Nucleoside monomer analog, synthesis method for oligonucleotide, and use

Abstract

The present invention provides a nucleoside monomer analog, a synthesis method for an oligonucleotide, and a use. In the nucleoside monomer, R is a base; R1 comprises one or more of methoxy, methoxyethyl, fluorine, hydroxyl, or hydrogen; R2 comprises one or more of hydroxyl, monophosphate, diphosphate, triphosphate, or a thiophosphate-modified phosphate; and R3 comprises one or more of methyl, ethyl, propyl, or isopropyl. The present invention can solve the problem in the prior art of low reversible end-capping activity of oligonucleotides, and is applicable to the field of oligonucleotide synthesis.

Description

Synthesis methods and applications of nucleoside monomer analogs and oligonucleotides

[0001] This application is based on and claims priority to Chinese application CN application number 2025100693535, filed on January 16, 2025, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the field of oligonucleotide synthesis, and more specifically, to a method for synthesizing nucleoside monomer analogs, oligonucleotides, and their applications. Background Technology

[0003] Oligonucleotides are fundamental tools for regulating gene expression in biomedical and life science research. As gene-targeted therapeutic drugs, they are used to treat various diseases, including genetic disorders, viruses, and tumors. Oligonucleotide drugs mainly include antisense oligonucleotides, small interfering RNA, microRNAs, and nucleic acid aptamers. Natural oligonucleotides are easily degraded in vivo, resulting in low drug-likeness. Therefore, drug oligonucleotides usually have specific modifying groups, such as phosphothioester bonds, fluorinated groups, methyl groups, and locked nucleic acids, to enhance their stability in vivo, improve their specificity, and reduce their toxic side effects.

[0004] The main method for synthesizing oligonucleotides is the phosphoramide method, but it has disadvantages such as low purity of the initial product, large amount of chemical solvents used, and difficulty in large-scale scale-up. Therefore, the emerging enzymatic method for synthesizing oligonucleotides has attracted much attention due to its green synthesis process, high coupling efficiency, and ease of scale-up.

[0005] Nucleotide analogs carrying reversible blocking groups are key raw materials for the enzymatic synthesis of oligonucleotides. The reversible end-capping groups enable the specific synthesis of oligonucleotides, preventing the introduction of erroneous sequences. Furthermore, the blocking groups can be removed without leaving a trace after synthesis to achieve the synthesis of the target oligonucleotide.

[0006] However, existing technologies have limited types of reversible blocking groups for oligonucleotides, and their activity is relatively low, resulting in low coupling efficiency with oligonucleotide chains and hindering the synthesis of target oligonucleotides. Summary of the Invention

[0007] The main objective of this invention is to provide a method for synthesizing nucleoside monomer analogs and oligonucleotides, and their applications, in order to solve the problem of low reversible end-capping activity of oligonucleotides in the prior art.

[0008] To achieve the above objectives, according to a first aspect of the present invention, a nucleoside monomer analog is provided, the structural formula of which is shown in 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 phosphoric acid; R3 includes one or more of methyl, ethyl, propyl, or isopropyl.

[0011] Furthermore, the bases include natural bases or non-natural bases; natural bases include adenine, guanine, cytosine, thymine, or uracil; non-natural bases include one or more of 3-deadenine, 7-deadenine, 2,6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamide uracil, 5-methylcytosine, 5-ethynyluracil, C7-modified deadenine, C7-modified deadenine, 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, the structural formula of which is shown in Formula II:

[0013] Wherein, 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 mercapto.

[0014] Furthermore, the bases include natural bases or non-natural bases; natural bases include adenine, guanine, cytosine, thymine, or uracil; non-natural bases include one or more of 3-deadenine, 7-deadenine, 2,6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamide uracil, 5-methylcytosine, 5-ethynyluracil, C7-modified deadenine, C7-modified deadenine, C5-modified cytosine, or C5-modified uracil, wherein the modification includes one or more of methyl modification, H modification, Cl modification, or F modification.

[0015] According to a third aspect of this application, a method for synthesizing an oligonucleotide having a reversible 3' end modified end is provided. The method includes: a) when the oligonucleotide chain substrate is a single strand, linking 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 reversible 3' end modified end.

[0016] Or b) When there are two oligonucleotide substrates, the oligonucleotide substrates include a first oligonucleotide substrate and a second oligonucleotide substrate; wherein, the 3' end of the first oligonucleotide substrate is connected to the above-mentioned nucleoside monomer analog or the above-mentioned nucleotide monomer analog; the first oligonucleotide substrate and the second oligonucleotide substrate are connected to obtain an oligonucleotide with a reversible 3' end modification.

[0017] Furthermore, the oligonucleotide chain substrate includes natural oligonucleotide chains composed of natural nucleotides, or non-natural oligonucleotide chains containing non-natural nucleotides.

[0018] Furthermore, non-natural oligonucleotide chains are composed of non-natural nucleotides.

[0019] Further, when R2 of the nucleoside monomer analog is a hydroxyl group, a monophosphate, a diphosphate, or a thiophosphate, the synthesis method includes: a-1) mixing the nucleoside monomer analog and a polyphosphate, and using a phosphokinase to catalyze the formation of a nucleoside monomer analog with R2 being a triphosphate; when the oligonucleotide substrate is a single strand, using a polymerase to catalyze the linking of the nucleoside monomer analog with R2 being a triphosphate to the 3' end of the oligonucleotide substrate, thereby obtaining an oligonucleotide with a reversibly modified 3' end; b-1) when there are two oligonucleotide substrates, wherein the 3' end of the first oligonucleotide substrate is connected to the aforementioned nucleoside monomer analog, and using a ligase to catalyze the linking of the first and second oligonucleotide substrates, thereby obtaining an oligonucleotide with a reversibly modified 3' end; wherein the phosphokinase includes one or more of acetate kinase, pyruvate kinase, adenosine kinase, polyphosphate kinase, nucleoside kinase, or nucleoside diphosphate kinase.

