Synthesis method and application of nucleoside monomer analogs and oligonucleotides

Abstract

The present invention provides a method for synthesizing a nucleoside monomer analog and an oligonucleotide and an application thereof. R on the nucleoside monomer is a base; R 1 Includes one or more of methoxy, methoxyethyl, fluorine, hydroxyl or hydrogen; R 2 Includes one or more of hydroxyl, monophosphate, diphosphate, triphosphate or thio-modified phosphate; R 3 It includes one or more of methyl, ethyl, propyl or isopropyl. It can solve the problem of low reversible end-capping activity of oligonucleotides in the prior art and is applicable to the field of oligonucleotide synthesis.

Description

Technical Field

[0001] The present invention relates to the field of oligonucleotide synthesis, and in particular to a synthesis method and application of a nucleoside monomer analog and an oligonucleotide. Background Art

[0002] Oligonucleotides are basic tools for regulating gene expression in biomedical and life science research. As gene-targeted therapeutic drugs, they are used to treat genetic diseases, viruses, tumors and other diseases. Oligonucleotide drugs mainly include antisense oligonucleotides, small interfering RNA, micro RNA, nucleic acid aptamers, etc. Natural oligonucleotides are easily degraded in the body and have low drugability. Therefore, drug oligonucleotides usually carry specific modification groups, such as phosphorothioate diester bonds, fluorinated, methyl and locked nucleic acids, to enhance the stability of oligonucleotides in the body, improve specificity, and reduce their toxic side effects.

[0003] The main method for synthesizing oligonucleotides is the phosphoramidite method, but it has disadvantages such as low purity of the initial product, large amount of chemical solvents used, and difficulty in large-scale amplification. Therefore, the emerging enzymatic method of synthesizing oligonucleotides has attracted much attention due to its many advantages such as green synthesis process, high coupling efficiency, and easy amplification.

[0004] Nucleotide analogs carrying reversible blocking groups are key raw materials for enzymatic synthesis of oligonucleotides. Reversible blocking groups are used to achieve specific synthesis of oligonucleotides and prevent the introduction of erroneous sequences. At the same time, the blocking groups can be removed seamlessly after the synthesis is completed to achieve the synthesis of the target oligonucleotide.

[0005] However, the types of reversible blocking groups of oligonucleotides in the prior art are relatively small and their activities are relatively low, resulting in low coupling efficiency with the oligonucleotide chain, thereby hindering the synthesis of the target oligonucleotide. Summary of the invention

[0006] The main purpose of the present invention is to provide a synthesis method and application of a nucleoside monomer analog and an oligonucleotide, so as to solve the problem of low reversible end-capping activity of oligonucleotides in the prior art.

[0007] In order to achieve the above object, according to the first aspect of the present invention, a nucleoside monomer analog is provided, and the structural formula of the nucleoside monomer analog is shown in Formula I;

[0008] Formula I;

[0009] Wherein, R is a base; R1 includes one or more of methoxy, methoxyethyl, fluorine, hydroxyl or hydrogen;

[0010] R2 includes one or more of hydroxyl, monophosphate, diphosphate, triphosphate or thio-modified phosphate; R3 includes one or more of methyl, ethyl, propyl or isopropyl.

[0011] Furthermore, the base includes a natural base or a non-natural base; the natural base includes adenine, guanine, cytosine, thymine or uracil; the non-natural base includes 3-deazaadenine, 7-deazaguanine, 2,6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamide uracil, 5-methylcytosine, 5-ethynyl uracil, C7-modified deazaadenine, C7-modified deazaguanine, C5-modified cytosine or C5-modified uracil, wherein the modification includes one or more of methyl modification, H modification, Cl modification or F modification.

[0012] According to a second aspect of the present invention, a nucleotide monomer analog is provided, and the structural formula of the nucleotide analog is shown in Formula II:

[0013] Formula II;

[0014] Among them, R is a base; R4 includes one or more of methoxy, methoxyethyl, fluorine, hydroxyl or hydrogen; R5 includes one or more of methyl, ethyl, propyl or isopropyl; R6 includes hydroxyl or thiol.

[0015] Furthermore, the base includes a natural base or a non-natural base; the natural base includes adenine, guanine, cytosine, thymine or uracil; the non-natural base includes 3-deazaadenine, 7-deazaguanine, 2,6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamide uracil, 5-methylcytosine, 5-ethynyl uracil, C7-modified deazaadenine, C7-modified deazaguanine, C5-modified cytosine or C5-modified uracil, wherein the modification includes one or more of methyl modification, H modification, Cl modification or F modification.

[0016] According to the third aspect of the present application, a method for synthesizing an oligonucleotide having a 3'-end reversibly modified end is provided, the method comprising: a) when the oligonucleotide chain substrate is a single strand, connecting the above-mentioned nucleoside monomer analog, or the above-mentioned nucleotide monomer analog, to the 3'-end of the oligonucleotide chain substrate to obtain an oligonucleotide having a 3'-end reversibly modified end;

[0017] Or b) when there are two oligonucleotide chain substrates, the oligonucleotide chain substrates include a first oligonucleotide chain substrate and a second oligonucleotide chain substrate; wherein the 3' end of the first oligonucleotide chain substrate is connected to the above-mentioned nucleoside monomer analogue, or the above-mentioned nucleotide monomer analogue; the first oligonucleotide chain substrate and the second oligonucleotide chain substrate are connected to obtain an oligonucleotide with a reversibly modified end at the 3' end.

[0018] Furthermore, the oligonucleotide chain substrate includes a natural oligonucleotide chain composed of natural nucleotides, or a non-natural oligonucleotide chain containing non-natural nucleotides.

[0019] Further, the non-natural oligonucleotide chain consists of non-natural nucleotides.

[0020] Furthermore, when R2 of the nucleoside monomer analog is a hydroxyl group, a monophosphate, a diphosphate or a thio-modified phosphate, the synthesis method comprises: a-1) mixing the nucleoside monomer analog and the polyphosphate, and using phosphokinase to catalyze to form a nucleoside monomer analog in which R2 is a triphosphate; when the oligonucleotide chain substrate is a single strand, using polymerase to catalyze the connection of the nucleoside monomer analog in which R2 is a triphosphate to the 3' end of the oligonucleotide chain substrate, thereby obtaining an oligonucleotide having a reversibly modified end at the 3' end; b-1) when there are two oligonucleotide chain substrates, wherein the 3' end of the first oligonucleotide chain substrate is connected with the above-mentioned nucleoside monomer analog, and using a ligase to catalyze the connection of the first oligonucleotide chain substrate and the second oligonucleotide chain substrate, thereby obtaining an oligonucleotide having a reversibly modified end at the 3' end; wherein the phosphokinase comprises one or more of acetate kinase, pyruvate kinase, adenylate kinase, polyphosphate kinase, nucleoside kinase or nucleoside diphosphate kinase.

