Nucleoside molecule with 3 '-hydroxyl blocking group as well as preparation method and application thereof

Abstract

The invention discloses a nucleoside molecule with a 3 '-hydroxyl blocking group as well as a preparation method and application of the nucleoside molecule. The nucleoside molecule has a structure as shown in a formula (I). The nucleoside molecule provided by the invention has better stability in a solution, and also has good stability in the solution during preparation and storage periods. The preparation and synthesis steps are few, the synthesis cost is low, and the cutting reagent is green and environment-friendly. Nucleoside molecules of the present invention are readily incorporated into enzymes as reversible terminators during sequencing, said 3 '-hydroxy blocking groups also enabling low predetermined phases, lower signal attenuation to improve data quality, which makes it possible to make longer reads from sequencing applications.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a nucleoside molecule with a 3'-hydroxyl blocking group and a preparation method and application thereof. Background Art

[0002] DNA sequencing is a basic method for biological and pharmaceutical research. To sequence nucleic acid molecules, sequencing by synthesis (SBS) or sequencing by ligation (SBL) can be used. Currently, most of the SBS nucleic acid sequencing platforms on the market, including second-generation and third-generation sequencing platforms, use DNA polymerase to add nucleotide analogs to the template (base extension) based on the principle of base complement pairing, and detect the signals emitted by the nucleic acid analogs after binding to the template or the changes in the physical and chemical signals brought about to achieve the determination of the nucleic acid sequence. Obtaining nucleotide analogs with stable structures, stable detectable signals, high reaction efficiency, and the ability to effectively inhibit the binding of the next position nucleotide to the template has always been a difficult problem to be solved or improved.

[0003] To ensure that only a single incorporation occurs, each labeled nucleotide (which is added to the growing chain to ensure that only one nucleotide is incorporated) includes a structural modification (a protecting group or blocking group). After the nucleotide with the protecting group is added, the protecting group is removed under reaction conditions that do not interfere with the integrity of the DNA being sequenced. The sequencing cycle can then continue with the incorporation of the next protected, labeled nucleotide.

[0004] In order to be suitable for DNA sequencing, nucleotides, usually nucleoside triphosphates, generally need 3'-hydroxyl protecting groups, to prevent the polymerase used to incorporate the nucleotide into the polynucleotide chain from continuing to replicate once the base on the nucleotide is added. There are many restrictions on the group type that can be added to the nucleotide and is still suitable. The protecting group should prevent other nucleotide molecules from being added to the polynucleotide chain, and it is easy to remove and not cause damage to the nucleotide chain. In addition, the modified nucleotide needs to be compatible with polymerase or another suitable enzyme for incorporating it into the polynucleotide chain. Therefore, the ideal protecting group must show long-term stability, can be effectively incorporated by polymerase, prevent nucleotide secondary incorporation or further incorporation, and have the ability to be removed under mild conditions (preferably under aqueous conditions) without destroying the polynucleotide structure.

[0005] Reversible protecting groups have been described previously. The use of ddNTPs as chain terminators in Sanger sequencing provided the basis for the initial development of reversible blocking groups attached to the 3'-end of nucleotides (Metzker et al., 1994; Canard and Sarfati, 1994). For example, Metzker et al. (Nucleic Acids Research, 22(20):4259-4267, 1994) disclosed the synthesis and use of eight 3'-modified 2-deoxyribonucleoside 5'-triphosphates (3'-modified dNTPs) and tested their incorporation activities in two DNA template assays. For example, the blocking groups of 3'-O-allyl-dNTP (Metzker et al., 1994; U.S. Patent 6,664,079; Ju et al., 2006; U.S. Patent 7,057,026; U.S. Patent 7,345,159; U.S. Patent 7,635,578; U.S. Patent 7,713,698) and 3'-O-azidomethyl-dNTP (U.S. Patent 7,057,026; Guo et al., 2008; Bentley et al., 2008; U.S. Patent 7,414,116; U.S. Patent 7,541,444; U.S. Patent 7,592,435; U.S. Patent 7,771,973) have been applied to SBS. The development of many reversible protecting groups and methods for deprotecting them under DNA-compatible conditions have been reported in these patents.

[0006] a. There are many synthesis steps and high costs. Some reversible blocking nucleotides require the use of hazardous chemicals during synthesis. For example, sodium azide (NaN3) is inevitably used in the synthesis of 3'-O-azidomethyl-dNTP, and sodium azide is highly toxic and explosive, and strict production operations are required (refer to Figure 1 ).

[0007] b. The cleavage reagents for the blocking groups are expensive or environmentally polluting. For example, the cleavage reagent for 3'-O-azidomethyl-dNTP requires tris(carboxyethyl)phosphine (TCEP). TCEP is a common phosphate ester that causes water pollution. Since TCEP is difficult to biodegrade and photodegrade, improper treatment will have an adverse impact on the ecosystem and human health if it exists in nature for a long time; therefore, the waste liquid of this cleavage reagent requires cumbersome post-treatment. For example, the cleavage reagent for 3'-O-allyl-dNTP requires Pd(II) complex and tris(hydroxypropyl)phosphine (THP). THP is also a phosphate ester that causes water pollution, so the cleaved reagent also needs to be properly post-treated; in addition, the cleavage reagent also uses precious metal Pd as a catalyst, which undoubtedly increases the application cost of this nucleotide (refer to Figure 2 ).

[0008] c. Reversible blocking nucleotides mentioned in the prior art, such as 3'-O-azidomethyl-dNTP, 3'-O-allyl-dNTP, and 3'-O-amino-dNTP, have mediocre stability and need to be stored at least below -20 °C to maintain stability for a certain period, thus increasing the storage and sample transportation costs and also affecting the read length of nucleotide sequencing.

[0009] Next-generation sequencing (NGS) technologies have facilitated many important biomedical discoveries. However, due to various reasons below, chemical improvements are still needed, including reducing the preparation cost and application cost of nucleotides, reducing environmental pollution, improving the stability of nucleotides to reduce the error rate, and increasing the sequencing length. To be effectively applied in NGS methods, reversible terminators are generally expected to exhibit multiple desirable properties. Therefore, new nucleosides and nucleotides need to be developed to meet these challenges. Summary of the Invention

[0010] The technical problem to be solved by the present invention is the defect in the prior art of lacking stable and safe reversible terminators for sequencing. The present invention provides a nucleoside molecule with a 3'-hydroxy blocking group, a preparation method thereof, and an application thereof. The nucleoside molecule of the present invention has better stability in solution and also has good stability in solution during formulation and storage. Moreover, the preparation synthesis steps are few, the synthesis cost is low, and the cleavage reagent is green and environmentally friendly. During the sequencing process, the nucleoside molecule of the present invention is easily incorporated by enzymes as a reversible terminator, and the 3'-hydroxy blocking group can also achieve a low predetermined phase and lower signal attenuation to improve data quality, which enables longer reads to be obtained from sequencing applications.

[0011] The present invention solves the above technical problems through the following technical solutions.

[0012] The first aspect of the present invention provides a nucleoside molecule having a structure shown in formula (I):

[0013]

[0014] Wherein, R' is H, a mono-phosphoryl group, a di-phosphoryl group, a tri-phosphoryl group, or a phosphoryl group analogue;

[0015] R" is H, -OH, a halogen, -CN, -NO2, -N3, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted carbocyclic group, an optionally substituted heterocyclic group, an optionally substituted acyl group, an optionally substituted amino group, or an optionally substituted thiol group;

[0016] Base is a nucleobase, and the nucleobase is optionally substituted with an N-protecting group or operably linked to a detectable label molecule;

[0017] Each R 1a and R 1b are independently H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, cyano, halogen, optionally substituted aryl or optionally substituted aralkyl;

[0018] Each R 2a , R 2b and R 2c are independently H, C1-C6 alkyl, C1-C6 haloalkyl, cyano or halogen; or, R 1a and R 2a together with the atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclic group.

[0019] In the present invention, the group attached to the 3'-hydroxy group of the sugar ring of the nucleoside molecule is the 3'-hydroxy blocking group, including R 1a , R 1b , R 2a , R 2b and R 2c and S.

[0020] In some embodiments, the phosphoacyl analog contains a modified phosphodiester linkage, such as phosphorothioate, dithiophosphate, C1-C6 alkyl phosphate, anilino phosphate or amino phosphate.

[0021] In some embodiments, R' is a mono-phosphoacyl, di-phosphoacyl or tri-phosphoacyl.

[0022] In some embodiments, the nucleobase is a nitrogenous base or a denitrogenated base; the nucleobase is preferably a purine, pyrimidine or denitrogenated purine; the nucleobase is for example adenine, guanine, cytosine, thymine or uracil.

[0023] In some embodiments, R” is H, -OH, halogen, -CN, -NO2, -N3, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclic group, optionally substituted heterocyclic group, optionally substituted acyl, optionally substituted amino or optionally substituted mercapto group; preferably, the “substituted” means substituted by one or more substituents independently selected from the following: halogen, -CN, hydroxy, amino, nitro, sulfonyl, -SO3H, sulfinyl, oxo, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, C3-C7 carbocyclic group, C3-C7 carbocyclic group-(C1-C6) alkylene, C1-C6 alkoxy-(C1-C6) alkylene, C6-C 10 aryl, C6-C 10 aryl-(C1-C6) alkylene, 5-10 membered heteroaryl, 5-10 membered heteroaryl-(C1-C6) alkylene, C1-C6 alkoxy-(C1-C6) alkylene, C6-C 10 aryloxy, mercapto, C1-C6 alkylthio, C6-C 10 arylthio, or amino-(C1-C6) alkylene.

[0024] In some embodiments, the aralkyl may be C6-C 10 aryl-substituted C1-C6 alkyl; such as benzyl, 2-phenylethyl, 3-phenylpropyl and naphthylmethyl.

