the reversible blocking group is attached to the base.

12. Methods for controlling nucleic acid synthesis, including: The modified nucleotides as described in claim 11 are incorporated into the nucleic acid molecule to be synthesized; Alternatively, it may include: sequentially incorporating a nucleotide with a reversible blocking group and the antibody-conjugated dye of claim 9 into the nucleic acid molecule to be synthesized; Preferably, the incorporation of the modified nucleotide and / or the incorporation of the nucleotide with the reversible blocking group is achieved by polymerase; Preferably, the method includes: using a polymerase to incorporate the modified nucleotide into the nucleic acid molecule to be synthesized; Alternatively, preferably, the method comprises: using a polymerase to incorporate the nucleotide with the reversible blocking group into the nucleic acid molecule to be synthesized; and then incorporating the antibody-conjugated dye of claim 9 into the nucleic acid molecule to be synthesized; Preferably, the method includes: using a polymerase to perform a nucleotide polymerization reaction under conditions that allow the polymerase to perform a nucleotide polymerization reaction, thereby incorporating the modified nucleotide into the 3' end of the nucleic acid molecule to be synthesized; Alternatively, preferably, the method comprises: performing a nucleotide polymerization reaction using a polymerase under conditions that allow the polymerase to perform a nucleotide polymerization reaction, thereby incorporating the nucleotide with the reversible blocking group into the 3' end of the nucleic acid molecule to be synthesized; and then incorporating the antibody-conjugated dye of claim 9 into the nucleic acid molecule to be synthesized. Preferably, the method further includes: removing the reversible blocking group and affinity reagent carried by the incorporated modified nucleotide and the modified dye attached to the affinity reagent, and carrying out the next round of nucleotide polymerization reaction; Alternatively, preferably, the method further includes: removing the reversible blocking group from the incorporated nucleotide with the reversible blocking group and the incorporated antibody-conjugated dye, and then proceeding to the next round of nucleotide polymerization.

13. A method for preparing a growing polynucleotide complementary to a target polynucleotide in a sequencing reaction, comprising a nucleotide polymerization reaction, incorporating the modified nucleotide of claim 11 into the growing complementary polynucleotide, or, sequentially incorporating a nucleotide with a reversible blocking group and the antibody-conjugated dye of claim 9 into the growing complementary polynucleotide, wherein, The incorporation of the modified nucleotide or the incorporation of the nucleotide with the reversible blocking group ensures that only one nucleotide molecule is polymerized in one round of polymerization. Preferably, the incorporation of the modified nucleotide and / or the incorporation of the nucleotide with the reversible blocking group is achieved by polymerase; Preferably, the method includes: using a polymerase to incorporate modified nucleotides into the grown complementary polynucleotides. middle; Alternatively, preferably, the method comprises: using a polymerase to incorporate the nucleotide with a reversible blocking group into the growing complementary polynucleotide; and then incorporating the antibody-conjugated dye of claim 9 into the growing complementary polynucleotide. Preferably, the method includes: performing a nucleotide polymerization reaction using a polymerase under conditions that allow the polymerase to perform a nucleotide polymerization reaction, thereby incorporating the modified nucleotide into the 3' end of the grown complementary polynucleotide; Alternatively, preferably, the method comprises: performing a nucleotide polymerization reaction using a polymerase under conditions that allow the polymerase to perform a nucleotide polymerization reaction, thereby incorporating the nucleotide with a reversible blocking group into the 3' end of the grown complementary polynucleotide; and then incorporating the antibody-conjugated dye of claim 9 into the grown complementary polynucleotide.

14. Nucleic acid intermediates, which are formed during the sequencing of the target polynucleotide, wherein, The nucleic acid intermediate is formed through the following steps: A complementary nucleotide to the target polynucleotide is incorporated into the growing nucleic acid chain to form the nucleic acid intermediate, wherein the incorporated complementary nucleotide is the modified nucleotide as described in claim 11; Alternatively, a nucleotide complementary to the target polynucleotide and the antibody-conjugated dye of claim 9 may be sequentially incorporated into the growing nucleic acid chain to form the nucleic acid intermediate, wherein the nucleotide carries a reversible blocking group. Alternatively, the nucleic acid intermediate is formed through the following steps: A complementary nucleotide to a target polynucleotide is incorporated into a growing nucleic acid chain to form the nucleic acid intermediate, wherein the incorporated complementary nucleotide is the modified nucleotide of claim 11, and at least one complementary nucleotide to the target polynucleotide is pre-incorporated into the growing nucleic acid chain, wherein the pre-incorporated at least one complementary nucleotide to the target polynucleotide is the modified nucleotide of claim 11, having had the reversible blocking group and affinity reagent and the modified dye linked to the affinity reagent removed; Alternatively, a nucleotide complementary to the target polynucleotide and the antibody-conjugated dye of claim 9 are sequentially incorporated into the growing nucleic acid chain to form the nucleic acid intermediate, wherein the incorporated complementary nucleotide has a reversible blocking group, and at least one nucleotide complementary to the target polynucleotide is pre-incorporated into the growing nucleic acid chain, the pre-incorporated at least one nucleotide complementary to the target polynucleotide being a nucleotide from which the reversible blocking group and the antibody-conjugated dye have been removed.