[0021] Furthermore, when R2 of the nucleoside monomer analogue is a triphosphate, the synthesis method includes: a-2) when there is a single oligonucleotide chain substrate, using a polymerase to catalyze the connection of the nucleoside monomer analogue whose R2 is a triphosphate to the 3' end of the oligonucleotide chain substrate, thereby obtaining an oligonucleotide with a reversibly modified end at the 3' end; b-2) when there are two oligonucleotide chain substrates, wherein the nucleoside monomer analogue is connected to the 3' end of the first oligonucleotide chain substrate, and using a ligase to catalyze the connection of the first oligonucleotide chain substrate and the second oligonucleotide chain substrate, thereby obtaining an oligonucleotide with a reversibly modified end at the 3' end.

[0022] Further, the synthesis method comprises: a-3) when the oligonucleotide chain substrate is a single strand, using a polymerase to catalyze the connection of a nucleotide monomer analog to the 3' end of the oligonucleotide chain substrate, thereby obtaining an oligonucleotide having a reversibly modified end at the 3' end;

[0023] b-3) When there are two oligonucleotide chain substrates, the 3' end of the first oligonucleotide chain substrate is connected to the above-mentioned nucleotide monomer analog, and the first oligonucleotide chain substrate and the second oligonucleotide chain substrate are connected by ligase to obtain an oligonucleotide with a reversibly modified 3' end.

[0024] Furthermore, the polymerase includes PUP polymerase; and the ligase includes T4 ligase.

[0025] According to the fourth aspect of the present application, a method for preparing an oligonucleotide is provided, the method comprising: removing the reversibly modified end at the 3' end of an oligonucleotide having a reversibly modified end at the 3' end synthesized by any of the above-mentioned synthesis methods to obtain an oligonucleotide.

[0026] Furthermore, the removal includes: mixing the oligonucleotide having the 3'-end reversibly modified end with the first solution to obtain a mixed system, and using the mixed system to achieve the removal of the 3'-end reversibly modified end.

[0027] Furthermore, the pH of the mixed system is 4-6, and the mixed system is maintained at a temperature of 20-40° C. for 5-10 minutes, thereby achieving the removal of the reversible modified end at the 3' end.

[0028] Furthermore, the first solution includes any one or more of the following: hydrochloric acid, sulfuric acid, phosphoric acid, disodium hydrogen phosphate, dipotassium hydrogen phosphate, citric acid or boric acid.

[0029] Furthermore, the concentration of the solute in the first solution is 0.01 mM~10M.

[0030] According to the fifth aspect of the present application, there is provided a method for synthesizing any one of the above-mentioned nucleoside monomer analogs, or any one of the above-mentioned nucleotide monomer analogs, or any one of the above-mentioned oligonucleotides having a 3'-end reversibly modified end, or a method for preparing any one of the above-mentioned oligonucleotides, and their use in preparing oligonucleotides.

[0031] By applying the technical solution of the present invention and utilizing a nucleoside monomer analogue having a structure of Formula I, the 3' reversibly modified end can be connected to an oligonucleotide chain, and an oligonucleotide having a 3' reversibly modified end can be successfully synthesized. Compared with the reversible end-capping commonly used in the prior art, the oligonucleotide has higher activity and a higher substrate conversion rate, and the target oligonucleotide can be synthesized after further removing the reversible modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0033] Figure 1A schematic diagram of UPLC detection results of two products of phosphoric acid connected to reaction system 1 according to Example 11 of the present invention is shown.

[0034] Figure 2 A schematic diagram of the UPLC detection results of the product of linked nucleoside monomers according to Example 12 of the present invention is shown.

[0035] Figure 3 A schematic diagram of the UPLC detection results of the product after removal of the 3'-end reversible blocked end according to Example 12 of the present invention is shown. DETAILED DESCRIPTION

[0036] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0037] Terminology explanation:

[0038] Nucleoside: A compound formed by the condensation of a base (purine or pyrimidine base) and a pentose sugar (including but not limited to ribose or deoxyribose). Nucleosides do not contain phosphate.

[0039] Nucleotide: A combination of a base (purine or pyrimidine), a pentose sugar (including but not limited to ribose or deoxyribose) and a phosphate, including nucleoside monophosphate, nucleoside diphosphate and nucleoside triphosphate.

[0040] Nucleoside monomer: refers to a single nucleoside with a length of 1 nt in this application.

[0041] Nucleotide monomer: refers to a single nucleotide with a length of 1 nt in this application.

[0042] As mentioned in the background art, in the prior art, the reversible blocking group at the 3' end of the oligonucleotide has low activity, resulting in low efficiency in connection with the oligonucleotide chain, which hinders the synthesis of the target oligonucleotide. Therefore, in the present application, the inventors attempt to develop a new nucleoside monomer that can connect the 3' end reversible blocking group to the oligonucleotide by enzymatic method, improve the conversion rate of the substrate, and have higher activity compared to the reversible blocking end capping of the prior art, and propose a series of protection schemes of the present application.

[0043] In a first typical embodiment of the present application, a nucleoside monomer analog is provided, and the structural formula of the nucleoside monomer analog is shown in Formula I;

[0044] Formula I;

[0045] Among them, R is a base; R1 includes one or more of methoxy, methoxyethyl, fluorine, hydroxyl or hydrogen; R2 includes one or more of hydroxyl, monophosphate, diphosphate, triphosphate or thio-modified phosphate; R3 includes one or more of methyl, ethyl, propyl or isopropyl.

[0046] Nucleoside monomer analogs include nucleoside monomer analogs and nucleotide monomer analogs; when the group at R2 is monophosphate, diphosphate, triphosphate or thio-modified phosphate, the nucleoside monomer is a nucleotide monomer analog; when the group at R2 is a hydroxyl group, the nucleoside monomer is a nucleoside monomer analog.