[0025] In some embodiments, the aryl may be phenyl, naphthyl, azulenyl or anthracenyl.

[0026] In some embodiments, the C1-C6 alkyl may be methyl, ethyl, isopropyl or tert-butyl.

[0027] In some embodiments, the C1-C6 haloalkyl may be trifluoromethyl, trichloromethyl, pentafluoroethyl or pentachloroethyl.

[0028] In some embodiments, each R 1a and R 1b is independently H or C1-C6 alkyl.

[0029] In some specific embodiments, R’ is triphosphoacyl.

[0030] In some specific embodiments, R” is H.

[0031] In some embodiments, at least one of R 1a and R 1b is H, for example R 1aand R 1b are both H.

[0032] In some embodiments, R 1a and R 1b at least one of which is a C1-C6 alkyl group.

[0033] In some embodiments, R 2a , R 2b and R 2c can independently be H, a C1-C6 alkyl group or a halogen.

[0034] In some embodiments, R 2a , R 2b and R 2c at least one of which is H, a C1-C6 alkyl group or a halogen; for example, R 2a , R 2b and R 2c are both H.

[0035] In some embodiments, the nucleobase is operably linked to a detectable label molecule; the operable linkage is achieved, for example, through a linking group.

[0036] In some specific embodiments, the nucleobase is selected from

[0037] In some embodiments, the nucleobase is a nucleobase operably linked to a detectable label molecule; the operable linkage is preferably a covalent linkage; the detectable label molecule can be a fluorescent dye molecule, a chemiluminescent molecule or a dye conjugate.

[0038] In some embodiments, the fluorescent dye molecule is preferably selected from coumarin, Alexa Fluor, Bodipy, fluorescein, tetramethylrhodamine, Cy3, Cy5, Texas Red and their derivatives.

[0039] In some embodiments, the fluorescent dye molecule is selected from

[0040] In some embodiments, the chemiluminescent molecule is preferably selected from acridinium esters, acridinium sulfonamides, luminol, isoluminol, coelenterazine, ruthenium terpyridine and dioxetane and their derivatives;

[0041] In some embodiments, the dye-conjugate refers to a molecule in which a dye molecule is linked to a biological or non-biological component. Such a biological or non-biological component is also referred to as the “conjugated molecule.” Examples of dye-conjugates include, but are not limited to, conjugates of antigens, antibodies, proteins, peptides, haptens.

[0042] In some embodiments, the covalent linkage can be made through a linking group, which can be a cleavable or non-cleavable linking group. Preferably, the linking group is a cleavable linking group. Using a cleavable linking group ensures that, if desired, the labeled molecule can be removed after detection, thus avoiding any interference signals with any labeled nucleotides or nucleosides incorporated subsequently.

[0043] The 3′-hydroxy blocking group and the cleavable linking group (and the linked label) can be removed under the same or substantially the same chemical reaction conditions. For example, the blocking group and the detectable label can be removed in a single chemical reaction. In other embodiments, the blocking group and the detectable label are removed in two separate steps.

[0044] In some further embodiments, the cleavable linking group comprises one or more of the following groups: azide, -O-C2-C6 alkenylene- (e.g., -O-allylidene-), disulfide, -methio-(C1-C6) alkylene-, 2-nitrobenzylidene or 4-nitrobenzylidene.

[0045] In some specific embodiments, the linking group is:

[0046] wherein X is -CH2-, -O- or -S-; Z is azide, phenylboronic acid pinacol ester, –O-C1-C6 alkyl, –O-C2-C6 alkenyl, –O-C2-C6 alkynyl, –S-C1-C6 alkyl, –S-C2-C6 alkenyl or –S-C2-C6 alkynyl; and n is 1, 2, 3, 4, or 5.

[0047] For more examples of linking groups, see US2016 / 0040025A1, WO2020 / 136170A1, which are incorporated herein by reference.

[0048] In some embodiments, when the detectable label molecule is a fluorescent dye molecule, the linking group can be selected from

[0049]

[0050] Wherein, Z is an azido group, –O-C1-C6 alkyl, –O-C2-C6 alkenyl or –O-C2-C6 alkynyl; n is 1, 2, 3, 4, or 5.

[0051] In some embodiments, when the detectable label molecule is a fluorescent dye molecule or a chemiluminescent molecule, Base can be represented as:

[0052] Wherein L is the linking group.

[0053] In some embodiments, the detectable label molecule may comprise a fluorescent label. Further, the detectable label molecule may further comprise an additional linking group structure. Those of ordinary skill in the art will understand that the label is covalently bonded to the linking group through the reaction of the functional group of the label (e.g., carboxyl group) with the functional group of the linking group (e.g., amino group).

[0054] In some embodiments, the detectable label molecule may be linked to the 3'-oxygen of the nucleoside molecule.

[0055] In some specific embodiments, the nucleoside molecule is a nucleoside triphosphate.

[0056] In some specific embodiments, the nucleoside molecule is

[0057] In some specific embodiments, the nucleoside molecule is

[0058]

[0059] In some specific embodiments, the nucleoside molecule is

[0060]

[0061] In any of the embodiments of the blocking groups described herein, when a group is described as "optionally substituted", it may be unsubstituted or substituted.

[0062] In any embodiment of the nucleotides or nucleosides having a 3'-hydroxy blocking group described herein, the nucleoside or nucleotide may be covalently linked to a detectable label molecule (such as a fluorophore or luminophore), optionally via a linking group. The linking group may be cleavable or non-cleavable. In some such embodiments, the detectable label (e.g., fluorophore) is covalently linked to the nucleobase of the nucleoside or nucleotide via a cleavable linking group. In some other embodiments, the detectable label (e.g., fluorophore) is covalently linked to the 3'-oxygen of the nucleoside or nucleotide via a cleavable linking group. In some embodiments, the 3'-hydroxy blocking group and the cleavable linking group (and the linked label) may be removed under the same or substantially the same chemical reaction conditions, e.g., the 3'-hydroxy blocking group and the detectable label may be removed in a single chemical reaction. In other embodiments, the 3'-hydroxy blocking group and the detectable label are removed in two separate steps.

[0063] A second aspect of the present invention provides a kit or composition, which comprises a nucleoside molecule as described in the first aspect.

[0064] In some embodiments, the kit or composition further comprises a template-dependent DNA polymerase such as KOD polymerase, a non-template-dependent DNA polymerase such as terminal deoxynucleotidyl transferase, and / or a reverse transcriptase.

[0065] A third aspect of the present invention provides a method for labeling a nucleic acid molecule, which comprises incorporating a nucleoside molecule as described in the first aspect into the nucleic acid molecule, wherein the Base in the nucleoside molecule is a nucleobase for linking a detectable label molecule.

[0066] A fourth aspect of the present invention provides a method for regulating nucleic acid synthesis, which comprises:

[0067] 1) adding a nucleoside molecule as described in the first aspect or a kit or composition as described in the second aspect to a reaction system for nucleic acid synthesis to block the extension of the nucleic acid chain;

[0068] Or,

[0069] 2) adding a cleavage reagent to a reaction system for nucleic acid synthesis containing a nucleoside molecule as described in the first aspect to allow the extension of the nucleic acid chain;

[0070] The cleavage reagent undergoes a cleavage reaction with the nucleoside molecule to convert the nucleoside molecule into a structure as shown in formula (II):

[0071] wherein the definitions of R', R" and Base are as described in the first aspect.

[0072] In some embodiments, the nucleic acid strand is single-stranded. In other embodiments, the nucleic acid strand is double-stranded. In still other embodiments, the nucleic acid strand comprises single-stranded nucleic acid and double-stranded nucleic acid.

[0073] In some embodiments, the method satisfies one or more of the following conditions:

[0074] (1) The nucleic acid synthesis is enzyme-catalyzed nucleic acid amplification;

[0075] (2) The cleavage reagent is selected from hydrogen peroxide, lanthanide metal salts, and bromides;

[0076] (3) The cleavage reagent further comprises an oxidizing agent.

[0077] In some embodiments, the method satisfies one or more of the following conditions:

[0078] (1) The lanthanide metal salts and bromides are selected from CeBr3, Ce(NO3)3-KBr, CeCl3-KBr, Ce(NH4)2(NO3)6-KBr, Ce(OTf)3-KBr, Ce(OAc)3-KBr, and combinations thereof;

[0079] (2) The oxidizing agent is 0.1%-30% (v / v) H2O2, NaIO4, Oxone, O2+UV, or NaI;

[0080] (3) The cleavage reaction refers to converting the group at the 3'-end of the sugar ring of the nucleoside molecule into a hydroxyl group at 25-65°C, for example, using CeBr3 as a catalyst and H2O2 as an oxidizing agent; preferably, the temperature of the cleavage reaction is preferably 40-65°C, such as 50°C;

[0081] (4) The molar ratio of the nucleoside molecule to the catalyst is 1:0.01-0.5;

[0082] (5) The molar ratio of the nucleoside molecule to the oxidizing agent is 1:1-50;

[0083] (6) The solvent in the reaction system is selected from acetonitrile, ethanol, tetrahydrofuran, water, and combinations thereof;

[0084] (7) The reaction time of the reaction system is 3-120 min.

[0085] In some preferred embodiments, after removing the group at the 3'-position of the nucleoside molecule, it further includes quenching with a Na2S2O3 solution.

[0086] In the present invention, the cleavage reagent uses CeBr3 as a catalyst and H2O2 as an oxidizing agent, which is inexpensive, easily available, has mild conditions, and is easy to operate, and is a very green and effective cleavage reagent at present.