[0020] Furthermore, when R2 of the nucleoside monomer analog is triphosphate, the synthesis method includes: a-2) when the oligonucleotide substrate is a single strand, using polymerase to catalyze the ligation of the nucleoside monomer analog with R2 being triphosphate to the 3' end of the oligonucleotide substrate, thereby obtaining an oligonucleotide with a reversible 3' end modification; b-2) when the oligonucleotide substrate is two strands, wherein the first oligonucleotide substrate has a nucleoside monomer analog attached to the 3' end, using ligase to catalyze the ligation of the first oligonucleotide substrate and the second oligonucleotide substrate, thereby obtaining an oligonucleotide with a reversible 3' end modification.

[0021] Further, the synthesis method includes: a-3) when the oligonucleotide chain substrate is a single strand, using polymerase to catalyze the nucleotide monomer analog to be linked to the 3' end of the oligonucleotide chain substrate, thereby obtaining an oligonucleotide with a reversible 3' end modification end;

[0022] b-3) When there are two oligonucleotide substrates, the first oligonucleotide substrate has the above-mentioned nucleotide monomer analog attached to its 3' end. The first oligonucleotide substrate and the second oligonucleotide substrate are ligated by ligase to obtain an oligonucleotide with a reversible 3' end modification.

[0023] Furthermore, polymerases include PUP polymerases; ligases include T4 ligases.

[0024] According to a fourth aspect of this application, a method for preparing an oligonucleotide is provided, the method comprising: removing the reversible modification end at the 3' end of an oligonucleotide having a reversible modification end synthesized by any of the above-mentioned synthetic methods, thereby obtaining an oligonucleotide.

[0025] Further, the removal includes: mixing the oligonucleotide with the reversible 3' end modification end with the first solution to obtain a mixed system, and using the mixed system to remove the reversible 3' end modification end.

[0026] 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 to achieve the removal of the 3' end reversible modification.

[0027] Further, 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.

[0028] Furthermore, the concentration of the solute in the first solution is 0.01 mM to 10 M.

[0029] According to the fifth aspect of this application, a method for synthesizing any of the above-mentioned nucleoside monomer analogs, or any of the above-mentioned nucleotide monomer analogs, or any of the above-mentioned oligonucleotides having a reversibly modified 3' end, or any of the above-mentioned oligonucleotides, is provided, and its application in the preparation of oligonucleotides.

[0030] By applying the technical solution of this invention, using a nucleoside monomer analog with the structure of Formula I, the 3' reversible modification end can be linked to the oligonucleotide chain, successfully synthesizing an oligonucleotide with a 3' reversible modification end. Compared with the reversible capping commonly used in the prior art, it has higher activity and higher substrate conversion rate. The target oligonucleotide can be synthesized after further removing the reversible modification. Attached Figure Description

[0031] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0032] Figure 1 shows a schematic diagram of the UPLC detection results of two products of reaction system 1 connected with phosphoric acid according to Example 11 of the present invention.

[0033] Figure 2 shows a schematic diagram of the UPLC detection results of the product of the linked nucleoside monomer according to Example 12 of the present invention.

[0034] Figure 3 shows a schematic diagram of the UPLC detection results of the product after removing the reversible closed end at the 3' end according to Embodiment 12 of the present invention. Detailed Implementation

[0035] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0036] Terminology Explanation:

[0037] Nucleosides are compounds formed by the condensation of a base (purine or pyrimidine base) with a pentose sugar (including but not limited to ribose or deoxyribose). Nucleosides do not contain phosphate.

[0038] Nucleotides are formed by the combination of a base (purine or pyrimidine base), a pentose sugar (including but not limited to ribose or deoxyribose), and a phosphate group, including nucleoside monophosphate, nucleoside diphosphate, and nucleoside triphosphate.

[0039] Nucleoside monomer: In this application, it refers to a single nucleoside with a length of 1 nt.

[0040] Nucleotide monomer: In this application, it refers to a single nucleotide with a length of 1 nt.

[0041] As mentioned in the background section, the reversible blocking group at the 3' end of oligonucleotides in the prior art has low activity, resulting in low efficiency of ligation with the oligonucleotide chain and hindering the synthesis of the target oligonucleotide. Therefore, in this application, the inventors attempted to develop a new nucleoside monomer that can link the reversible blocking group at the 3' end to the oligonucleotide via enzymatic methods, thereby improving the substrate conversion rate and achieving higher activity compared to the reversible blocking capping of the prior art. This application proposes a series of protection schemes.

[0042] In a first typical embodiment of this application, a nucleoside monomer analog is provided, the structural formula of which is shown in Formula I;

[0043] Wherein, 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 phosphoric acid; R3 includes one or more of methyl, ethyl, propyl or isopropyl.

[0044] Nucleoside monomer analogs include nucleoside monomer analogs and nucleotide monomer analogs; when the group at R2 is a monophosphate, diphosphate, triphosphate or thiophosphate, 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.