[0047] In the prior art system of enzymatically synthesized oligonucleotides, preliminary studies of this application have found that for certain specific reactions, the efficiency of the reaction is associated with the reversible blocking of the 3' end, resulting in poor commonly used blocking activity, low substrate conversion rate, and difficulty in subsequently synthesizing the target oligonucleotide.

[0048] The present application utilizes the nucleoside monomer analogue of the structural formula shown in Formula I to couple with the oligonucleotide chain substrate in the system of enzymatic synthesis of oligonucleotides, so that its 3' end is successfully connected to the reversible closed end, and the conversion rate of the substrate is high. It plays a role in the synthesis of oligonucleotides, prevents the connection of wrong sequences, and can be removed by simple acid hydrolysis later. The oligonucleotide product with the group removed can be used as the oligonucleotide chain substrate for the next round of coupling of nucleoside monomers, realizing the enzymatic specific synthesis of target oligonucleotides of specific sequences.

[0049] That is, the present application has developed a new reversible end-capping different from the prior art. When the reaction has the phenomenon of low substrate conversion rate caused by the above-mentioned association with the 3'-end reversible end-capping, the present application provides a variety of reversible end-capping options, which can overcome this association phenomenon and help synthesize various types of target oligonucleotides. When the R2 group of the above-mentioned nucleoside monomer analog is a triphosphate group, the nucleoside monomer is a nucleotide monomer analog, and its structural formula is shown in Formula III. Under the catalytic action of the polymerase, with a single oligonucleotide chain substrate as a template, the nucleotide monomer shown in Formula III can be directly coupled with the oligonucleotide chain substrate to synthesize an oligonucleotide with a 3'-end reversible blocked end.

[0050] Formula III.

[0051] In a preferred embodiment, the base includes a natural base or a non-natural base. The natural base includes natural adenine, natural guanine, natural cytosine, natural thymine or natural uracil; the non-natural base includes 3-deazaadenine, 7-deazaguanine, 2,6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamide uracil, 5-methylcytosine, 5-ethynyl uracil, C7 modified deazaadenine, C7 modified deazaguanine, C5 modified cytosine or C5 modified uracil, wherein the modification includes one or more of methyl modification, H modification, Cl modification or F modification.

[0052] In a second typical embodiment of the present application, a nucleotide monomer analog is provided, and the structural formula of the nucleotide analog is shown in Formula II:

[0053] Formula II;

[0054] Among them, R is a base; R4 includes one or more of methoxy, methoxyethyl, fluorine, hydroxyl or hydrogen; R5 includes one or more of methyl, ethyl, propyl or isopropyl; R6 includes hydroxyl or thiol.

[0055] By using the nucleotide monomer analogues as shown in Formula II in the present application, an oligonucleotide with a reversibly modified end at the 3' end can be synthesized. In the case of a single oligonucleotide chain substrate, the above-mentioned nucleotide monomer analogues are mixed with a polymerase, and the oligonucleotide chain is used as a synthetic chain to catalyze the coupling of the nucleotide monomer to the oligonucleotide chain, so that an oligonucleotide with a reversibly modified end at the 3' end can be obtained, and then the target oligonucleotide can be further synthesized.

[0056] Or in the case of two oligonucleotide chain substrates, one of the substrates has the above-mentioned nucleotide monomer analog connected to its 3' end, and the other 3' end does not contain the above-mentioned nucleotide monomer, and the ligase can be used to catalyze the formation of a phosphodiester bond between the two substrates, and finally an oligonucleotide with a reversibly modified end at the 3' end can be formed, and then the target oligonucleotide can be further synthesized. The above two methods are simple to operate, and both can synthesize oligonucleotides with reversible modification groups at the 3' end in one step, and are easy to remove, which is conducive to the synthesis of target oligonucleotides.

[0057] In a preferred embodiment, the base includes a natural base or a non-natural base; the natural base includes adenine, guanine, cytosine, thymine or uracil; the non-natural base includes 3-deazaadenine, 7-deazaguanine, 2,6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamide uracil, 5-methylcytosine, 5-ethynyl uracil, C7-modified deazaadenine, C7-modified deazaguanine, C5-modified cytosine or C5-modified uracil. One or more of the modifications include methyl modification, H modification, Cl modification, and F modification.

[0058] In a second typical embodiment of the present application, a method for synthesizing an oligonucleotide having a 3'-end reversibly modified end is provided, the method comprising: a) when the oligonucleotide chain substrate is a single strand, connecting the above-mentioned nucleoside monomer analog, or the above-mentioned nucleotide monomer analog, to the 3'-end of the oligonucleotide chain substrate to obtain an oligonucleotide having a 3'-end reversibly modified end;

[0059] Or b) when there are two oligonucleotide chain substrates, the oligonucleotide chain substrates include a first oligonucleotide chain substrate and a second oligonucleotide chain substrate; wherein the 3' end of the first oligonucleotide chain substrate is connected to the above-mentioned nucleoside monomer analogue, or the above-mentioned nucleotide monomer analogue; the first oligonucleotide chain substrate and the second oligonucleotide chain substrate are connected to obtain an oligonucleotide with a reversibly modified end at the 3' end.

[0060] When there are two oligonucleotide chain substrates, the last nucleoside monomer at the 3' end of one of the oligonucleotide chain substrates is connected with a reversible end cap, i.e. the first oligonucleotide chain substrate mentioned above, and the nucleoside monomer is connected to the remaining oligonucleotide chain in front of it through a phosphate bond, and the reversible end cap can be connected to the 3' end of the oligonucleotide chain substrate by the method of the present application or chemical synthesis method or other means known to those skilled in the art, and those skilled in the art can flexibly select according to actual needs.

[0061] Under the catalytic action of the ligase, the above-mentioned first oligonucleotide chain substrate with a reversibly blocked end at the 3' end is connected to the second oligonucleotide chain substrate to obtain an oligonucleotide with a reversibly modified end at the 3' end.

[0062] In a preferred embodiment, the oligonucleotide chain substrate includes a natural oligonucleotide chain composed of natural nucleotides, or a non-natural oligonucleotide chain containing non-natural nucleotides.

[0063] In a preferred embodiment, the non-natural oligonucleotide chain consists of non-natural nucleotides.