[0087] An example of the deprotection in the present invention is that when the 3'-hydroxy blocking group is 3'-O-methylthiomethyl and the Base is dNTP, at room temperature, using CeBr3 as a catalyst and H2O2 as an oxidant, 3'-O-methylthiomethyl-dNTP can be converted into 3'-OH nucleotide or nucleoside molecule methylthiomethylene in a short time.

[0088] The 3'-O-methylthiomethylene blocking group described herein can be removed or cleaved under various chemical conditions. For

[0089] the blocking group, the non-limiting cleavage conditions include hydrogen peroxide, lanthanide metal salts and bromides, and the lanthanide metal salts and bromides include any one of the combinations of metals and bromides such as CeBr3, Ce(NO3)3-KBr, CeCl3-KBr, Ce(NH4)2(NO3)6-KBr, Ce(OTf)3-KBr, Ce(OAc)3-KBr, etc.

[0090] In some preferred embodiments, after removing the group at the 3'-position of the nucleoside molecule, it further includes quenching with a Na2S2O3 solution.

[0091] In some embodiments, the reaction system satisfies one or more of the following conditions:

[0092] (1) The molar ratio of the nucleoside molecule to the catalyst is 1:0.01 - 0.5;

[0093] (2) The molar ratio of the nucleoside molecule to the oxidant is 1:1 - 50;

[0094] (3) The reaction system further includes a solvent, and the solvent is selected from acetonitrile, ethanol, tetrahydrofuran, water and their combinations;

[0095] (4) The reaction time of the reaction system is 3 - 120 min.

[0096] The fourth aspect of the present invention provides a reaction system for group cleavage, and the reaction system includes a molecule connecting the group and a cleavage reagent; the cleavage reagent reacts with the molecule to separate the group from the molecule;

[0097] The group is methylthiomethyl.

[0098] In some embodiments, the cleavage reagent is selected from hydrogen peroxide, lanthanide metal salts and bromides.

[0099] In some preferred embodiments, the cleavage reagent further includes an oxidant.

[0100] In some more preferred embodiments, the reaction system satisfies one or more of the following conditions:

[0101] (1) The lanthanide metal salt and bromide are selected from CeBr3, Ce(NO3)3-KBr, CeCl3-KBr, Ce(NH4)2(NO3)6-KBr, Ce(OTf)3-KBr, Ce(OAc)3-KBr, and combinations thereof;

[0102] (2) The oxidizing agent is 0.1%-30% (v / v) H2O2, NaIO4, Oxone, O2+UV, or NaI;

[0103] (3) The reaction temperature of the reaction system is 25-65 °C, preferably 40-65 °C, such as 50 °C;

[0104] (4) The reaction time of the reaction system is 3-120 min;

[0105] (5) The reaction system further includes a quenching agent, such as a Na2S2O3 solution;

[0106] (6) The molar ratio of the molecule to the lanthanide metal salt and bromide is 1:0.01-0.5;

[0107] (7) The molar ratio of the molecule to the oxidizing agent is 1:1-50;

[0108] (8) The solvent in the reaction system is selected from acetonitrile, ethanol, tetrahydrofuran, water, and combinations thereof.

[0109] The fifth aspect of the present invention provides a method for preparing the nucleoside molecule as described in the first aspect; it includes any of the following schemes:

[0110] Scheme 1:

[0111] The method includes the following steps:

[0112] Reacting the intermediate product as shown in formula (b) in a solvent with a deprotecting reagent to obtain the product as shown in formula (c)

[0113] wherein, R b is tert-butyldimethylsilyl;

[0114] The solvent is preferably an alcohol solvent and / or an ether solvent, such as methanol and / or tetrahydrofuran;

[0115] The deprotecting reagent is preferably NH4F or tetrabutylammonium fluoride;

[0116] Scheme 2: It includes the following steps:

[0117] Mix the compound shown in formula (II) with dimethyl sulfoxide, acetic acid and acetic anhydride, and through reaction, obtain the intermediate product shown in formula (b);

[0118]

[0119] Preferably, R” is H, and Base is

[0120] Scheme Three:

[0121] The method includes the following steps: In the presence of proton sponge, react the product shown in formula (c) obtained in Scheme One with an anhydrous N,N-dimethylformamide solution of POCl3, bis-tri-n-butyl pyrophosphate and tri-n-butylamine in trimethyl phosphate to obtain the product shown in formula (d)

[0122] Alternatively, react the product shown in formula (c) obtained in Scheme One with tetrabutylammonium pyrophosphate and 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one in an N,N-dimethylformamide solution to obtain the product shown in formula (d) where R 1 is triphosphate acyl group;

[0123] Scheme Four:

[0124] The method includes the following steps: In an organic solvent, react the product shown in formula (d) obtained in Scheme Three with ammonia water, or react the product shown in formula (d) obtained in Scheme Three with an iodine (pyridine / water) solution to obtain the product shown in formula (e)

[0125] where, Base 1 is

[0126] The organic solvent is preferably an alcohol solvent, such as methanol.

[0127] The sixth aspect of the present invention provides a method for determining the sequence of a polynucleotide, and the method includes the following steps:

[0128] 1) Add the nucleoside molecule described in the first aspect or the kit or composition described in the second aspect to the reaction system for polynucleotide synthesis, so that the nucleoside molecule is incorporated into the complementary strand of the polynucleotide to block the extension of the hybrid double strand formed by the free nucleotide and the polynucleotide template; the nucleoside molecule contains a detectable labeling molecule;

[0129] 2) Obtain the type of the incorporated nucleoside molecule by detecting the detectable labeling molecule contained in the nucleoside molecule.

[0130] In some embodiments, the method further includes, after obtaining the type of the incorporated nucleoside molecule, converting the group at the 3'-end of the sugar ring of the nucleoside molecule into a hydroxyl group.

[0131] In some embodiments, the polynucleotide is a single-stranded polynucleotide.

[0132] In some preferred embodiments, the polynucleotide is DNA or RNA.

[0133] In some preferred embodiments, the conversion of the group at the 3'-end of the sugar ring of the nucleoside molecule into a hydroxyl group is achieved through a cleavage reaction as described in the third aspect.

[0134] In some preferred embodiments, the conversion of the group at the 3'-end of the sugar ring of the nucleoside molecule into a hydroxyl group is achieved through a cleavage reaction, and the reaction system used in the cleavage reaction is as described in the fourth aspect.

[0135] In some embodiments, the assay is a synthesis-while-sequencing assay.

[0136] The seventh aspect of the present invention provides a method for synthesizing or labeling a polynucleotide, the method including adding the nucleoside molecule as described in the first aspect or the kit or composition as described in the second aspect to a reaction system for polynucleotide synthesis, and binding the nucleoside molecule to the 3'-end of the polynucleotide;

[0137] When the method is used for labeling a polynucleotide, the nucleoside molecule contains a detectable labeling molecule.

[0138] In some embodiments, the polynucleotide is a single-stranded polynucleotide. In some embodiments, the polynucleotide is DNA or RNA.

[0139] The eighth aspect of the present invention provides an application of the nucleoside molecule as described in the first aspect or the kit or composition as described in the second aspect in determining the sequence of a nucleic acid.

[0140] In some embodiments, the nucleic acid is DNA.

[0141] In some embodiments, the assay is a synthesis-while-sequencing assay.

[0142] An example of a sequencing application of the present invention is to use the nucleoside molecules of the present invention to prepare a growing polynucleotide complementary to a target polynucleotide (such as a single-stranded polynucleotide), which includes incorporating the nucleoside molecules described herein into the growing complementary polynucleotide, wherein the incorporation of the nucleotide prevents the introduction of any subsequent nucleotides into the growing complementary polynucleotide. In some embodiments, the incorporation of the nucleotide is accomplished by a polymerase, terminal deoxynucleotidyl transferase (TdT), or reverse transcriptase. In one embodiment, the incorporation is accomplished by a polymerase (such as a DNA polymerase).

[0143] The nucleoside molecules of the present invention can be used as reversible terminators for SBS-based DNA sequencing technology. This sequencing cycle generally includes three steps: incorporation, imaging, and deprotection. The term "deprotection" can be used synonymously with "cleavage", so the three steps can also be described as incorporation, imaging, and cleavage. For this procedure, cycle efficiency, cycle time, and sensitivity are important factors. Cycle efficiency depends on deprotection and incorporation efficiency and determines the read length of the CRT. For whole-genome sequencing, the nucleoside molecules disclosed herein can be used, which can exhibit efficient deprotection characteristics. Reversible terminators are generally not good substrates for commercially available DNA polymerases. The modified nucleic acid analogs of the present invention can be used to improve this technology by providing a substrate that incorporates as well as or better than natural nucleotides when using commercially available DNA polymerases.

[0144] Based on common general knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0145] The reagents and raw materials used in the present invention are all commercially available.

[0146] The positive and progressive effects of the present invention are as follows:

[0147] The nucleoside molecules of the present invention have better stability in solution and also have good stability in solution during formulation and storage. Moreover, the preparation and synthesis steps are few, the synthesis cost is low, and the cleavage reagent is green and environmentally friendly. During the sequencing process, the nucleoside molecules of the present invention are easily incorporated by enzymes as reversible terminators, and the 3'-hydroxy blocking group can also achieve a low predetermined phase and low signal attenuation to improve data quality, which enables longer reads from sequencing applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0148] Figure 1 It is a comparison of the synthesis routes of 3'-O-azidomethyl-dATP and 3'-O-methylthiomethyl-dATP.

[0149] Figure 2 It is the route of cleaving the blocking group using different reagents.

[0150] Figure 3 Schematic diagram of the deprotection process and reaction mechanism of 3'-O-methylthiomethyl-dNTP.

[0151] Figure 4 Schematic diagram of the incorporation effect of nucleotides containing reversible terminators by different polymerases.