[0045] In the existing enzymatic synthesis of oligonucleotides, preliminary studies in this application have found that for certain specific reactions, the efficiency of the reaction is related to the reversible 3' end capping, resulting in poor capping activity, low substrate conversion, and difficulty in subsequently synthesizing the target oligonucleotide.

[0046] This application utilizes nucleoside monomer analogs of Formula I to couple with oligonucleotide substrates in enzymatic oligonucleotide synthesis systems, successfully attaching a reversibly closed end to the 3' end with high substrate conversion. This plays a crucial role in oligonucleotide synthesis, preventing the ligation of erroneous sequences, which can be easily removed subsequently using acid hydrolysis. The oligonucleotide product, after de-grouping, can be used as the substrate for the next round of nucleoside monomer coupling, achieving the specific enzymatic synthesis of target oligonucleotides with specific sequences.

[0047] This application develops a novel reversible capping method, different from existing technologies. When the reaction exhibits the low substrate conversion rate caused by the aforementioned correlation with the reversible 3' capping, this application provides multiple options for reversible capping, overcoming this correlation and facilitating the synthesis of various types of target oligonucleotides. When the R2 group of the aforementioned 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 catalysis of polymerase, using a single oligonucleotide chain substrate as a template, the nucleotide monomer shown in Formula III can be directly coupled to the oligonucleotide chain substrate to synthesize an oligonucleotide with a reversibly capped 3' end.

[0048] In a preferred embodiment, the bases include natural bases or non-natural bases. Natural bases include natural adenine, natural guanine, natural cytosine, natural thymine, or natural uracil; non-natural bases include one or more of 3-deadenine, 7-deadenine, 2,6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamide uracil, 5-methylcytosine, 5-ethynyluracil, C7-modified deadenine, C7-modified deadenine, C5-modified cytosine, or C5-modified uracil, wherein the modification includes one or more of methyl, H, Cl, or F modification.

[0049] In a second typical embodiment of this application, a nucleotide monomer analog is provided, the structural formula of which is shown in Formula II:

[0050] Wherein, 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 mercapto.

[0051] Using the nucleotide monomer analogs shown in Formula II of this application, oligonucleotides with a reversibly modified 3' end can be synthesized. In the case of a single oligonucleotide chain substrate, the above-mentioned nucleotide monomer analogs are mixed with polymerase, and the oligonucleotide chain is used as the synthetic chain to catalyze the coupling of the nucleotide monomer to the oligonucleotide chain, thereby obtaining an oligonucleotide with a reversibly modified 3' end, which can then be further used to synthesize the target oligonucleotide.

[0052] Alternatively, in the case of two oligonucleotide substrates, one substrate has the aforementioned nucleotide monomer analog attached to its 3' end, while the other does not. A ligase can catalyze the formation of a phosphodiester bond between the two substrates, ultimately forming an oligonucleotide with a reversibly modified 3' end, which can then be used to synthesize the target oligonucleotide. Both methods are simple to operate, capable of synthesizing oligonucleotides with reversibly modified 3' ends in one step, and the modified groups are easily removed, facilitating the synthesis of the target oligonucleotide.

[0053] In a preferred embodiment, the bases include natural bases or non-natural bases; natural bases include adenine, guanine, cytosine, thymine, or uracil; non-natural bases include one or more of 3-deadenine, 7-deadenine, 2,6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamide uracil, 5-methylcytosine, 5-ethynyluracil, C7-modified deadenine, C7-modified deadenine, C5-modified cytosine, or C5-modified uracil, wherein the modification includes methyl modification, H modification, Cl modification, and F modification.

[0054] In a second typical embodiment of this application, a method for synthesizing an oligonucleotide with a reversible 3' end modification is provided. The method includes: a) when the oligonucleotide chain substrate is a single strand, linking 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 with a reversible 3' end modification.

[0055] Or b) When there are two oligonucleotide substrates, the oligonucleotide substrates include a first oligonucleotide substrate and a second oligonucleotide substrate; wherein, the 3' end of the first oligonucleotide substrate is connected to the above-mentioned nucleoside monomer analog or the above-mentioned nucleotide monomer analog; the first oligonucleotide substrate and the second oligonucleotide substrate are connected to obtain an oligonucleotide with a reversible 3' end modification.

[0056] When there are two oligonucleotide substrates, the last nucleoside monomer at the 3' end of one of the oligonucleotide substrates is reversibly capped, namely the first oligonucleotide substrate mentioned above. This nucleoside monomer is connected to the remaining oligonucleotide chain before it by a phosphate ester bond. The reversible capping can be connected to the 3' end of the oligonucleotide substrate by the method of this application, chemical synthesis, or other means known to those skilled in the art. Those skilled in the art can flexibly choose according to actual needs.

[0057] Under the catalysis of ligase, the above-mentioned first oligonucleotide substrate with reversible end capping and second oligonucleotide substrate are ligated to obtain an oligonucleotide with a reversible 3' end modification.

[0058] In a preferred embodiment, the oligonucleotide chain substrate comprises a natural oligonucleotide chain composed of natural nucleotides, or a non-natural oligonucleotide chain containing non-natural nucleotides.

[0059] In a preferred embodiment, the non-natural oligonucleotide chain is composed of non-natural nucleotides.