[0064] Non-natural nucleotides (xeno-nucleic acid, XNA) are a class of nucleic acid molecules with non-natural backbones or nucleic acid bases. Preferably, non-natural nucleotides include: ribonucleotides with one or more of the following modifications: ribose 2' modification, ribose backbone modification, base modification or phosphate backbone modification; preferably, ribose 2' modification includes 2'-methoxy modification, 2'-fluorine modification, 2'-hydrogen modification, 2'-methoxyethyl modification, 2'-FANA modification, locked nucleic acid modification or hexitol nucleic acid modification; preferably, ribose backbone modification includes replacing ribose in the nucleotide with ribuloNA, TNA, tPhoNA or dXNA; preferably, base modification includes deazaadenine C7 modification, deazaguanosine C7 modification, cytosine C5 modification or uridine C5 modification; phosphate backbone modification includes PS modification.

[0065] In a preferred embodiment, when R2 of the nucleoside monomer analog is hydroxyl, monophosphate, diphosphate or thio-modified phosphate, the synthesis method comprises: mixing the nucleoside monomer analog and polyphosphate, and using phosphokinase to catalyze to form a nucleoside monomer analog in which R2 is triphosphate; a-1) when there is a single oligonucleotide chain substrate, using polymerase to catalyze the connection of the nucleoside monomer analog in which R2 is triphosphate to the 3' end of the oligonucleotide chain substrate, thereby obtaining an oligonucleotide having a reversibly modified end at the 3' end; b-1) when there are two oligonucleotide chain substrates, wherein the 3' end of the first oligonucleotide chain substrate is connected to the above-mentioned nucleoside monomer analog, and using ligase to catalyze the connection of the first oligonucleotide chain substrate and the second oligonucleotide chain substrate, thereby obtaining an oligonucleotide having a reversibly modified end at the 3' end; the phosphokinase comprises one or more of acetate kinase, pyruvate kinase, adenylate kinase, polyphosphate kinase, nucleoside kinase or nucleoside diphosphate kinase. When R2 of the above nucleoside monomer analog is one or more of hydroxyl, monophosphate, diphosphate or thio-modified phosphate, polyphosphate kinase is used to connect the R2 end of the nucleoside monomer analog to phosphate to form a nucleoside monomer analog with three phosphates in R2.

[0066] The above-mentioned synthesis method of the oligonucleotide chain substrate includes: mixing the above-mentioned nucleoside monomer analogs in which R2 is one or more of hydroxyl, monophosphate, diphosphate or thio-modified phosphate with a phosphate donor, Tris-HCl, and MgCl2, maintaining the pH value of the reaction system at 6-9, adding kinase, reacting at 0-80°C for 0.1-100h, adding acetonitrile of 1 volume of the reaction system to inactivate the enzyme after the reaction, and the supernatant after centrifugation contains a nucleoside monomer in which R2 has three phosphates.

[0067] The system containing the nucleoside monomer with three phosphate groups in R2, the oligonucleotide chain substrate, Tris-HCl, MnCl2 and NaCl are mixed, the pH value of the reaction system is maintained at 6-9, a polymerase is added, and the reaction is carried out at 0-100° C. for 0.1-100 hours. After the reaction is completed, acetonitrile with a volume of 1 times the reaction system is added to inactivate the enzyme, and the supernatant after centrifugation contains an oligonucleotide with a reversibly modified end at the 3' end.

[0068] In a preferred embodiment, when R2 of the nucleoside monomer analog is triphosphate, the synthesis method comprises: a-2) when there is a single oligonucleotide chain substrate, using a polymerase to catalyze the connection of the nucleoside monomer analog in which R2 is triphosphate to the 3' end of the oligonucleotide chain substrate, thereby obtaining an oligonucleotide having a reversibly modified end at the 3' end; b-2) when there are two oligonucleotide chain substrates, wherein the 3' end of the first oligonucleotide chain substrate is connected to the above-mentioned nucleoside monomer analog, and using a ligase to catalyze the connection of the first oligonucleotide chain substrate and the second oligonucleotide chain substrate, thereby obtaining an oligonucleotide having a reversibly modified end at the 3' end.

[0069] When R2 of the nucleoside monomer analog is triphosphate, the nucleoside monomer can directly react with polymerase and oligonucleotide chain substrate, and couple to the oligonucleotide chain substrate to achieve the synthesis of oligonucleotide with 3' reversibly modified end in one step.

[0070] In some preferred embodiments, the synthesis method includes: when the oligonucleotide chain substrate is a single strand, a nucleoside monomer analog having three phosphates in R2, an oligonucleotide chain substrate, Tris-HCl, MnCl2, and NaCl are mixed, the pH value of the reaction system is maintained at 6 to 9, a polymerase is added, and the reaction is carried out at 1 to 100° C. for 0.1 to 100 hours. After the reaction is completed, acetonitrile with a volume of 1 times the reaction system is added to inactivate the enzyme, and the supernatant after centrifugation contains an oligonucleotide with a reversibly modified end at the 3' end.

[0071] When there are two oligonucleotide chain substrates, the 3' end of the first oligonucleotide chain substrate is connected with the above-mentioned R2 nucleoside monomer analog with three phosphates; the first oligonucleotide chain substrate, the second oligonucleotide chain substrate, RNA ligase, MgCl2, ATP, and Tris-HCl are mixed, the pH of the reaction system is maintained at 7-7.8, and incubated at 1-50°C for 1-50h: after the reaction is completed, acetonitrile with 1 volume of the reaction system is added to inactivate the enzyme, and the supernatant after centrifugation contains an oligonucleotide with a reversibly modified end at the 3' end.

[0072] In a preferred embodiment, the synthesis method comprises:

[0073] a-3) when the oligonucleotide chain substrate is a single strand, using a polymerase to catalyze the connection of a nucleotide monomer analog to the 3' end of the oligonucleotide chain substrate, thereby obtaining an oligonucleotide having a reversibly modified end at the 3' end;

[0074] b-3) When there are two oligonucleotide chain substrates, the 3' end of the first oligonucleotide chain substrate is connected to the above-mentioned nucleotide monomer analog, and the first oligonucleotide chain substrate and the second oligonucleotide chain substrate are connected by ligase to obtain an oligonucleotide with a reversibly modified 3' end.