[0152] Figure 5 Schematic diagram of the nucleotide incorporation effect of KOD polymerase on reversible terminator nucleotides in different nucleobase forms.

[0153] Figure 6 Results of the stability test of different 3'-protected dNTP raw materials.

[0154] Figure 7 1H NMR spectrum of product 1e.

[0155] Figure 8 31P NMR spectrum of product 1e.

[0156] Figure 9 1H NMR spectrum of product 2d.

[0157] Figure 10 31P NMR spectrum of product 2d.

[0158] Figure 11 1H NMR spectrum of product 3e.

[0159] Figure 12 31P NMR spectrum of product 3e.

[0160] Figure 13 1H NMR spectrum of product 4e.

[0161] Figure 14 31P NMR spectrum of product 4e. Detailed implementation manners

[0162] Except as described above, when used in the specification and claims of this application, unless otherwise specifically indicated, the following terms have the meanings shown below:

[0163] The term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0164] The term "alkyl" refers to a straight-chain or branched-chain, saturated monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C 1-6 or C1-C6, C 1-4 or C1-C4). Alkyl includes, but is not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0165] The term "alkenyl" or "olefinic group" refers to a group having a specified number of carbon atoms (e.g., C 2-6a straight-chain or branched, unsaturated monovalent hydrocarbon group having from C1-C6 (or C2-C6), having a carbon-carbon sp 2 double bond.

[0166] The term "alkynyl" refers to a straight-chain or branched, unsaturated monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C 2-6 or C2-C6), having a carbon-carbon sp3 triple bond. Alkynyl groups include, but are not limited to: etc.

[0167] The term "aryl" refers to an aromatic group having a specified number of ring carbon atoms (e.g., C6-10 or C6-C10). Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulyl, or anthracenyl.

[0168] The term "heteroaryl" refers to a cyclic group having a specified number of ring atoms (e.g., 5-10 membered, 5-6 membered, 8-10 membered), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified heteroatom type (e.g., one, two, or more of N, O, and S), which may be monocyclic or polycyclic (e.g., fused, spiro, and bridged rings), and at least one ring is aromatic (complies with Hückel's rule). The heteroaryl group is connected to other fragments in the molecule through an aromatic or non-aromatic ring. Heteroaryl groups include, but are not limited to, furyl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, pyrimidinyl, indolyl, or 1,2,3,4-tetrahydroisoquinolinyl, etc.

[0169] The term "carbocyclic group" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon group composed only of carbon and hydrogen atoms, which includes fused or bridged ring systems and has a specified number of carbon atoms (e.g., 3-6, 3-8, 3-15, C3-C7).

[0170] The term "heterocyclic group" refers to a saturated monocyclic, spiro, bridged, or fused cyclic group having a specified number of ring atoms (e.g., 5-8 membered, 3-10 membered, 3-12 membered, 6-10 membered, 6-12 membered, five to eight membered), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified heteroatom type (1, 2, or 3 of N, O, and S), and when polycyclic, each ring is saturated. Heterocycloalkyl groups include, but are not limited to, oxetanyl, azetidinyl, pyrrolidinyl, piperazinyl, piperidinyl, morpholinyl.

[0171] The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by one or more halogen atoms, and the definition of alkyl is as described above. Haloalkyl can be substituted with 1, 2, or 3 halo groups. The term "haloalkyl" includes perfluoroalkyl. Examples of haloalkyl include trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, etc.

[0172] The term "alkoxy" means -O-alkyl, where alkyl is defined as described above.

[0173] In the term "haloalkoxy", alkoxy is defined as described above.

[0174] The term "aralkyl" means an alkyl group substituted by an aryl group, where "aryl" and "alkyl" are defined as described above.

[0175] The term "sulfonyl" means -S(O)2H.

[0176] The term "sulfinyl" means -S(O)H.

[0177] The term "heteroalkyl" means one or more carbon atoms (excluding any terminal carbon atoms) in a straight-chain or branched-chain alkyl group having a specified number of carbon atoms (e.g., C 1-6 or C1-C6, C 1-4 or C1-C4), each independently replaced by the same or different heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.), where "alkyl" is defined as described above.

[0178] The term "alkylene" means a divalent straight-chain or branched-chain alkyl group, where "alkyl" is defined as described above.

[0179] The term "alkenylene" means a divalent straight-chain or branched-chain alkenyl group, where "alkenyl" is defined as described above.

[0180] The term "aryloxy" means -O-aryl, where "aryl" is defined as described above.

[0181] The term "arylthio" means -S-aryl, where "aryl" is defined as described above.

[0182] The term "aminoalkylene" means an alkyl group substituted by an amino group, where "alkylene" is defined as described above.

[0183] The term "one or more" means 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0184] The term "phosphoryl" is used in its ordinary meaning as understood by those skilled in the art and includes its protonated form.

[0185] In this text, when a group is described as "optionally substituted", it can be unsubstituted or substituted. The "substitution" is selected from one or more of the following groups as used herein. The substituent is derived from an unsubstituted parent group, where one or more hydrogen atoms have been exchanged for another atom or group. Unless otherwise specified, when a group is considered "substituted", it means that the group is substituted with one or more substituents independently selected from the following: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C7 carbocyclic group (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), C3-C7-carbocyclic-C1-C6-alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 3- to 10-membered heterocyclic group (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 3- to 10-membered heterocyclic-C1-C6-alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), aryl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), aryl(C1-C6)alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 5- to 10-membered heteroaryl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 5- to 10-membered heteroaryl(C1-C6)alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), halogen, -CN, hydroxy, C1-C6 alkoxy, C1-C6 alkoxy(C1-C6)alkyl (i.e., ether), aryloxy, sulfhydryl and mercapto, halo(C1-C6)alkyl (e.g., –CF3), halo(C1-C6)alkoxy (e.g., –OCF3), (C1-C6)alkylthio, arylthio, amino, amino(C1-C6)alkyl, nitro, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl, -SO3H, sulfinyl, -OSO2C 1-4Alkyl and oxo(=O). Whenever a group is described as "optionally substituted", the group may be substituted with the above substituents.

[0186] Those skilled in the art will understand that, according to the convention used in the art, the in the structural formula of the group described in the present invention means that the corresponding group R is connected to other fragments and groups in the compound through this site.

[0187] The term "nucleotide" consists of a nucleobase, a sugar, and one or more phosphate groups. They are the monomeric units of nucleic acid sequences. In RNA, the sugar is ribose, while in DNA, the sugar is deoxyribose, i.e., a sugar lacking the hydroxyl group present in ribose. The nitrogenous bases are derivatives of purines or pyrimidines. Purines are adenine (A) and guanine (G), and pyrimidines are cytosine (C) and thymine (T) [or in the case of RNA, uracil (U)]. The C-1 atom of deoxyribose is bonded to the N-1 of pyrimidine or the N-9 of purine. Nucleotides are also phosphate esters or nucleosides, where esterification occurs at the hydroxyl group attached to C-5 of the sugar. Nucleotides are usually monophosphate, diphosphate, or triphosphate. "Nucleoside" is structurally similar to a nucleotide but does not include the phosphate ester unit. Common abbreviations include "dNTP" for deoxynucleotide triphosphate.

[0188] The term "nucleobase" refers to a nitrogenous base or a modified deazabase that can base-pair with a complementary nitrogenous base of a template nucleic acid. Exemplary nucleobases include adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), inosine (I), and their derivatives. References to thymine in this document should be understood to also refer to uracil, unless otherwise apparent from the context. As used herein, the terms "nucleobase", "nucleoside base", and "base" are used interchangeably.

[0189] The term "derivative" or "analogue" means a compound or molecule whose core structure is the same as or very similar to the core structure of the parent compound, but which has chemical or physical modifications, such as different or additional side groups, or 2' and / or 3' blocking groups, which allow the derivatized nucleotide or nucleoside to be linked to another molecule. For example, the base can be a deazapurine. The derivative should be able to perform Watson-Crick pairing. "Derivative" and "analogue" also refer to synthetic nucleotide or nucleoside derivatives having modified base units and / or modified sugar units.

[0190] Nucleotide analogs may also include modified phosphodiester linkages, including phosphorothioate, dithiophosphonate, alkyl-phosphate, phosphoramidite, and phosphoroamidate linkages. The analogs should be capable of Watson-Crick base pairing. For example, deoxyadenosine analogs include didanosine (ddI) and vidarabine, and adenosine analogs include BCX4430; deoxycytidine analogs include cytarabine, gemcitabine, emtricitabine (FTC), lamivudine (3TC), and zalcitabine (ddC); guanosine and deoxyguanosine analogs include abacavir, acyclovir, and entecavir; thymidine and deoxythymidine analogs include stavudine (d4T), telbivudine, and zidovudine (azidothymidine, or AZT); and deoxyuridine analogs include idoxuridine and trifluridine. As used herein, the terms "derivative," "analog," and "modified" are used interchangeably and are encompassed by the terms "nucleotide" and "nucleoside" as defined herein.

[0191] Phosphoacyl analogs may include modified phosphodiester linkages, such as phosphorothioacyl, dithiophosphorothioacyl, C1-C6 alkylphosphoacyl, anilinophosphoacyl, or phosphoroamidate.

[0192] The term "incorporation" means to become part of a nucleic acid molecule. In SBS, incorporation of an RT occurs when the polymerase adds an RT to a growing DNA strand by forming a phosphodiester or modified phosphodiester bond between the 3'-position of the pentose of one nucleotide (i.e., the 3'-nucleotide on the DNA strand) and the 5'-position of the pentose on an adjacent nucleotide, i.e., the RT is added to the DNA strand.