[0060] Xeno-nucleic acid (XNA) is a class of nucleic acid molecules with a non-natural backbone or nucleic acid bases. Preferably, the non-natural nucleotide includes ribonucleotides with one or more of the following modifications: ribose 2' position modification, ribose backbone modification, base modification, or phosphate backbone modification; preferably, ribose 2' position 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 the ribose in the nucleotide with ribuloNA, TNA, tPhoNA, or dXNA; preferably, base modification includes deadenine C7 modification, deadenguanosine C7 modification, cytosine C5 modification, or uridine C5 modification; phosphate backbone modification includes PS modification.

[0061] In a preferred embodiment, when R2 of the nucleoside monomer analog is a hydroxyl, monophosphate, diphosphate, or thio-modified phosphate, the synthesis method includes: mixing the nucleoside monomer analog and a polyphosphate, and using a phosphokinase to catalyze the formation of a nucleoside monomer analog with R2 being a triphosphate; a-1) when the oligonucleotide substrate is a single strand, using a polymerase to catalyze the linking of the nucleoside monomer analog with R2 being a triphosphate to the 3' end of the oligonucleotide substrate, thereby obtaining an oligonucleotide with a reversibly modified 3' end; b-1) when the oligonucleotide substrate is two strands, wherein the 3' end of the first oligonucleotide substrate is connected to the aforementioned nucleoside monomer analog, and using a ligase to catalyze the linking of the first oligonucleotide substrate and the second oligonucleotide substrate, thereby obtaining an oligonucleotide with a reversibly modified 3' end; the phosphokinase includes one or more of acetate kinase, pyruvate kinase, adenosine kinase, polyphosphate kinase, nucleoside kinase, or nucleoside diphosphate kinase. When the R2 of the above-mentioned 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, forming a nucleoside monomer analog with R2 having three phosphates.

[0062] The above-mentioned method for synthesizing oligonucleotide chain substrates includes: mixing one or more nucleoside monomer analogs of R2 being hydroxyl, monophosphate, diphosphate, or thiomodified phosphate with a phosphate donor, Tris-HCl, and MgCl2; maintaining the pH of the reaction system at 6-9; adding a kinase; reacting at 0-80°C for 0.1-100 h; after the reaction is completed, adding acetonitrile inactivating enzyme at one volume of the reaction system; and the supernatant after centrifugation containing nucleoside monomers of R2 having three phosphates.

[0063] The system containing the nucleoside monomer with R2 having three phosphates, oligonucleotide chain substrate, Tris-HCl, MnCl2, and NaCl were mixed. The pH of the reaction system was maintained at 6–9. Polymerase was added, and the reaction was carried out at 0–100℃ for 0.1–100 h. After the reaction was completed, acetonitrile inactivating enzyme was added at a volume equal to that of the reaction system. The supernatant after centrifugation contained oligonucleotides with a reversibly modified 3' end.

[0064] In a preferred embodiment, when R2 of the nucleoside monomer analog is triphosphate, the synthesis method includes: a-2) when the oligonucleotide substrate is a single strand, using a polymerase to catalyze the ligation of the nucleoside monomer analog with R2 being triphosphate to the 3' end of the oligonucleotide substrate, thereby obtaining an oligonucleotide with a reversible 3' end modification; b-2) when the oligonucleotide substrate is two strands, wherein the 3' end of the first oligonucleotide substrate is connected to the above-mentioned nucleoside monomer analog, and using a ligase to catalyze the ligation of the first oligonucleotide substrate and the second oligonucleotide substrate, thereby obtaining an oligonucleotide with a reversible 3' end modification.

[0065] When the R2 of the above-mentioned nucleoside monomer analog is triphosphate, the nucleoside monomer can be directly mixed with polymerase and oligonucleotide chain substrate to react and couple to the oligonucleotide chain substrate, thereby achieving the synthesis of oligonucleotides with a reversible 3' end modification in one step.

[0066] In some preferred embodiments, the synthesis method includes: when the oligonucleotide substrate is a single strand, mixing a nucleoside monomer analog of R2 with three phosphates, the oligonucleotide substrate, Tris-HCl, MnCl2, and NaCl, maintaining the pH of the reaction system at 6–9, adding polymerase, reacting at 1–100°C for 0.1–100 h, adding acetonitrile inactivating enzyme at one volume of the reaction system after the reaction is completed, and the supernatant after centrifugation containing oligonucleotides with a reversibly modified 3' end.

[0067] When there are two oligonucleotide substrates, the 3' end of the first oligonucleotide substrate is attached to the above-mentioned R2 nucleoside monomer analog with three phosphates; the first oligonucleotide substrate, the second oligonucleotide 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℃ for 1-50h; after the reaction is completed, acetonitrile inactivating enzyme with 1 volume of the reaction system is added, and the supernatant after centrifugation contains oligonucleotides with reversibly modified 3' ends.

[0068] In a preferred embodiment, the synthesis method includes:

[0069] a-3) When the oligonucleotide substrate is a single strand, polymerase is used to catalyze the nucleotide monomer analog to be linked to the 3' end of the oligonucleotide substrate, thereby obtaining an oligonucleotide with a reversible 3' end modification.

[0070] b-3) When there are two oligonucleotide substrates, the first oligonucleotide substrate has the above-mentioned nucleotide monomer analog attached to its 3' end. The first oligonucleotide substrate and the second oligonucleotide substrate are ligated by ligase to obtain an oligonucleotide with a reversible 3' end modification.

[0071] The synthesis method of oligonucleotides with a reversibly modified 3' end involving nucleotide monomer analogs in this application is, as described above, the synthesis method when R2 of the nucleoside monomer analog in this application is a triphosphate.