[0075] The method for synthesizing an oligonucleotide with a 3'-end reversibly modified end in which a nucleotide monomer analogue participates in the present application is the method for synthesizing the oligonucleotide when R2 of the nucleoside monomer analogue in the present application is triphosphate as mentioned above.

[0076] In a preferred embodiment, the polymerase comprises PUP polymerase; and the ligase comprises T4 ligase.

[0077] The PUP polymerase is derived from fission yeast, and its NCBI accession number is NP_594901.1; the T4 ligase is derived from Escherichia phage T4, and its NCBI accession number is NP_049790.1.

[0078] The polymerase adds a nucleoside monomer with a cap to the 3' end of the nucleic acid chain, so that the extended nucleic acid chain has a 3' reversible cap. The ligase adds a short oligonucleotide chain with a cap to the 3' end of the nucleic acid chain, so that the extended oligonucleotide chain has a 3' reversible cap.

[0079] In a third typical embodiment of the present application, a method for preparing an oligonucleotide is provided, the method comprising: removing the reversibly modified end at the 3' end of the oligonucleotide having a 3' reversibly modified end synthesized by the above-mentioned synthesis method to obtain the oligonucleotide.

[0080] Using the synthesis method of the present application, the synthesized oligonucleotide with a reversibly modified end at the 3' end can be removed by simple acid hydrolysis in the subsequent step, thereby achieving reversible modification. The oligonucleotide with the modified end at the 3' end removed can also be used as a substrate for the next round of coupling with a nucleoside monomer. Using the nucleoside monomer analog or nucleotide monomer analog of the present application, the reversible modification of the 3' end of the oligonucleotide can be achieved, and the target oligonucleotide of a specific sequence can be specifically synthesized by enzymatic method.

[0081] In a preferred embodiment, the removal comprises: mixing the oligonucleotide having a 3'-end reversibly modified end with the first solution to obtain a mixed system, and removing the 3'-end reversibly modified end using the mixed system.

[0082] In a preferred embodiment, the pH of the mixed system is 4-6, and the mixed system is maintained at a temperature of 20-40°C for 5-10 minutes, thereby achieving the removal of the 3'-end reversible modification end. The removal conditions include pH 4-6, including but not limited to 4, 5, or 6; temperature 20-40°C, including but not limited to 20°C, 25°C, 30°C, 35°C or 40°C, and time 5-10 minutes, including but not limited to 5, 6, 7, 8, 9 or 10 minutes.

[0083] In a preferred embodiment, the first solution includes any one or more of the following: hydrochloric acid, sulfuric acid, phosphoric acid, disodium hydrogen phosphate, dipotassium hydrogen phosphate, citric acid or boric acid.

[0084] In a preferred embodiment, the concentration of the solute in the first solution is 0.01 mM~10M.

[0085] The present application utilizes simple acid hydrolysis to achieve the removal of the reversibly modified end of the 3' end of the nucleoside monomer coupled to the oligonucleotide chain. By controlling the conditions of the acid hydrolysis reaction for removal, the effective removal of the reversibly modified end can be promoted, thereby facilitating the subsequent synthesis of the target oligonucleotide.

[0086] In a fourth typical embodiment of the present application, a method for synthesizing any of the above nucleoside monomers, or any of the above oligonucleotides having a 3'-end reversibly modified end, or any of the above oligonucleotide preparation methods is provided, and their use in preparing oligonucleotides.

[0087] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0088] Unless otherwise specified, the reagents in the examples of this application are conventional commercially available products.

[0089] Example 1

[0090] Add the reaction system to a clean container so that the oligonucleotide chain substrate is 50 µM, add PUP polymerase (derived from fission yeast PUP polymerase, NCBI accession number is NP_594901.1) to make its concentration 0.2 mg / mL, add 10mM MnCl2, 50mM NaCl, 50mM Tris-HCl, adjust the pH to 8.0, and add nucleoside monomer analogs to make their concentration 700 µM. The structural formula of the nucleoside monomer analogs in this example is as follows:

[0091] ;

[0092] After reacting at 37°C for 2 h, acetonitrile of 1 volume of the reaction system was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000 rpm, and the supernatant was subjected to UPLC detection.

[0093] The oligonucleotide chain substrates of this example are substrate 1, substrate 2 and substrate 3 (SEQ ID NO: 1), and their sequences and the results of UPLC detection of the supernatant after synthesis and connection of nucleoside monomer analogs are shown in Table 1.

[0094] The UPLC detection results of this example show that the 3' ends of different oligonucleotide chain substrates are all coupled with one nucleoside monomer analog, indicating that the nucleoside monomer analog shown in the above structural formula can be used for enzymatic synthesis of oligonucleotides.

[0095] Table 1

[0096]

[0097] In Table 1, “++++” indicates that the product was detected with a conversion rate of 0.1% to 30%; “+++++” indicates that the product was detected with a conversion rate of 30% to 90%.

[0098] In the present application, "m" after A, C, G or U represents 2' methoxy modification of the ribonucleotide, "f" represents 2' fluoro modification of the ribonucleotide, and "s" represents thio modification of the 5' phosphate of the ribonucleotide.

[0099] Example 2

[0100] Add the reaction system into a clean container so that the two oligonucleotide chain substrate fragments are 50 mM. The two oligonucleotide chain substrate fragments of this embodiment are shown in the following structural formula, wherein the 3'OH end of the first oligonucleotide chain substrate fragment is connected to the reversibly blocked modified end (nucleoside monomer analog) of the present application, i.e., the framed portion in the following structural formula:

[0101] First oligonucleotide strand substrate fragment:

[0102] ;

[0103] Second oligonucleotide strand substrate fragment:

[0104] .

[0105] RNA ligase (from Escherichia phage T4, NCBI accession number NP_049790.1) was added to a concentration of 0.2 mg / mL, 10 mM MgCl2, 250 mM ATP, 50 mM Tris-HCl were added, the pH was adjusted to 7.5, and incubated at 16°C for 16 h: 1 volume of acetonitrile was added to the reaction system to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000 rpm, and the supernatant was detected by LCMS.

[0106] Since the 3'OH end of the first oligonucleotide fragment has a reversible end-capping modification, theoretically only a molecular weight of 2618.83±2 can be detected, and the molecular weight of the product of two or more oligonucleotide fragments cannot be detected. After mass spectrometry, only a molecular weight of 2618.79 was detected, indicating that the connection was successful.