[0193] The term "labeling molecule" means any atom or molecule that can be used to provide a detectable and / or quantifiable signal. Suitable labels include radioisotopes, fluorophores, chromophores, mass labels, electron-dense particles, magnetic particles, spin labels, molecules that emit chemiluminescence, electrochemically active molecules, enzymes, cofactors, and enzyme substrates. In some embodiments, the detection label is a molecule containing a charged group (e.g., a molecule containing a cationic group or a molecule containing an anionic group), a fluorescent molecule (e.g., a fluorescent dye), a fluorogenic molecule, or a metal. Optionally, the detection label is a fluorogenic label. A fluorogenic label can be any label that is capable of emitting light in its unquenched form (e.g., when not quenched by another agent). When excited by an appropriate excitation wavelength, the fluorescent moiety emits light energy (i.e., fluoresces) at a specific emission wavelength. When the fluorescent moiety and the quencher moiety are in close proximity, the light energy emitted by the fluorescent moiety is absorbed by the quencher moiety. In some embodiments, the fluorogenic dye is fluorescein, rhodamine, phenoxazine, acridine, coumarin, or a derivative thereof. In some embodiments, the fluorogenic dye is carboxyfluorescein. Non-fluorogenic labels can also be used, including but not limited to, redox labels, reducing tags, sulfur- or thiol-containing molecules, substituted or unsubstituted alkyl groups, fluorescent proteins, non-fluorescent dyes, and luminescent proteins.

[0194] As used herein, the terms "reversible," "removable," and "cleavable" when referring to a blocking group have the same meaning.

[0195] The blocking group in the term "reversible terminator" is also referred to as a "reversible blocking group," "removable blocking group," "cleavable linker," "blocking unit," "blocking group," "reversible terminator blocking group," etc. A reversible blocking group is a chemical unit that is typically attached to the nucleotide sugar (e.g., deoxyribose) at the 3'-O position of the sugar moiety, which prevents the addition of a nucleotide at this position by a polymerase. The reversible blocking group can be cleaved by an enzyme (e.g., a phosphatase or an esterase), a chemical reaction, heat, light, etc., to provide a hydroxyl group at the 3'-position of the nucleoside or nucleotide, such that the addition of a nucleotide by a polymerase becomes possible.

[0196] The term "dNTP" includes naturally occurring deoxyribonucleoside triphosphates and their analogs, including analogs having a 3'-O cleavable blocking group.

[0197] The term "nucleic acid" means a polymer of nucleotide monomers. As used herein, the term can refer to single-stranded or double-stranded forms. The monomers that make up nucleic acids and oligonucleotides are capable of specifically binding to native polynucleotides by regular patterns of monomer-to-monomer interactions (such as Watson-Crick type base pairing, base stacking, Hoogsteen or reverse Hoogsteen type base pairing, etc.) to form duplex or triplex forms. Such monomers and their internucleoside linkages can be naturally occurring or can be analogs thereof, such as naturally occurring or non-naturally occurring analogs. Non-naturally occurring analogs can include peptide nucleic acids, locked nucleic acids, phosphorothioate internucleoside linkages, bases containing linking groups that permit attachment of labels, such as fluorophores or haptens, etc. The size of nucleic acids generally ranges from a few monomer units (e.g., 5 - 40, in which case they are usually referred to as "oligonucleotides") up to hundreds of thousands or more monomer units. Whenever a nucleic acid or oligonucleotide is represented by a sequence of letters (upper or lower case), e.g., "ATGCCTG", it should be understood that the nucleotides are in 5' to 3' order from left to right, and that "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, and "T" represents thymidine, "I" represents deoxyinosine, "U" represents uridine, unless otherwise specified or obvious from the context. Unless otherwise indicated, the terminology and atomic numbering conventions will follow those disclosed in Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York, 1999). Generally, nucleic acids contain natural nucleosides (e.g., deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribose counterparts for RNA) linked by phosphodiester linkages; however, they can also contain non-natural nucleotide analogs, such as modified bases, sugars, or internucleoside linkages. For those skilled in the art, when an enzyme has specific oligonucleotide or nucleic acid substrate activity requirements (such as single-stranded DNA, RNA / DNA duplexes, etc.), the selection of suitable oligonucleotide or nucleic acid substrate compositions is within the knowledge of an ordinary skilled person.

[0198] As used herein, room temperature means "25 - 30 °C".

[0199] In the prior art, the cleavage reagents for blocking groups are expensive or environmentally polluting. For example, the cleavage reagent for 3'-O-azidomethyl-dNTP requires the use of tris(carboxyethyl)phosphine (TCEP). TCEP is a common phosphate ester that causes water pollution. Since TCEP is difficult to biodegrade and photodegrade, improper treatment will have an adverse impact on the ecosystem and human health if it exists in nature for a long time. Therefore, the waste liquid of this cleavage reagent needs to be properly post-treated. For example, the cleavage reagent for 3'-O-allyl-dNTP requires the use of Pd(II) complex and tris(hydroxypropyl)phosphine (THP). THP is also a phosphate ester that causes water pollution. Therefore, the cleaved reagent also needs to be properly post-treated. In addition, the cleavage reagent also uses precious metal Pd as a catalyst, which undoubtedly increases the application cost of this nucleotide. The cleavage agent for 3'-O-methylthiomethyl-dNTP uses CeBr3 as a catalyst and H2O2 as an oxidant, which is cheap, easily available, mild in conditions and easy to operate. It is a very green, environmentally friendly and safe method for cleaving nucleotide reversible blockers, and has application potential in both sequencing and enzymatic synthesis of oligonucleotides.

[0200] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. The experimental methods without specific conditions in the following examples are carried out according to conventional methods and conditions, or selected according to the product instructions.

[0201] Example 1: Preparation of 3'-O-methylthiomethyl-dATP

[0202]

[0203] (i) 3.5 g of 1a was added to a solution composed of 14.7 mL of DMSO (dimethyl sulfoxide), 6.7 mL of AcOH, and 21.59 mL of Ac2O, and stirred at room temperature for 48 hours. Then, a sample was taken for TLC spotting, and it was found that the product spot (Rf = 0.4, ethyl acetate / hexane = 10 / 1). The solution was neutralized with saturated NaHCO3, extracted with dichloromethane (3×100 mL), washed with saturated NaHCO3 again, dried over anhydrous Na2SO4, and concentrated under vacuum. The product 1b (2.9 g, yield 73%) as a white powder was obtained by purification through a normal-phase column (ethyl acetate / hexane = 2:1 - 9:1).

[0204] (ii) 500 mg of 1b was dissolved in 5 mL of methanol, 600 mg of NH4F was added, and the reaction was carried out at room temperature for 20 h. After the solution was rotary evaporated, it was extracted with dichloromethane. The organic phase was dried over anhydrous Na2SO4 and rotary evaporated under vacuum to collect. A white powdery substance (product 1c) (200 mg) was obtained through a normal-phase column (hexane / ethyl acetate = 1:4 - 1:10).

[0205] (iii) 1c (6 mg, 0.014 mmol) and proton sponge (6 mg; 0.028 mmol) were dried under vacuum in a flask for 5 h and then dissolved in PO(OMe)3 at -10 °C. POCl3 (2.6 μL, 0.028 mmol) was added dropwise at -10 °C and the mixture was stirred for 2 h under a nitrogen atmosphere. In a new flask, an anhydrous DMF (0.28 mL) solution of bis-tri-n-butyl pyrophosphate (66 mg, 0.14 mmol) and tri-n-butylamine (28 μL) was added. After stirring at -10 °C for 10 min, the mixed solution was added to the first flask and stirred for 2 h to obtain product 1d.

[0206] (iv) NH3·H2O (25%, 5 mL) was added to a solution of product 1d in MeOH (10 mL), and the mixture was stirred at room temperature for 2 days. LCMS showed that the reaction was complete. The mixture was concentrated under vacuum and purified by preparative RP-HPLC to obtain 3'-O-SMe-dATP (i.e., product 1e). LCMS (ESI): for the molecular ion [M-H], the calculated mass was 550.00 and the measured mass was 550.11. The NMR data of this compound are as Figure 7 and Figure 8 shown.

[0207] Example 2: Preparation of 3'-O-methylthiomethyl-dTTP

[0208]

[0209] (i) Acetic acid (2.6 mL, 45 mmol) and acetic anhydride (8.6 mL, 90 mmol) were added to a stirred solution of 2a (1.07 g, 3 mmol) in DMSO (10 mL). The reaction mixture was stirred at room temperature for 48 h until the reaction was complete as monitored by TLC. Then, saturated sodium bicarbonate solution was slowly added to the above mixed solution with vigorous stirring, and then extracted with ethyl acetate (3 × 30 mL). The combined organic layers were dried over Na2SO4 and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (ethyl acetate / hexane: 1:2) to obtain product 2a (0.94 g, 72%).

[0210] (ii) Dissolve 2b (240 mg, 0.52 mmol) in anhydrous THF (10 mL), and add a THF solution of tetrabutylammonium fluoride (1.0 M, 1.04 mL, 1.04 mmol, 1.5 eq.). The reaction mixture was stirred at room temperature for 4 h. After the reaction mixture was concentrated in vacuo, saturated NaHCO3 solution (50 mL) was added, and then the mixture was extracted with dichloromethane (3 x 20 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The resulting crude mixture was purified by flash column chromatography (dichloromethane / methanol: 20 / 1) to give 2c (120 mg, 66%).