15. A method for determining the sequence of a target polynucleotide, comprising: 1) Detecting the incorporation of a nucleotide complementary to the target polynucleotide in the grown nucleic acid chain, wherein at least one complementary nucleotide incorporated is the modified nucleotide of claim 11 or a nucleotide with a reversible blocking group, and the nucleotide with the reversible blocking group is bound to the antibody-conjugated dye of claim 9, and, 2) Determine the type of nucleotide incorporated; Preferably, the incorporation of the nucleotide complementary to the target polynucleotide is achieved by polymerase; Preferably, before introducing the next complementary nucleotide, the reversible blocking group and affinity reagent carried by the incorporated complementary nucleotide, as well as the modifying dye attached to the affinity reagent, are removed. Alternatively, preferably, the reversible blocking group carried by the incorporated complementary nucleotide and the bound antibody-conjugated dye are removed before the next complementary nucleotide is introduced. Preferably, the reversible blocking group, the affinity reagent, and the modified dye attached to the affinity reagent are removed simultaneously; Alternatively, preferably, the reversible blocking group and the antibody-conjugated dye are removed simultaneously.

16. The method of claim 15, further comprising the steps of: (a) Provides a variety of different nucleotides, wherein at least one nucleotide is the modified nucleotide of claim 11; (b) Incorporating the various different nucleotides into the complementary sequence of the target polynucleotide. (c) Detect the nucleotides obtained in step (b) to determine the type of incorporated nucleotides; (d) Remove the reversible blocking groups and affinity reagents carried by the nucleotides obtained in step (b), as well as the modifying dyes attached to the affinity reagents; and (e) Optionally repeat steps (a)-(d) once or more; This allows for the determination of the sequence of the target polynucleotide; Alternatively, it may include the following steps: (a) Provides a variety of different nucleotide and antibody-conjugated dyes, wherein the nucleotide is a nucleotide with a reversible blocking group, and the antibody-conjugated dye is as described in claim 9; (b) The various different nucleotides and the antibody-conjugated dye are sequentially incorporated into the target polynucleotide. In complementary sequences, (c) Detect the nucleotides obtained in step (b) to determine the type of incorporated nucleotides; (d) Remove the reversible blocking groups and antibody-conjugated dyes carried by the nucleotides obtained in step (b); and (e) Optionally repeat steps (a)-(d) once or more; This allows for the determination of the sequence of the target polynucleotide; Preferably, the incorporation of the various nucleotides is achieved by polymerase.

17. The method of claim 15, further comprising the steps of: (1) Provide a first nucleotide, a second nucleotide, a third nucleotide and a fourth nucleotide, wherein at least one of the four nucleotides is the modified nucleotide as described in claim 11; (2) Contact the four nucleotides with the target polynucleotide; remove the nucleotides not incorporated into the grown nucleic acid chain; detect the nucleotides incorporated into the grown nucleic acid chain; remove the reversible blocking groups and affinity reagents carried by the nucleotides incorporated into the grown nucleic acid chain and the modifying dyes linked to the affinity reagents; Optionally, it also includes (3): repeating (1)-(2) once or more; Alternatively, it may include the following steps: (1) Provides a first nucleotide, a second nucleotide, a third nucleotide and a fourth nucleotide and an antibody-conjugated dye, wherein each of the four nucleotides contains a reversible blocking group, and the antibody-conjugated dye is as described in claim 9; (2) Contact the four nucleotides with the target polynucleotide; remove the nucleotides that are not incorporated into the growing nucleic acid chain; contact the antibody-conjugated dye with the target polynucleotide; remove the unbound antibody-conjugated dye; detect the nucleotides incorporated into the growing nucleic acid chain; remove the reversible blocking groups carried by the nucleotides incorporated into the growing nucleic acid chain and the bound antibody-conjugated dye. Optionally, it also includes (3): repeating (1)-(2) once or more; Preferably, the contacting of the four nucleotides with the target polynucleotide is carried out in the presence of a polymerase.