[0107] Example 3

[0108] Add the reaction system to a clean container, so that the oligonucleotide chain substrate (5'HO-Cms-Ams-Gm-Am-3'OH, that is, substrate 1 in Example 1) is 50 µM, add PUP polymerase to make its concentration 0.2 mg / mL, add 10mM MnCl2, 50mM NaCl, 50mM Tris-HCl, adjust the pH to 8.0, and add nucleotide monomer analogs to make their concentration 700 µM. The structural formula of the nucleoside monomer analog in this example is as follows:

[0109] ;

[0110] After reacting at 37°C for 4 hours, acetonitrile of 1 volume of the reaction system was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000 rpm, and the supernatant was subjected to UPLC detection. The product of an oligonucleotide with a nucleoside monomer coupled to the 3' end was detected, and the conversion rate was 20%, indicating that the nucleoside monomer analog of this example can be used for enzymatic synthesis of oligonucleotides.

[0111] Example 4

[0112] Add the reaction system to a clean container, so that the oligonucleotide chain substrate (5'HO-Cms-Ams-Gm-Am-3'OH, that is, substrate 1 in Example 1) is 50 µM, add PUP polymerase to make its concentration 0.2 mg / mL, add 10mM MnCl2, 50mM NaCl, 50mM Tris-HCl, adjust the pH to 8.0, and add nucleotide monomer analogs to make their concentration 700 µM. The structural formula of the nucleoside monomer analog in this example is as follows:

[0113] ;

[0114] After reacting at 37°C for 4 hours, acetonitrile of 1 volume of the reaction system was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000 rpm, and the supernatant was subjected to UPLC detection. The product of an oligonucleotide with a nucleoside monomer analog coupled to the 3' end was detected, and the conversion rate was 9%, indicating that the nucleoside monomer analog of this example can be used for enzymatic synthesis of oligonucleotides.

[0115] Example 5

[0116] Add the reaction system to a clean container, so that the oligonucleotide chain substrate (5'HO-Cms-Ams-Gm-Am-3'OH, that is, substrate 1 in Example 1) is 50 µM, add PUP polymerase to make its concentration 0.2 mg / mL, add 10mM MnCl2, 50mM NaCl, 50mM Tris-HCl, adjust the pH to 8.0, and add nucleotide monomer analogs to make their concentration 700 µM. The structural formula of the nucleoside monomer analog in this example is as follows:

[0117] ;

[0118] After reacting at 37°C for 4 hours, acetonitrile of 1 volume of the reaction system was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000 rpm, and the supernatant was subjected to UPLC detection. The product of an oligonucleotide with a nucleoside monomer analog coupled to the 3' end was detected, and the conversion rate was 1%, indicating that the nucleotide monomer analog of this example can be used for enzymatic synthesis of oligonucleotides.

[0119] Example 6

[0120] Add the reaction system to a clean container so that the oligonucleotide chain substrate (SEQ ID NO: 1) is 50 µM, add PUP polymerase to a concentration of 0.2 mg / mL, add 10 mM MnCl2, 50 mM NaCl, 50 mM Tris-HCl, adjust the pH to 8.0, and add nucleotide monomer analogs to a concentration of 700 µM. The structural formula of the nucleoside monomer analog in this example is as follows:

[0121] ;

[0122] After reacting at 37°C for 2 hours, acetonitrile of 1 volume of the reaction system was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000 rpm, and the supernatant was subjected to UPLC detection. The product of an oligonucleotide with a nucleoside monomer analog coupled to the 3' end was detected, and the conversion rate was 43%, indicating that the nucleoside monomer analog of this example can be used for enzymatic synthesis of oligonucleotides.

[0123] Example 7

[0124] Add the reaction system to a clean container, so that the oligonucleotide chain substrate (5'HO-Cms-Ams-Gm-Am-3'OH, that is, substrate 1 in Example 1) is 50 µM, add PUP polymerase to make its concentration 0.2 mg / mL, add 10mM MnCl2, 50mM NaCl, 50mM Tris-HCl, adjust the pH to 8.0, and add nucleotide monomer analogs to make their concentration 700 µM. The structural formula of the nucleoside monomer analog in this example is as follows:

[0125] ;

[0126] After reacting at 37°C for 16 hours, acetonitrile of 1 volume of the reaction system was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000 rpm, and the supernatant was subjected to UPLC detection. The product of an oligonucleotide with a nucleoside monomer analog coupled to the 3' end was detected, and the conversion rate was 0.6%, indicating that the nucleoside monomer analog of this example can be used for enzymatic synthesis of oligonucleotides.

[0127] Example 8

[0128] Add the reaction system to a clean container so that the oligonucleotide chain substrate (SEQ ID NO: 1) is 50 µM, add PUP polymerase to a concentration of 0.2 mg / mL, add 10 mM MnCl2, 50 mM NaCl, 50 mM Tris-HCl, adjust the pH to 8.0, and add nucleotide monomer analogs to a concentration of 700 µM. The structural formula of the nucleoside monomer analog in this example is as follows:

[0129] ;

[0130] After reacting at 37°C for 1 hour, acetonitrile of 1 volume of the reaction system was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000 rpm, and the supernatant was subjected to UPLC detection. The product of an oligonucleotide with a nucleoside monomer analog coupled to the 3' end was detected, and the conversion rate was 83%, indicating that the nucleoside monomer analog of this example can be used for enzymatic synthesis of oligonucleotides.

[0131] Example 9

[0132] Add the reaction system to a clean container, so that the oligonucleotide chain substrate (5'HO-Cms-Ams-Gm-Am-3'OH, that is, substrate 1 in Example 1) is 50 µM, add PUP polymerase to make its concentration 0.2 mg / mL, add 10mM MnCl2, 50mM NaCl, 50mM Tris-HCl, adjust the pH to 8.0, and add nucleoside monomer analogs to make their concentration 700 µM. The structural formula of the nucleoside monomer analogs in this example is as follows:

[0133] ;

[0134] After reacting at 37°C for 2 hours, acetonitrile of 1 volume of the reaction system was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000rpm. The supernatant was subjected to MS detection, and the result showed that the product molecular weight of 1693.3 was detected, which was the oligonucleotide chain substrate coupled with 1 molecule of nucleoside monomer analog, and the product coupled with 2 or more nucleoside monomers was not detected. The nucleoside monomer shown in the formula was effectively capped.