[0211] (iii) 2c (50 mg, 0.14 mmol), tetrabutylammonium pyrophosphate (197 mg, 0.36 mmol, 2.5 eq.) and 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (44 mg, 0.22 mmol, 1.5 eq.) were dried under vacuum separately overnight at room temperature. Tetrabutylammonium pyrophosphate was dissolved in N,N-dimethylformamide (DMF, 1 mL) under argon, and then tributylamine (1 mL) was added. Under argon, this mixture was injected into a solution of 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (DMF, 2 mL). After stirring for 1 h, the reaction mixture was added to the solution of 2c and further stirred at room temperature for 1 h. Then an iodine solution (0.02 M iodine / pyridine / water) was injected into the reaction mixture until a brown color was observed. After 10 min, water (30 mL) was added and the reaction mixture was stirred at room temperature for another 2 h. The resulting solution was extracted with ethyl acetate (2 × 30 mL). The aqueous layer was concentrated in vacuo to about 20 mL and transferred to two centrifuge tubes (50 mL). To each tube, brine (1.5 mL) and absolute ethanol (35 mL) were added, and then shaken vigorously. After standing at -80 °C for 2 h, centrifugation (4200 rpm, 10 min) was performed to obtain the crude product as a white precipitate. The supernatant was decanted, and the white precipitate was diluted with 5 mL of water and purified by ion exchange chromatography using a TEAB (pH 8.0; 0.1 - 1.0 M) gradient at 4 °C. The crude product was further purified by reverse-phase HPLC to give 2d. LCMS (ESI): For the molecular ion [M-H], the calculated mass was 540.99 and the measured mass was 541.00. The NMR data of this compound are as Figure 9 and Figure 10 shown.

[0212] Example 3: Preparation of 3'-O-Methylthiomethyl-dGTP

[0213]

[0214] (i) Acetic acid (2.1 mL, 36 mmol) and acetic anhydride (5.456 mmol) were added to a solution of 3a (1.33 g, 2.1 mmol) in DMSO (10 mL). The mixture was stirred at room temperature until the reaction was complete as monitored by TLC (24 h). The mixture was then slowly added to a sodium bicarbonate solution with vigorous stirring and extracted with ethyl acetate (3 x 30 mL). The combined organic layers were dried over Na2SO4 and filtered. The filtrate was concentrated to dryness under reduced pressure and the desired compound was purified by silica gel column chromatography (ethyl acetate / hexane: 1:2) to give the pure product 3b (1.24 g, 86%) as a white solid.

[0215] (ii) 3b (240 mg, 0.52 mmol) was dissolved in anhydrous THF (10 mL), and a THF solution of tetrabutylammonium fluoride (1.0 M, 1.04 mL, 1.04 mmol, 1.5 eq.) was added. The reaction mixture was stirred at room temperature for 4 h. After the reaction mixture was concentrated in vacuo, saturated NaHCO3 solution (50 mL) was added, and then the mixture was extracted with dichloromethane (3 x 20 mL). The organic layer was further dried over anhydrous Na2SO4, filtered and concentrated. The resulting crude mixture was purified by flash column chromatography (dichloromethane / methanol: 20 / 1) to give 2c (120 mg, 66%). 3b (264 mg, 0.38 mmol), ammonium hydroxide (10 mL) and methanol (10 mL) were stirred at room temperature until the reaction was complete (4 h). The reaction was monitored by thin layer chromatography (TLC). After evaporation of the solvent under reduced pressure, the crude solid was treated with 3% trichloroacetic acid in dichloromethane for 10 min. Then, the mixture was slowly added to a solution of sodium carbonate with vigorous stirring and extracted with ethyl acetate (3 x 30 mL). The combined organic layers were dried over Na2SO4 and filtered. The filtrate was concentrated to dryness under reduced pressure and the desired compound was purified by silica gel column chromatography (dichloromethane / methanol: 20 / 1) to give 3c (70 mg, 51%). (iii) 3c (62 mg, 0.17 mmol), tetrabutylammonium pyrophosphate (238 mg, 0.44 mmol, 2.5 eq.) and 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (53 mg, 0.27 mmol, 1.5 eq.) were dried individually under vacuum overnight at room temperature. Tetrabutylammonium pyrophosphate was dissolved in dimethylformamide (DMF, 1 mL) under argon, and then tributylamine (1 mL) was added. The mixture was injected into a solution of 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (DMF, 2 mL) under argon. After stirring for 1 h, the reaction mixture was added to a solution of 3c and further stirred at room temperature for 1 h to give 3d.

[0216] (iv) Inject iodine solution (0.02 M iodine / pyridine / water) into the reaction mixture until a brown color is observed. After 10 minutes, add water (30 mL) and stir the reaction mixture at room temperature for another 2 hours. Extract the resulting solution with ethyl acetate (2 × 30 mL). Concentrate the aqueous layer under vacuum to approximately 20 mL and transfer it to two centrifuge tubes (50 mL). Add brine (1.5 mL) and absolute ethanol (35 mL) to each tube and then shake vigorously. After standing at -80 °C for 2 hours, centrifuge (4200 rpm, 10 minutes) to obtain the crude product as a white precipitate. Decant the supernatant, dilute the white precipitate with 5 mL of water, and purify it by ion-exchange chromatography using a TEAB (pH 8.0; 0.1 - 1.0 M) gradient at 4 °C. Further purify the crude product by reverse-phase HPLC to obtain 3e. LCMS (ESI): For the molecular ion [M-H], the calculated mass is 566.00 and the measured mass is 566.09. The NMR data of this compound are as Figure 11 and Figure 12 shown.

[0217] Example 4: Preparation of 3'-O-methylthiomethyl-dCTP

[0218]

[0219] (i) Add acetic acid (2.9 mL) and acetic anhydride (9.3 mL) to a solution of 4a (1.5 g, 3.4 mmol) in DMSO (6.5 mL) and stir at room temperature for 48 hours. Then slowly add sodium bicarbonate solution (50 mL) to the mixture with vigorous stirring and extract with ethyl acetate (3 x 50 mL). Combine the organic layers, dry over Na2SO4 and filter. Concentrate the filtrate to dryness under reduced pressure and purify the desired compound by silica gel column chromatography (ethyl acetate / n-hexane: 8:2) to obtain the pure product 3b (1.2 g, 70%) as a white solid.

[0220] (ii) Dissolve 500 mg of 4b in 9 mL of methanol and 3 mL of tetrahydrofuran, add 1.8 g of NH4F, react at room temperature for 48 h, spin-dry the solution, extract with dichloromethane, dry the organic phase over anhydrous Na2SO4 and spin-dry under vacuum to collect. Obtain a white powdery substance (product 4c) (200 mg, 48%) by a normal-phase column (ethyl acetate / hexane = 2:8 - 7:3)

[0221] (iii) Compound 4c (60 mg, 0.14 mmol) and Proton Sponge (40 mg, 0.19 mmol) were dried overnight in a P2O5 vacuum desiccator and then dissolved in trimethyl phosphate (1 mL) cooled in an ice bath. Freshly distilled POCl3 (19 μL, 0.2 mmol) was added dropwise at 0 °C and the mixture was stirred for 2 h. A solution of tetrabutylammonium pyrophosphate (255 mg, 0.47 mmol) and tributylamine (27.6 μL, 0.12 mmol) in anhydrous DMF (1.5 mL) was added at room temperature and the mixture was stirred for an additional 30 min. A solution of triethylammonium bicarbonate (TEAB) (0.1 M; pH 8.0; 7.5 mL) was added and the mixture was stirred at room temperature for 1 h to afford Compound 4d.

[0222] (iv) Concentrated NH4OH (7.5 mL) was added and stirring was continued overnight at room temperature. The mixture was concentrated in vacuo and the crude product was purified by anion exchange chromatography using a TEAB gradient (pH 8.0; 0.1 - 1.0 M), followed by further purification by reverse phase HPLC to afford 4e. LCMS (ESI): calculated mass for the molecular ion [M - H] is 525.99, measured mass is 525.00. The NMR data for this compound are as Figure 13 and Figure 14 shown.

[0223] Example 5: 3’-OH Blocking Group Stability Experiment

[0224] In this example, a stability experiment of 3’-O-methylthiomethyl thymidine triphosphate was carried out in parallel with standard 3’-O-azidomethyl thymidine triphosphate and 3’-O-amino thymidine triphosphate in an incorporation buffer. The specific experimental procedure is as follows:

[0225]

[0226] 1 mL of 0.1 mM of each 3’-protected thymidine triphosphate in 100 mL of ethanolamine buffer (pH 9.8), 100 mM NaCl and 2.5 mM EDTA solution was placed at room temperature for 10 days. At set time points, 100 μL aliquots were taken and analyzed by HPLC to determine the percentage of the remaining blocked nucleotide and the unblocked nucleotide formed finally.

[0227] The results of the stability tests for 3’-blocked nucleotides with SMe methylthiomethyl protecting group and the standard azidomethyl blocking group and amino are shown in Figure 6As a result, it shows that the content of the 3'-O-methylthiomethyl-dTTP solution remains basically unchanged after being stored at room temperature for 10 days. It is observed that the 3'-blocked nucleotide with an SMe blocking group is the most stable in solution. Such improved stability extends the shelf life of the incorporated mixed reagent and enables the possibility of room temperature transportation, greatly reducing the transportation volume and cost. The stability improvement provided by the methylthiomethyl protecting group will also result in a lower pre-phasing rate during the sequencing run.

[0228] The reversibly blocked nucleotides mentioned in the prior art, such as 3'-O-azidomethyl-dNTP, 3'-O-allyl-dNTP, and 3'-O-amino-dNTP, have general stability and need to be stored at least below -20°C to maintain stability for a certain period of time, thus increasing the storage and sample transportation costs and also affecting the read length of nucleotide sequencing. For example, compared with the azidomethyl-protected 3'-OH under the same conditions for the same period of time, the methylthiomethylene blocking group in this example can confer at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 2500%, or 3000% improved stability, thereby reducing the pre-phasing value and obtaining a longer sequencing read length.