[0072] In a preferred embodiment, the polymerase includes PUP polymerase; the ligase includes T4 ligase.

[0073] The PUP polymerase mentioned above was derived from *Fission Yeast* NCBI accession number NP_594901.1; the T4 ligase mentioned above was derived from *Escherichia phage* T4, NCBI accession number NP_049790.1.

[0074] Polymerase adds a capped nucleoside monomer to the 3' end of a nucleic acid chain, thus elongating the nucleic acid chain with a reversible 3' cap. Ligase adds a capped oligonucleotide short chain to the 3' end of a nucleic acid chain, forming an elongated oligonucleotide chain with a reversible 3' cap.

[0075] In a third typical embodiment of this application, a method for preparing an oligonucleotide is provided, the method comprising: removing the reversible modification end at the 3' end of an oligonucleotide synthesized by the above-described synthesis method to obtain the oligonucleotide.

[0076] The synthetic method described in this application yields oligonucleotides with a reversibly modified 3' end, which can be removed by simple acid hydrolysis, thus achieving reversible modification. The oligonucleotides without the 3' modification can also be used as substrates for subsequent coupling with nucleoside monomers. The nucleoside monomer analogs or nucleotide monomer analogs described in this application enable reversible modification of the 3' end of oligonucleotides, allowing for the enzymatic synthesis of target oligonucleotides with specific sequences.

[0077] In a preferred embodiment, the removal includes: mixing an oligonucleotide having a reversible 3' end modification with a first solution to obtain a mixed system, and using the mixed system to remove the reversible 3' end modification.

[0078] In a preferred embodiment, the pH of the mixture is 4–6, and the mixture is maintained at a temperature of 20–40°C for 5–10 minutes to achieve the removal of the reversible 3' end modification. The removal conditions include a pH of 4–6, including but not limited to 4, 5, or 6; a temperature of 20–40°C, including but not limited to 20°C, 25°C, 30°C, 35°C, or 40°C; and a time of 5–10 minutes, including but not limited to 5, 6, 7, 8, 9, or 10 minutes.

[0079] In a preferred embodiment, the first solution comprises any one or more of the following: hydrochloric acid, sulfuric acid, phosphoric acid, disodium hydrogen phosphate, dipotassium hydrogen phosphate, citric acid, or boric acid.

[0080] In a preferred embodiment, the concentration of the solute in the first solution is 0.01 mM to 10 M.

[0081] This application utilizes simple acid hydrolysis to achieve the reversible removal of the 3' end modification of the nucleoside monomer coupled to the oligonucleotide chain. By controlling the conditions of the acid hydrolysis reaction, the effective removal of this reversible modification can be promoted, facilitating the subsequent synthesis of the target oligonucleotide.

[0082] In a fourth typical embodiment of this application, a method for synthesizing any of the above-mentioned nucleoside monomers, or any of the above-mentioned oligonucleotides having a reversible 3' end modification, or any of the above-mentioned oligonucleotides preparation methods, is provided for use in the preparation of oligonucleotides.

[0083] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0084] Unless otherwise specified, the reagents used in the embodiments of this application are all commercially available products.

[0085] Example 1

[0086] Add the reaction mixture to a clean container to make the oligonucleotide substrate 50 μM. Add PUP polymerase (derived from fission yeast PUP polymerase, NCBI accession number NP_594901.1) to a concentration of 0.2 mg / mL. Add 10 mM MnCl2, 50 mM NaCl, and 50 mM Tris-HCl to adjust the pH to 8.0. Add the nucleoside monomer analog to a concentration of 700 μM. The structural formula of the nucleoside monomer analog in this example is shown below:

[0087] After reacting at 37℃ for 2 hours, acetonitrile inactivating enzyme was added to the reaction system, and the denatured protein was removed by centrifugation at 12000 rpm. The supernatant was then analyzed by UPLC.

[0088] The oligonucleotide substrates in this embodiment 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 linkage of nucleoside monomer analogs are shown in Table 1.

[0089] The UPLC detection results of this embodiment show that the 3' end of different oligonucleotide chain substrates is coupled with a nucleoside monomer analog, indicating that the nucleoside monomer analog shown in the above structural formula can be applied to the enzymatic synthesis of oligonucleotides.

[0090] Table 1

[0091] 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%.

[0092] In this application, "m" after A, C, G or U indicates a 2' methoxy modification of the ribonucleotide, "f" indicates a 2' fluoro modification of the ribonucleotide, and "s" indicates a thio modification of the 5' phosphate of the ribonucleotide.

[0093] Example 2

[0094] Add the reaction system to a clean container to make the concentration of the two oligonucleotide substrate fragments 50 mM. The two oligonucleotide substrate fragments in this embodiment are shown in the following structural formulas. The 3'OH end of the first oligonucleotide substrate fragment is connected to the reversible blocking modification end (nucleoside monomer analog) of this application, that is, the part framed in the following structural formula:

[0095] First oligonucleotide chain substrate fragment:

[0096] Second oligonucleotide chain substrate fragment:

[0097] RNA ligase (derived 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, and 50 mM Tris-HCl were added to adjust the pH to 7.5, and the mixture was incubated at 16 °C for 16 h. Acetonitrile was added to the reaction system to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000 rpm. The supernatant was analyzed by LCMS.

[0098] Because the 3'OH end of the first oligonucleotide fragment is reversibly capped, theoretically only a molecular weight of 2618.83±2 can be detected, and the molecular weight of products with two or more oligonucleotide fragments linked together cannot be detected. Mass spectrometry analysis revealed a molecular weight of only 2618.79, indicating successful ligation.