[0135] Example 10

[0136] The nucleoside monomers of this embodiment include three nucleotide monomer analogs as shown below:

[0137] First nucleoside monomer analog ;

[0138] Second nucleoside monomer analog ;

[0139] Third nucleoside monomer analog ;

[0140] Add the reaction system to three clean containers respectively, so that the oligonucleotide chain substrate (5'HO-Cms-Ams-Gm-Am-3'OH, i.e., substrate 1 in Example 1) is 50 µM, add polymerase to make its concentration 0.2 mg / mL, add 10mM MnCl2, 50mM NaCl, 50mM Tris-HCl, adjust the pH to 8.0, and add the above three nucleotide monomer analogs respectively to make their concentration 700 µM.

[0141] After reacting at 37°C for 16 h, acetonitrile of 1 volume of the reaction system was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000 rpm, and the supernatant was detected by mass spectrometry.

[0142] The theoretical molecular weights of the oligonucleotides synthesized by the three reaction systems were 1706.53, 1720.37, and 1720.37, respectively. Mass spectrometry detected peaks of molecular weights of 1705.17, 1719.42, and 1719.40, respectively, indicating that the three nucleoside monomers shown in the above structural formulas can be used for enzymatic synthesis of oligonucleotides.

[0143] Embodiment 11

[0144] In a clean container, a nucleoside monomer analog is added to a concentration of 2000 μM, i.e., reaction system 1. The nucleoside monomer analog of this example is shown in the following formula:

[0145] ;

[0146] 15mM MnCl2, 0.5mg / ml (NaPO3)6, 100mM Tris-HCl were added to reaction system 1, the pH was adjusted to 8.0, polyphosphate kinase (the polyphosphate kinase was derived from Meiothermus ruber, PDB: 5LC9) was added to make its concentration 0.5mg / mL, and after reacting at 30℃ for 16h, 1 volume of acetonitrile was added to the reaction sample to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000rpm. The conversion was detected by UPLC. The schematic diagram of UPLC results is shown in the figure. Figure 1 As shown, two products 1 and product 2 produced by the present system and the nucleoside monomer analogs of the present application were detected.

[0147] Reaction system 2 was added to a clean container so that the oligonucleotide chain substrate (5'HO-Cms-Ams-Gm-Am-3'OH, i.e., substrate 1 in Example 1) was 50 µM, PUP polymerase was added to make its concentration 0.2 mg / mL, 10 mM MnCl2, 50 mM NaCl, 50 mM Tris-HCl were added, the pH was adjusted to 8.0, and the above-mentioned reaction system 1 was added so that the concentration of reaction system 2 was 700 µM. After reacting at 37°C for 2 h, acetonitrile of 1 volume of the reaction system was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000 rpm. The supernatant was subjected to UPLC detection, and the generation of the target product coupled with an oligonucleoside chain of product 2 was detected, indicating that the nucleoside monomer of this example can be applied to the enzymatic synthesis of oligonucleotides.

[0148] Example 12

[0149] Add the reaction system to a clean container so that the oligonucleotide chain substrate (5'HO-Cms-Ams-Gm-Am-3'OH, i.e., substrate 1 in Example 1) is 50 µM, add PUP polymerase to a concentration of 0.2 mg / mL, add 10 mM MnCl2, 50 mM NaCl, 50 mM Tris-HCl, adjust the pH to 8.0, and add 35 µM of the fourth nucleoside monomer analog and 665 µM of the fifth nucleoside monomer analog (currently capped, 3' end is -NH2) with the following structural formula:

[0150] Quaternary nucleoside monomer analogs ;

[0151] Fifth nucleoside monomer analog ;

[0152] After reacting at 37°C for 16 hours, acetonitrile of 1 volume of the reaction system was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12,000 rpm. The supernatant was subjected to UPLC detection. It was detected that only 6% of the oligonucleotide chain substrate remained, 28% of the oligonucleotide product coupled with a fifth nucleoside monomer analog was generated, and 36% of the oligonucleotide product coupled with a fourth nucleoside monomer analog was generated. The UPLC detection results are shown in the figure below. Figure 2 As shown, the coupling efficiency of the polymerase PUP for the fourth nucleoside monomer analog is higher, far exceeding the coupling efficiency for the existing blocked fifth nucleoside monomer analog.

[0153] 1M NaOAc and 0.7M NaNO2 were added to the above reaction system, the pH was adjusted to 4-6, and the reaction was incubated at 30°C for 5-10 minutes to remove the 3'-end modification group on the nucleoside monomer analog. After the reaction, the solution was sent to UPLC for detection. The results were as follows: Figure 3 As shown. This indicates that the fourth modified monomer has good polymerase catalytic activity and can be effectively removed to achieve reversible modification of the oligonucleotide. The oligonucleotide with the reversible modification group removed can be used as the oligonucleotide chain substrate for the next round of coupling of nucleoside monomers.

[0154] Embodiment 13

[0155] Add the reaction system to a clean container, so that the oligonucleotide chain substrate (5'HO-Ams-Cfs-Am-Af-Af-3'OH, i.e., substrate 2 in Example 1) is 50 µM, add PUP polymerase to make its concentration 0.2 mg / mL, add 10 mM MnCl2, 50 mM NaCl, 50 mM Tris-HCl, adjust the pH to 8.0, add the fifth nucleoside monomer analog (currently capped, 3' end is -NH2) to make its concentration 300 µM, add the fourth nucleoside monomer analog to make its concentration 300 µM, the structural formula is as follows:

[0156] a fourth nucleoside monomer analog; ;

[0157] a fifth nucleoside monomer analog; ;

[0158] After reacting at 37°C for 2 hours, acetonitrile of 1 volume of the reaction system was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12,000 rpm. The supernatant was tested by UPLC, and the results showed that 67% of the fourth nucleoside monomer analog 2 product was generated and 30% of the fifth nucleoside monomer analog coupled product was generated. The polymerase PUP has a higher coupling efficiency for the fourth nucleoside monomer analog, far exceeding the coupling efficiency for the existing capped fifth nucleoside monomer analog.