18. The method of claim 15, further comprising the following steps: (a) Providing a double strand, a nucleotide comprising at least one modification as described in claim 11, a polymerase, and A mixture of excision reagents; the duplex comprises a grown nucleic acid strand and a nucleic acid strand to be sequenced; (b) Perform the reaction comprising the steps (i), (ii) and (iii), optionally repeating it once or more: Step (i): Using a polymerase, the modified nucleotide is incorporated into the growing nucleic acid chain to form a nucleic acid intermediate containing a reversible blocking group, an affinity reagent, and a modified dye attached to the affinity reagent. Step (ii): Detect the nucleic acid intermediate; Step (iii): Use an excision reagent to excise the reversible blocking group and affinity reagent contained in the nucleic acid intermediate, as well as the modifying dye attached to the affinity reagent; Preferably, the same reagent is used for the excision of the reversible blocking group and the excision of the affinity reagent and the modified dye attached to the affinity reagent; Alternatively, it may include the following steps: (a) Provides a mixture comprising a duplex, a nucleotide with a reversible blocking group, an antibody-conjugated dye, a polymerase, and an excision reagent; said duplex comprising a grown nucleic acid strand and a nucleic acid strand to be sequenced; said antibody-conjugated dye as described in claim 9; (b) Perform the reaction comprising the steps (i), (ii) and (iii), optionally repeating it once or more: Step (i): Using a polymerase, the nucleotide with the reversible blocking group is incorporated into the growing nucleic acid chain, followed by incorporation of the antibody-conjugated dye into the growing nucleic acid chain, ultimately forming a nucleic acid intermediate containing the reversible blocking group and the antibody-conjugated dye. Step (ii): Detect the nucleic acid intermediate; Step (iii): Use an excision reagent to excise the reversible blocking group and antibody-conjugated dye contained in the nucleic acid intermediate; Preferably, the same excision reagent is used for the excision of the reversible blocking group and the excision of the antibody-conjugated dye.

19. The method according to claim 18, wherein, The double chain is fixed to the support; Preferably, the grown nucleic acid chain is a primer; Preferably, the primers are annealed to the nucleic acid strand to be sequenced to form the double strand; Preferably, the double strand, the modified nucleotide, and the polymerase together form a reaction system; Alternatively, preferably, the double strand, the nucleotide with a reversible blocking group, the antibody-conjugated dye, Together with polymerase, they form a reaction system; Preferably, under conditions that allow the polymerase to perform nucleotide polymerization, the polymerase is used to incorporate the modified nucleotide into the growing nucleic acid chain to form a nucleic acid intermediate comprising a reversible blocking group, an affinity reagent, and a modified dye linked to the affinity reagent. Alternatively, preferably, under conditions that allow polymerase to perform nucleotide polymerization, polymerase is used to incorporate the nucleotide with the reversible blocking group into the growing nucleic acid chain, and then the antibody-conjugated dye is incorporated into the growing nucleic acid chain, ultimately forming a nucleic acid intermediate containing the reversible blocking group and the antibody-conjugated dye. Preferably, before any step of detecting the nucleic acid intermediate, the solution phase of the reaction system in the previous step is removed, leaving the double strands immobilized on the support; Preferably, the excision reagent is in contact with the duplex or the grown nucleic acid strand in the reaction system; Preferably, the excision reagent is capable of excising the reversible blocking group and affinity reagent carried by the modified nucleotide incorporated into the growing nucleic acid chain, as well as the modified dye attached to the affinity reagent, without affecting the phosphodiester bonds on the double-stranded backbone. Alternatively, preferably, the excision reagent is capable of excising the reversible blocking groups carried by the nucleotides incorporated into the growing nucleic acid chain and the bound antibody-modified dyes, without affecting the phosphodiester bonds on the double-stranded backbone. Preferably, after any step of removing the reversible blocking group and affinity reagent contained in the nucleic acid intermediate and the modified dye attached to the affinity reagent, the solution phase of the reaction system in this step is removed; Alternatively, preferably, after any step of removing the reversible blocking group and antibody-conjugated dye contained in the nucleic acid intermediate, the solution phase of the reaction system in that step is removed; Preferably, after step (ii), the method further includes: determining the type of nucleotide modified by the nucleic acid chain incorporated into the growing nucleic acid chain in step (i) based on the signal detected in step (ii), and determining the type of nucleotide at the corresponding position in the nucleic acid chain to be sequenced based on the base complementary pairing principle; Alternatively, preferably, after step (ii), the method further includes: determining the type of nucleotide with reversible blocking groups incorporated into the grown nucleic acid chain in step (i) based on the signal detected in step (ii), and determining the type of nucleotide at the corresponding position in the nucleic acid chain to be sequenced based on the base complementary pairing principle.

20. A kit comprising the compound of any one of claims 1-3 or its stereoisomer or salt thereof, or comprising the modified dye of any one of claims 4-8, or comprising the antibody-conjugated dye of claim 9, or comprising the modified nucleotide of any one of claims 10-11.

21. The kit according to claim 20, wherein, The kit further comprises: reagents for processing nucleic acid molecules; primers for initiating nucleotide polymerization; polymerase for performing nucleotide polymerization; one or more buffer solutions; one or more washing solutions; or any combination thereof.

22. Use of the compound of any one of claims 1-3 or its stereoisomer or salt thereof, or the modified dye of any one of claims 4-8, or the antibody-conjugated dye of claim 9, or the modified nucleotide of any one of claims 10-11, or the kit of any one of claims 20-21 for determining the sequence of a target polynucleotide.

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