[0099] Example 3

[0100] Add the reaction mixture to a clean container to make the oligonucleotide substrate (5'HO-Cms-Ams-Gm-Am-3'OH, i.e., substrate 1 in Example 1) 50 μM. Add PUP polymerase to make the concentration 0.2 mg / mL. Add 10 mM MnCl2, 50 mM NaCl, and 50 mM Tris-HCl to adjust the pH to 8.0. Add the nucleotide monomer analog to make the concentration 700 μM. The structural formula of the nucleoside monomer analog in this example is shown below:

[0101] After reacting at 37°C for 4 hours, acetonitrile was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12,000 rpm. The supernatant was then analyzed by UPLC. An oligonucleotide product with a nucleoside monomer coupled to the 3' end was detected, with a conversion rate of 20%, indicating that the nucleoside monomer analog in this embodiment can be used for enzymatic synthesis of oligonucleotides.

[0102] Example 4

[0103] Add the reaction mixture to a clean container to make the oligonucleotide substrate (5'HO-Cms-Ams-Gm-Am-3'OH, i.e., substrate 1 in Example 1) 50 μM. Add PUP polymerase to make the concentration 0.2 mg / mL. Add 10 mM MnCl2, 50 mM NaCl, and 50 mM Tris-HCl to adjust the pH to 8.0. Add the nucleotide monomer analog to make the concentration 700 μM. The structural formula of the nucleoside monomer analog in this example is shown below:

[0104] After reacting at 37°C for 4 hours, acetonitrile was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000 rpm. The supernatant was then analyzed by UPLC. An oligonucleotide product with a nucleoside monomer analog coupled to the 3' end was detected, with a conversion rate of 9%, indicating that the nucleoside monomer analog in this embodiment can be used for enzymatic synthesis of oligonucleotides.

[0105] Example 5

[0106] Add the reaction mixture to a clean container to make the oligonucleotide substrate (5'HO-Cms-Ams-Gm-Am-3'OH, i.e., substrate 1 in Example 1) 50 μM. Add PUP polymerase to make the concentration 0.2 mg / mL. Add 10 mM MnCl2, 50 mM NaCl, and 50 mM Tris-HCl to adjust the pH to 8.0. Add the nucleotide monomer analog to make the concentration 700 μM. The structural formula of the nucleoside monomer analog in this example is shown below:

[0107] After reacting at 37°C for 4 hours, acetonitrile was added to the reaction system to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000 rpm. The supernatant was then analyzed by UPLC. An oligonucleotide product with a nucleoside monomer analog coupled to the 3' end was detected, with a conversion rate of 1%, indicating that the nucleotide monomer analog of this embodiment can be used for enzymatic synthesis of oligonucleotides.

[0108] Example 6

[0109] Add the reaction mixture to a clean container to make the oligonucleotide substrate (SEQ ID NO:1) 50 μM, add PUP polymerase to make the concentration 0.2 mg / mL, add 10 mM MnCl2, 50 mM NaCl, and 50 mM Tris-HCl, adjust the pH to 8.0, and add the nucleotide monomer analog to make the concentration 700 μM. The structural formula of the nucleoside monomer analog in this embodiment is shown below:

[0110] After reacting at 37°C for 2 hours, acetonitrile was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12,000 rpm. The supernatant was then analyzed by UPLC. An oligonucleotide product with a nucleoside monomer analog coupled to the 3' end was detected, with a conversion rate of 43%, indicating that the nucleoside monomer analog in this embodiment can be used for enzymatic synthesis of oligonucleotides.

[0111] Example 7

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

[0113] After reacting at 37°C for 16 hours, acetonitrile was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12,000 rpm. The supernatant was then analyzed by UPLC. An oligonucleotide product with a nucleoside monomer analog coupled to the 3' end was detected, with a conversion rate of 0.6%, indicating that the nucleoside monomer analog in this embodiment can be used for enzymatic synthesis of oligonucleotides.

[0114] Example 8

[0115] Add the reaction mixture to a clean container to make the oligonucleotide substrate (SEQ ID NO:1) 50 μM, add PUP polymerase to make the concentration 0.2 mg / mL, add 10 mM MnCl2, 50 mM NaCl, and 50 mM Tris-HCl, adjust the pH to 8.0, and add the nucleotide monomer analog to make the concentration 700 μM. The structural formula of the nucleoside monomer analog in this embodiment is shown below:

[0116] After reacting at 37°C for 1 hour, acetonitrile was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12,000 rpm. The supernatant was then analyzed by UPLC. An oligonucleotide product with a nucleoside monomer analog coupled to the 3' end was detected, with a conversion rate of 83%, indicating that the nucleoside monomer analog in this embodiment can be used for enzymatic synthesis of oligonucleotides.

[0117] Example 9

[0118] Add the reaction mixture to a clean container to make the oligonucleotide substrate (5'HO-Cms-Ams-Gm-Am-3'OH, i.e., substrate 1 in Example 1) 50 μM. Add PUP polymerase to make the concentration 0.2 mg / mL. Add 10 mM MnCl2, 50 mM NaCl, and 50 mM Tris-HCl to adjust the pH to 8.0. Add the nucleoside monomer analog to make the concentration 700 μM. The structural formula of the nucleoside monomer analog in this example is shown below:

[0119] After reacting at 37℃ for 2 hours, acetonitrile was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000 rpm. MS analysis of the supernatant revealed a product molecular weight of 1693.3, indicating a nucleoside monomer analog coupled to an oligonucleotide substrate. No products coupled to two or more nucleoside monomers were detected. The nucleoside monomers shown in the figure were effectively capped.