[0229] Example 6: Cleavage of 3'-O-methylthiomethyl-dTTP

[0230]

[0231] Dissolve 2d (1 μmol) in a 2 mM aqueous solution, and successively add CeBr3 (0.1 μmol) and an aqueous H2O2 solution (2 μmol), and stir the reaction at room temperature for 30 min. After the reaction is completed, quench the reaction with a Na2S2O3 solution (0.1 M) to obtain the target product 2e (yield: 65%). The reaction process is as Figure 3 shown.

[0232] Example 7: Cleavage of 3'-O-methylthiomethyl-dATP

[0233]

[0234] Dissolve 1e (1 μmol) in a 2 mM aqueous solution, and successively add CeBr3 (0.1 μmol) and an aqueous H2O2 solution (10 μmol), and stir the reaction at room temperature for 30 min. After the reaction is completed, quench the reaction with a Na2S2O3 solution (0.1 M) to obtain the target product 1f (yield: 80%).

[0235] Example 8: Cleavage of 3’-O-methylthiomethyl-dGTP

[0236]

[0237] Dissolve 3e (0.5 μmol) in 2 mM aqueous solution, and successively add CeBr3 (0.1 μmol) and H2O2 aqueous solution (10 μmol), then stir and react at 50 °C for 10 min. After the reaction is completed, quench the reaction with Na2S2O3 solution (0.1 M) to obtain the target product 3f (yield: 75%).

[0238] Example 9: Cleavage of 3’-O-methylthiomethyl-dCTP

[0239]

[0240] Dissolve 4e (0.5 μmol) in 2 mM aqueous solution, and successively add CeBr3 (0.1 μmol) and H2O2 aqueous solution (10 μmol), then stir and react at 65 °C for 10 min. After the reaction is completed, quench the reaction with Na2S2O3 solution (0.1 M) to obtain the target product 4f (yield: 85%).

[0241] Example 10: Incorporation of 3’-reversible nucleoside triphosphates with natural nucleobases by polymerase

[0242] It is known that terminal deoxynucleotidyl transferase (TdT) has the ability to incorporate natural deoxynucleotides as a polymerase. To determine the incorporation of 3’-methylthiomethyl-modified reversible nucleotides, add the following reagents to the reaction chamber: 0.2 μM primer, 5 U TdT, 12.5 mM Tris-HCl, 0.05% Triton X-100, 1 mM CoCl2, pH 7.2. Next, incubate the extension reaction mixture with 5 μM of 3’-O-SMe-dTTP reversible nucleotide in a thermal cycler at 37 °C for 5 min. Then analyze the reaction using a 15% TBE-urea denaturing gel. The primer used to initiate the synthesis is as follows:

[0243] 5’-AGCCAAGCGGTCGCGATGAT-3’ (SEQ ID NO:1)

[0244] The known KOD DNA polymerase, as a common high-fidelity DNA polymerase, has strong DNA extension ability and 3'-5' exonuclease proofreading activity. To evaluate the efficacy of KOD polymerase incorporating multiple reversibly modified nucleotides with a 3'-methylthiomethyl group, an appropriate buffer was supplemented for the extension reaction, which included 0.2 μM primer-template, 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton, 4 mM MgSO4, and 2 μg KOD polymerase, pH 8.8. The extension reaction mixture was incubated with 5 μM of the 3'-O-SMe-dTTP reversibly modified nucleotide in a thermal cycler at 40 °C for 1 min. Then, a denaturing gel was used to analyze the reaction. The primer-template used to initiate the synthesis is given below:

[0245] 5’-CGTGTAAGCGTAATAGGATCCCGACTCACTATGGACG-3’(SEQ ID NO:2)

[0246] 3’-CGCATTATCCTAGGGCTGAGTGATACCTGCAATGTGC-5’(SEQ ID NO:3)

[0247] As Figure 4 shown, it was demonstrated that the 3'-methylthiomethyl modified nucleotides can be incorporated as effective reversible terminators by KOD DNA polymerase and TdT enzyme, and the incorporation efficiency of KOD DNA polymerase is higher. To determine whether this is the case for reversible terminator nucleoside triphosphates of different bases, 3'-O-methylthiomethyl-dATP, -dTTP, -dCTP, -dGTP were synthesized. Using the optimal conditions, the extension reaction mixture was incubated with 1 mM of 3'-O-methylthiomethyl-dATP, -dTTP, -dCTP, -dGTP in a thermal cycler at 40 °C for 1 minute, and then the reaction was analyzed using a 15% TBE-urea denaturing gel. As Figure 5 shown, it was demonstrated that KOD polymerase effectively incorporated the 3'-O-methylthiomethyl reversible terminators in each nucleobase form.

[0248] Example 11: Sequencing Application

[0249] The reversible terminators of the present invention can be used in DNA sequencing methods based on many means, including but not limited to:

[0250] · Sequencing-by-synthesis (SBS). See Metzker, 2005, which is incorporated herein by reference.

[0251] ·DNA polymerase-based strategies, including cyclic reversible termination (CRT),

[0252] single-nucleotide addition (SNA), and real-time sequencing. See Metzker,

[0253] 2005; Metzker, 2010, Sara Goodwin, 2016; etc., which are incorporated herein by reference.

[0254] ·Massively parallel sequencing using unlabeled nucleotides. See WO2020 / 097607, which is incorporated herein by reference.

[0255] The sequencing elements include: i) a set of nucleic acids; ii) a nucleotide polymerase; iii) a sample template strand capable of hybridizing nucleic acids or nucleic acid analogs; iv) a set of labeled analogs of nucleotides; which includes the following parts: a ribose or deoxyribose ring, a base, a 3'-blocking group, a cleavable methylthio group, and a detectable label. For example, it may have the structure shown in any of compounds (1)-(6), where the Base linked to the 1'-position of the sugar ring can be replaced with A, G, C, T as needed, and the dye can be AF647, AF660, AF488, or AF532 as needed, and is coupled to the linker at the base end through an NHS ester.

[0256] The specific steps are as follows:

[0257] (i) Hybridize the primer extension product with the template nucleic acid immobilized on the surface to form a primer-template hybrid;

[0258] Among them, the primer extension product can be obtained according to Example 10.

[0259] (ii) Add nucleoside molecules containing any structure of a detectable molecule as described herein, and a nucleic acid polymerase to incorporate the primer-template hybrid in step (i) into the growing primer strand.

[0260] (iii) Wash the solid surface to remove unincorporated components;

[0261] (iv) Detect the incorporated reporter group;

[0262] (v) Remove the cleavable capping group; the cleavable capping group is a blocking group, such as a methylthio group.

[0263] (vi) Repeat steps (ii) to (v) at least once, ten times, fifty times, one hundred times, one thousand times to identify the base sequence of the target nucleic acid.

[0264] When these sequencing results were compared with a commercial run with a standard azidomethyl blocking group, an almost identical error rate was observed and the phasing would improve, with the phasing being greatly reduced due to the stability of this reversible nucleotide.

Claims

1. A nucleoside molecule having a structure as shown in formula (I): Wherein, R’ is H, a mono-phosphoryl group, a di-phosphoryl group, a tri-phosphoryl group or a phosphoryl group analogue; R” is H, -OH, halogen, -CN, -NO2, -N3, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclic group, optionally substituted heterocyclic group, optionally substituted acyl group, optionally substituted amino group or optionally substituted thiol group; Base is a nucleobase, and the nucleobase is optionally substituted by an N-protecting group; Each R 1a and R 1b independently is H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, cyano, halogen, optionally substituted aryl or optionally substituted aralkyl; Each R 2a , R 2b and R 2c is independently H, C1-C6 alkyl, C1-C6 haloalkyl, cyano or halogen; or, R 1a and R 2a together with the atom to which it is attached form an optionally substituted 5- to 8-membered heterocyclic group.

2. The nucleoside molecule according to claim 1, wherein The nucleoside molecule satisfies one or more of the following conditions: (1) The nucleobase is a nitrogen-containing base or a deazabase; the nucleobase is preferably a purine, pyrimidine or deazapurine; the nucleobase is, for example, adenine, guanine, cytosine, thymine or uracil; (2) Each R 1a and R 1b is independently H or C1-C6 alkyl; (3)R 2a 、R 2b and R 2c at least one of which is H, C1-C6 alkyl or halogen; (4) The aryl is phenyl, naphthyl, azulyl or anthryl; (5) The aralkyl group is a C6-C 10 aryl-substituted C1-C6 alkyl group; for example, benzyl, 2-phenylethyl, 3-phenylpropyl, and naphthylmethyl; (6) The phosphoryl group analogue contains a modified phosphodiester linkage, such as phosphorothioyl, dithiophosphorothioyl, C1-C6 alkylphosphoryl, anilinophosphoryl or aminophosphoryl; (7) R’ is a mono-phosphoryl group, a di-phosphoryl group or a tri-phosphoryl group; (8)R” is H, -OH, halogen, -CN, -NO2, -N3, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C6-C 10 aryl, optionally substituted 5-10 membered heteroaryl, optionally substituted C3-C7 carbocyclic group, optionally substituted 3-10 membered heterocyclic group, optionally substituted acyl group, optionally substituted amino group or optionally substituted mercapto group; preferably, the "substituted" means substituted by one or more substituents independently selected from the following: halogen, -CN, hydroxy group, amino group, nitro group, sulfonyl group, -SO3H, sulfinyl group, oxo group, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, C3-C7 carbocyclic group, C3-C7 carbocyclic group-(C1-C6) alkylene, C1-C6 alkoxy-(C1-C6) alkylene, C6-C 10 aryl, C6-C 10 aryl-(C1-C6) alkylene, 5-10 membered heteroaryl, 5-10 membered heteroaryl-(C1-C6) alkylene, C1-C6 alkoxy-(C1-C6) alkylene, C6-C 10 aryloxy, mercapto group, C1-C6 alkylthio, C6-C 10 arylthio or amino-(C1-C6) alkylene; (9) The nucleobase is operably linked to a detectable label molecule; the operable linkage is achieved, for example, through a linking group; (10) The nucleobase is selected from 3. The nucleoside molecule according to claim 1 or 2, characterized in that, The nucleoside molecule satisfies one or more of the following conditions: (1) The R’ is a tri-phosphoryl group; (2) The R” is H; (3)R 1a and R 1b are both H; (4)R 2a , R 2b and R 2c All are H; (5) The C1-C6 alkyl is methyl, ethyl, isopropyl or tert-butyl; (6) The C1-C6 haloalkyl is trifluoromethyl, trichloromethyl, pentafluoroethyl or pentachloroethyl.