[0159] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: using the nucleoside monomers of the present application, the 3' end of the oligonucleotide chain substrate can be connected to the reversible blocked end under the enzymatic synthesis system, and the reversible blocked end can be effectively removed by simple acid hydrolysis in the subsequent process. The oligonucleotide with the reversible modification group removed can be used as the oligonucleotide chain substrate for the next round of coupling of nucleoside monomers, proving that the nucleoside monomers of the present application can be used to achieve the enzymatic specific synthesis of target oligonucleotides of specific sequences.

[0160] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nucleoside monomer analog, characterized in that: The structural formula of the nucleoside monomer analog is shown in Formula I; Formula I; Wherein, R is a base; R1 is methoxy, methoxyethyl, fluorine, hydroxyl or hydrogen; R2 is hydroxyl, monophosphate, diphosphate, triphosphate or thio-modified phosphate; R3 is methyl, ethyl, propyl or isopropyl.

2. The nucleoside monomer analog according to claim 1, characterized in that The bases include natural bases or unnatural bases; The natural base is adenine, guanine, cytosine, thymine or uracil; The non-natural base is 3-deazaadenine, 7-deazaguanine, 2,6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamidouracil, 5-methylcytosine, 5-ethynyluracil, C7-modified deazaadenine, C7-modified deazaguanine, C5-modified cytosine or C5-modified uracil; Wherein, the modification is methyl modification, H modification, Cl modification or F modification.

3. A nucleotide monomer analogue, characterized in that: The structural formula of the nucleotide analog is shown in Formula II: Formula II; Wherein, R is a base; R4 is methoxy, methoxyethyl, fluorine, hydroxyl or hydrogen; R5 is methyl, ethyl, propyl or isopropyl; R6 is a hydroxyl group or a mercapto group.

4. The nucleotide monomer analogue according to claim 3, characterized in that The bases include natural bases or unnatural bases; The natural base is adenine, guanine, cytosine, thymine or uracil; The non-natural base is 3-deazaadenine, 7-deazaguanine, 2,6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamidouracil, 5-methylcytosine, 5-ethynyluracil, C7-modified deazaadenine, C7-modified deazaguanine, C5-modified cytosine or C5-modified uracil; Wherein, the modification is methyl modification, H modification, Cl modification or F modification.

5. A method for synthesizing an oligonucleotide having a 3'-end reversibly modified end, characterized in that: The synthesis method comprises: a) when the oligonucleotide chain substrate is a single strand, connecting the nucleoside monomer analogue according to any one of claims 1 to 2, or the nucleotide monomer analogue according to any one of claims 3 to 4, to the 3' end of the oligonucleotide chain substrate to obtain the oligonucleotide with a 3' reversibly modified end; or b) when there are two oligonucleotide chain substrates, the oligonucleotide chain substrates include a first oligonucleotide chain substrate and a second oligonucleotide chain substrate; wherein the 3' end of the first oligonucleotide chain substrate is connected to the nucleoside monomer analogue according to any one of claims 1 to 2, or the nucleotide monomer analogue according to any one of claims 3 to 4; The first oligonucleotide chain substrate and the second oligonucleotide chain substrate are connected by using a ligase to obtain the oligonucleotide with a 3'-end reversibly modified end.

6. The synthesis method according to claim 5, characterized in that The oligonucleotide chain substrate includes a natural oligonucleotide chain composed of natural nucleotides, or a non-natural oligonucleotide chain containing non-natural nucleotides.

7. The synthesis method according to any one of claim 5, characterized in that When R2 of the nucleoside monomer analog is hydroxyl, monophosphate, diphosphate or thio-modified phosphate, the synthesis method comprises: a-1) mixing the nucleoside monomer analog and polyphosphate, and using phosphokinase to catalyze the formation of the nucleoside monomer analog wherein R2 is triphosphate; When the oligonucleotide chain substrate is a single strand, the nucleoside monomer analogue in which R2 is a triphosphate is catalyzed by a polymerase to be connected to the 3' end of the oligonucleotide chain substrate, thereby obtaining the oligonucleotide having a reversibly modified end at the 3' end; Wherein, the phosphokinase is acetate kinase, pyruvate kinase, adenylate kinase, polyphosphate kinase, nucleoside kinase or nucleoside diphosphate kinase.

8. The synthesis method according to claim 5, characterized in that When R2 of the nucleoside monomer analog is triphosphate, the synthesis method comprises: a-2) When the oligonucleotide chain substrate is a single strand, the nucleoside monomer analogue wherein R2 is triphosphate is connected to the 3' end of the oligonucleotide chain substrate using a polymerase to obtain the oligonucleotide having a reversibly modified 3' end.

9. The synthesis method according to claim 5, characterized in that The synthesis method comprises: a-3) When the oligonucleotide chain substrate is a single strand, the nucleotide monomer analogue according to any one of claims 3 to 4 is catalyzed by a polymerase to connect to the 3' end of the oligonucleotide chain substrate, thereby obtaining the oligonucleotide having a reversibly modified 3' end.

10. The synthesis method according to any one of claims 9, characterized in that: The polymerase is PUP polymerase; The ligase is RNA ligase or DNA ligase.

11. A method for preparing an oligonucleotide, characterized in that: The preparation method comprises: removing the reversibly modified end at the 3' end of the oligonucleotide having a 3' reversibly modified end synthesized by the synthesis method according to any one of claims 5 to 10, to obtain the oligonucleotide.

12. The preparation method according to claim 11, characterized in that: The removal comprises: The oligonucleotide having a 3'-end reversibly modified end is mixed with the first solution to obtain a mixed system, The mixed system is used to achieve the removal of the 3'-end reversibly modified end.

13. The preparation method according to claim 12, characterized in that: The pH of the mixed system is 4-6, and the mixed system is maintained at a temperature of 20-40° C. for 5-10 minutes, thereby achieving the removal of the reversible modified end at the 3' end.

14. The preparation method according to claim 13, characterized in that: The first solution is any one of the following solutions: hydrochloric acid, sulfuric acid, phosphoric acid, disodium hydrogen phosphate, dipotassium hydrogen phosphate, citric acid or boric acid.

15. The preparation method according to claim 14, characterized in that: The concentration of the solute in the first solution is 0.01 mM~10M.

16. Use of the method for synthesizing the nucleoside monomer analogue according to any one of claims 1 to 2, or the nucleotide monomer analogue according to any one of claims 3 to 4, or the oligonucleotide having a 3'-end reversibly modified end according to any one of claims 5 to 10 in preparing oligonucleotides.

Images