[0120] Example 10

[0121] The nucleoside monomers in this embodiment include the following three nucleotide monomer analogs:

[0122] First nucleoside monomer analog

[0123] Second nucleoside monomer analog

[0124] Third nucleoside monomer analog

[0125] The reaction system was added to three clean containers to make the oligonucleotide substrate (5'HO-Cms-Ams-Gm-Am-3'OH, i.e., substrate 1 in Example 1) 50 μM, polymerase was added to make the concentration 0.2 mg / mL, 10 mM MnCl2, 50 mM NaCl, and 50 mM Tris-HCl were added, the pH was adjusted to 8.0, and the above three nucleotide monomer analogs were added to make the concentration 700 μM.

[0126] After reacting at 37℃ for 16 hours, acetonitrile was added to the reaction system to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000 rpm. The supernatant was then analyzed by mass spectrometry.

[0127] The theoretical molecular weights of the oligonucleotides synthesized in the three reaction systems were 1706.53, 1720.37, and 1720.37, respectively. Mass spectrometry detected molecular weight peaks 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.

[0128] Example 11

[0129] In a clean container, add the nucleoside monomer analog to a concentration of 2000 μM, i.e., reaction system 1. The nucleoside monomer analog in this embodiment is shown in the following formula:

[0130] In reaction system 1, 15 mM MnCl2, 0.5 mg / ml (NaPO3)6, and 100 mM Tris-HCl were added to adjust the pH to 8.0. Polyphosphokinase (derived from Meiothermus ruber, PDB:5LC9) was added to a concentration of 0.5 mg / mL. After reacting at 30 °C for 16 h, acetonitrile was added to the reaction sample to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000 rpm. The transformation was detected by UPLC. The schematic diagram of the UPLC results is shown in Figure 1. Two products, product 1 and product 2, generated in this system, as well as the nucleoside monomer analog of this application, were detected.

[0131] In a clean container, reaction system 2 was added to make the oligonucleotide substrate (5'HO-Cms-Ams-Gm-Am-3'OH, i.e., substrate 1 in Example 1) 50 μM. PUP polymerase was added to make its concentration 0.2 mg / mL. 10 mM MnCl2, 50 mM NaCl, and 50 mM Tris-HCl were added to adjust the pH to 8.0. The above reaction system 1 was added to make the concentration of reaction system 2 700 μM. After reacting at 37°C for 2 h, acetonitrile was added to inactivate the enzyme. The supernatant was centrifuged at 12000 rpm to remove the denatured protein. The supernatant was analyzed by UPLC and the formation of the target product coupled with an oligonucleotide chain of product 2 was detected, indicating that the nucleoside monomer of this example can be used for enzymatic synthesis of oligonucleotides.

[0132] Example 12

[0133] Add the reaction mixture to a clean container to make the oligonucleotide substrate (5'HO-Cms-Ams-Gm-Am-3'OH, i.e., substrate 1 in Example 1) 50 μM. Add PUP polymerase to make the concentration 0.2 mg / mL. Add 10 mM MnCl2, 50 mM NaCl, and 50 mM Tris-HCl to adjust the pH to 8.0. Add 35 μM of the fourth nucleoside monomer analog and 665 μM of the fifth nucleoside monomer analog (with existing end caps, 3' end -NH2) as shown below:

[0134] Fourth nucleoside monomer analog

[0135] Fifth nucleoside monomer analog

[0136] After reacting at 37℃ for 16 h, acetonitrile was added to inactivate the enzyme, and the denatured protein was removed by centrifugation at 12000 rpm. The supernatant was then analyzed by UPLC. Only 6% of the oligonucleotide substrate remained, with 28% of the oligonucleotide product conjugated to a fifth nucleoside monomer analog and 36% conjugated to a fourth nucleoside monomer analog. The UPLC results are shown in Figure 2. The polymerase PUP showed significantly higher conjugation efficiency for the fourth nucleoside monomer analog, far exceeding its efficiency for existing capped fifth nucleoside monomer analogs.

[0137] 1M NaOAc and 0.7M NaNO2 were added to the above reaction system to adjust the pH to 4–6, and the mixture was incubated at 30°C for 5–10 min to remove the 3'-terminal modification group from the nucleoside monomer analog. The solution after the reaction was analyzed by UPLC, and the results are shown in Figure 3. This indicates that the fourth modifying monomer has good polymerase catalytic activity and can effectively remove the modification group, achieving reversible modification of the oligonucleotide. The oligonucleotide with the reversibly removed modification group can be used as the substrate for the next round of coupling with the nucleoside monomer.

[0138] Example 13

[0139] Add the reaction mixture to a clean container to make the oligonucleotide substrate (5'HO-Ams-Cfs-Am-Af-Af-3'OH, i.e., substrate 2 in Example 1) 50 μM. Add PUP polymerase to make the concentration 0.2 mg / mL. Add 10 mM MnCl2, 50 mM NaCl, and 50 mM Tris-HCl to adjust the pH to 8.0. Add the fifth nucleoside monomer analog (existing end-capped, 3' end is -NH2) to make the concentration 300 μM. Add the fourth nucleoside monomer analog to make the concentration 300 μM. The structural formula is shown below:

[0140] Fourth nucleoside monomer analog;

[0141] Fifth nucleoside monomer analog;

[0142] After reacting at 37℃ for 2 hours, acetonitrile was added to inactivate the enzyme, and the supernatant was centrifuged at 12000 rpm to remove denatured protein. UPLC analysis of the supernatant revealed 67% production of the fourth nucleoside monomer analog 2 and 30% production of the product conjugated with the fifth nucleoside monomer analog. The polymerase PUP showed significantly higher conjugation efficiency for the fourth nucleoside monomer analog, far exceeding its efficiency for existing capped fifth nucleoside monomer analogs.