4. The nucleoside molecule according to claim 3, wherein, The nucleoside molecule satisfies one or more of the following conditions: (1) The linking group is a cleavable linking group or a non-cleavable linking group, and the cleavable linking group preferably contains one or more of the following groups: azide, -O-C2-C6 alkenylene- (for example, -O-allylidene-), dithio group, -methylthio-(C1-C6) alkylene-, 2-nitrobenzyl or 4-nitrobenzyl; or, the linking group is Wherein, X is -CH2-, -O- or -S-; Z is azide, phenylboronic acid pinacol ester, –O-C1-C6 alkyl, –O-C2-C6 alkenyl, –O-C2-C6 alkynyl, –S-C1-C6 alkyl, –S-C2-C6 alkenyl or –S-C2-C6 alkynyl; n is 1, 2, 3, 4, or 5; (2) The detectable label molecule is a fluorescent dye molecule, a chemiluminescent molecule or a dye-conjugate; for example, when the detectable label molecule is a fluorescent dye molecule or a chemiluminescent molecule, Base is: wherein L is the connecting group; The fluorescent dye molecule is preferably selected from coumarin, AlexaFluor, Bodipy, fluorescein, tetramethylrhodamine, Cy3, Cy5, Texas Red and their derivatives; or preferably selected from The chemiluminescent molecule is preferably selected from acridinium ester, acridinium sulfonamide, luminol, isoluminol, coelenterazine, tris(2,2'-bipyridyl)ruthenium(II) and dioxetane and their derivatives; Preferably, the nucleoside molecule is any one of the following compounds: More preferably, the nucleoside molecule is any one of the following compounds:

5. A kit or composition, characterized in that, The kit or composition contains the nucleoside molecule as described in any one of claims 1-4; Preferably, the kit or composition further contains a template-dependent DNA polymerase such as KOD polymerase, a non-template-dependent DNA polymerase such as terminal deoxynucleotidyl transferase, and / or reverse transcriptase.

6. A method for labeling nucleic acid molecules, characterized in that, The method includes incorporating the nucleoside molecule as described in any one of claims 1-4 into the nucleic acid molecule, wherein the Base in the nucleoside molecule is a nucleobase linked to a detectable label molecule.

7. A method for regulating nucleic acid synthesis, characterized in that, The method includes: 1) Adding the nucleoside molecule as described in any one of claims 1-4 or the kit or composition as described in claim 5 to the reaction system for nucleic acid synthesis to block the extension of the nucleic acid chain; Or, 2) Adding a cleavage reagent to the reaction system for nucleic acid synthesis containing the nucleoside molecule as described in any one of claims 1-4 to cause the extension of the nucleic acid chain; The cleavage reagent undergoes a cleavage reaction with the nucleoside molecule to convert the nucleoside molecule into the structure shown in formula (II); wherein the definitions of R’, R” and Base are as defined in claims 1-4; Preferably, the nucleic acid chain is single-stranded or double-stranded.

8. The method according to claim 7, characterized in that, The method satisfies one or more of the following conditions: (1) The nucleic acid synthesis is an enzyme-catalyzed nucleic acid amplification; (2) The cleavage reagent is selected from hydrogen peroxide, lanthanide metal salts, and bromides; (3) The cleavage reagent further includes an oxidant; Preferably, it satisfies one or more of the following conditions: (1) The lanthanide metal salts and bromides are selected from CeBr3, Ce(NO3)3-KBr, CeCl3-KBr, Ce(NH4)2(NO3)6-KBr, Ce(OTf)3-KBr, Ce(OAc)3-KBr, and combinations thereof; (2) The oxidant is 0.1%-30% (v / v) H2O2, NaIO4, Oxone, O2+UV, or NaI; (3) The cleavage reaction refers to converting the group at the 3'-position of the sugar ring of the nucleoside molecule into a hydroxyl group at 25-65 °C, for example, using CeBr3 as a catalyst and H2O2 as an oxidant; preferably, the temperature of the cleavage reaction is preferably 40-65 °C, such as 50 °C; (4) The molar ratio of the nucleoside molecule to the catalyst is 1:0.01-0.5; (5) The molar ratio of the nucleoside molecule to the oxidant is 1:1-50; (6) The solvent in the reaction system is selected from acetonitrile, ethanol, tetrahydrofuran, water, and combinations thereof; (7) The reaction time of the reaction system is 3-120 min; More preferably, after removing the group at the 3'-position of the nucleoside molecule, it further includes quenching with a Na2S2O3 solution.

9. A reaction system for group cleavage, characterized in that, The reaction system contains a molecule linking the group and a cleavage reagent; the cleavage reagent undergoes a cleavage reaction with the molecule to separate the group from the molecule; The group is a methylthiomethyl group; Preferably, the cleavage reagent is selected from hydrogen peroxide, lanthanide metal salts, and bromides; More preferably, the cleavage reagent further includes an oxidant; Even more preferably, the reaction system satisfies one or more of the following conditions: (1) The lanthanide metal salts and bromides are selected from CeBr3, Ce(NO3)3-KBr, CeCl3-KBr, Ce(NH4)2(NO3)6-KBr, Ce(OTf)3-KBr, Ce(OAc)3-KBr, and combinations thereof; (2) The oxidizing agent is 0.1%-30% (v / v) H2O2, NaIO4, Oxone, O2+UV, or NaI; (3) The reaction temperature of the reaction system is 25-65 °C, preferably 40-65 °C, such as 50 °C; (4) The reaction time of the reaction system is 3-120 min; (5) The reaction system further includes a quenching agent, such as a Na2S2O3 solution; (6) The molar ratio of the molecule to the lanthanide metal salts and bromides is 1:0.01-0.5; (7) The molar ratio of the molecule to the oxidizing agent is 1:1-50; (8) The solvent in the reaction system is selected from acetonitrile, ethanol, tetrahydrofuran, water, and combinations thereof.

10. A method for preparing the nucleoside molecule according to any one of claims 1-4; it includes any of the following schemes: Scheme One: The method includes the following steps: React the intermediate product shown in formula (b) with a deprotection reagent in a solvent to obtain the product shown in formula (c) Among them, R b is tert-butyldimethylsilyl; The solvent is preferably an alcohol solvent and / or an ether solvent, such as methanol and / or tetrahydrofuran; The deprotecting reagent is preferably NH4F or tetrabutylammonium fluoride; Scheme Two: includes the following steps: Mix the compound shown in formula (II) with dimethyl sulfoxide, acetic acid, and acetic anhydride, and react to obtain the intermediate product shown in formula (b); Preferably, R” is H, and Base is Scheme Three: The method includes the following steps: Reacting the product shown in formula (c) obtained from Scheme 1 with an anhydrous N,N-dimethylformamide solution of POCl3, bis-tri-n-butyl pyrophosphate, and tri-n-butylamine in trimethyl phosphate in the presence of proton sponge to obtain the product shown in formula (d). Alternatively, the product shown in formula (c) obtained from Scheme 1 is reacted with tetrabutylammonium pyrophosphate and 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one in an N,N-dimethylformamide solution to obtain the product shown in formula (d). wherein R 1 is a triphosphate acyl group; Scheme Four: The method comprises the following steps: In an organic solvent, reacting the product shown by formula (d) obtained in Scheme III with ammonia water, or reacting the product shown by formula (d) obtained in Scheme III with an iodine (pyridine / water) solution, to obtain the product shown by formula (e). Among them, Base 1 is The organic solvent is preferably an alcohol solvent, such as methanol.

11. A method for determining the sequence of a polynucleotide, characterized in that, The method includes the following steps: 1) Add the nucleoside molecule according to any one of claims 1-4 or the kit or composition according to claim 5 to the reaction system for polynucleotide synthesis, so that the nucleoside molecule is incorporated into the complementary strand of the polynucleotide to block the extension of the hybrid double strand formed by the free nucleotide and the polynucleotide template; the nucleoside molecule contains a detectable labeling molecule; 2) Obtain the type of the incorporated nucleoside molecule by detecting the detectable labeling molecule contained in the nucleoside molecule; Preferably, after obtaining the type of the incorporated nucleoside molecule, the method further includes converting the group at the 3'-end of the sugar ring of the nucleoside molecule into a hydroxyl group; and / or, the polynucleotide is a single-stranded polynucleotide, preferably, the polynucleotide is DNA or RNA; Preferably, the determination is a synthesis-while-determining; More preferably, the conversion of the group at the 3'-end of the sugar ring of the nucleoside molecule into a hydroxyl group is achieved by a cleavage reaction as described in claim 7 or 8.

12. A method for synthesizing or labeling a polynucleotide, characterized in that, The method includes adding the nucleoside molecule according to any one of claims 1-4 or the kit or composition according to claim 5 to the reaction system for polynucleotide synthesis, so that the nucleoside molecule binds to the 3'-end of the polynucleotide; When the method is used to label a polynucleotide, the nucleoside molecule contains a detectable labeling molecule; Preferably, the polynucleotide is a single-stranded polynucleotide; preferably, the polynucleotide is DNA or RNA.

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