[0143] As can be seen from the above description, the above embodiments of the present invention achieve the following technical effects: using the nucleoside monomer of the present application, in the enzymatic synthesis system, the 3' end of the oligonucleotide chain substrate can be connected to a reversible blocking end, and the reversible blocking 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 with the nucleoside monomer, proving that the nucleoside monomer of the present application can be used to achieve the enzymatic specific synthesis of target oligonucleotides with specific sequences.

[0144] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection 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; Where 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 phosphoric acid; R3 includes one or more of methyl, ethyl, propyl, or isopropyl.

2. The nucleoside monomer analog according to claim 1, characterized in that, The bases include natural bases or non-natural bases; The natural bases include adenine, guanine, cytosine, thymine, or uracil; The non-natural bases include one or more of the following: 3-deadenine, 7-deadenine, 2,6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamide uracil, 5-methylcytosine, 5-ethynyluracil, C7-modified deadenine, C7-modified deadenine, C5-modified cytosine, or C5-modified uracil. The modification includes one or more of methyl modification, H modification, Cl modification or F modification.

3. A nucleotide monomer analog, characterized in that, The structural formula of the nucleotide analog is shown in Formula II: Where R is a base; R4 includes one or more of methoxy, methoxyethyl, fluorine, hydroxy, or hydrogen; R5 includes one or more of methyl, ethyl, propyl, or isopropyl; R6 includes hydroxyl or thiol groups.

4. The nucleotide monomer analog according to claim 3, characterized in that, The bases include natural bases or non-natural bases; The natural bases include adenine, guanine, cytosine, thymine, or uracil; The non-natural bases include one or more of the following: 3-deadenine, 7-deadenine, 2,6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamide uracil, 5-methylcytosine, 5-ethynyluracil, C7-modified deadenine, C7-modified deadenine, C5-modified cytosine, or C5-modified uracil. The modification includes one or more of methyl modification, H modification, Cl modification or F modification.

5. A method for synthesizing an oligonucleotide having a reversibly modified 3' end, characterized in that, The synthesis method includes: a) When the oligonucleotide chain substrate is a single strand, the nucleoside monomer analog of any one of claims 1 to 2, or the nucleotide monomer analog of any one of claims 3 to 4, is linked to the 3' end of the oligonucleotide chain substrate to obtain the oligonucleotide having a reversible 3' end modification end; or b) When there are two oligonucleotide substrates, the oligonucleotide substrates include a first oligonucleotide substrate and a second oligonucleotide substrate; wherein, the 3' end of the first oligonucleotide substrate is connected to a nucleoside monomer analog as described in any one of claims 1 to 2, or a nucleotide monomer analog as described in any one of claims 3 to 4. The first oligonucleotide substrate and the second oligonucleotide substrate are linked together to obtain the oligonucleotide with a reversible 3' end modification.

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 claims 5, characterized in that, When R2 of the nucleoside monomer analog is a hydroxyl group, a monophosphate, a diphosphate, or a thio-modified phosphate, the synthesis method includes: a-1) The nucleoside monomer analog and polyphosphate are mixed and catalyzed by phosphokinase to form the nucleoside monomer analog with R2 as triphosphate; When the oligonucleotide chain substrate is a single strand, a nucleoside monomer analog with R2 being a triphosphate is catalyzed by polymerase to be linked to the 3' end of the oligonucleotide chain substrate, thereby obtaining the oligonucleotide with a reversible 3' end modification. The phosphokinases include one or more of the following: acetate kinase, pyruvate kinase, adenosine 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 includes: a-2) When the oligonucleotide substrate is a single strand, the nucleoside monomer analog with R2 being a triphosphate is catalyzed by polymerase to be linked to the 3' end of the oligonucleotide substrate, thereby obtaining the oligonucleotide with a reversible 3' end modification.

9. The synthesis method according to claim 5, characterized in that, The synthesis method includes: a-3) When the oligonucleotide substrate is a single strand, a polymerase is used to catalyze the nucleotide monomer analog of any one of claims 3 to 4 to the 3' end of the oligonucleotide substrate, thereby obtaining the oligonucleotide with a reversible 3' end modification.

10. The synthesis method according to any one of claims 7 to 9, characterized in that, The polymerase includes PUP polymerase; The ligase includes RNA ligase or DNA ligase.

11. A method for preparing an oligonucleotide, characterized in that, The preparation method includes: removing the reversible modification end at the 3' end of the oligonucleotide 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 includes: The oligonucleotide with the reversibly modified 3' end was mixed with the first solution to obtain a mixed system. The removal of the reversible modification at the 3' end is achieved using the hybrid system.

13. The preparation method according to claim 12, characterized in that, The pH of the mixture is 4-6. The mixture is maintained at a temperature of 20-40°C for 5-10 minutes to remove the reversible modification at the 3' end.

14. The preparation method according to claim 13, characterized in that, The first solution includes any one or more 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 to 10 M.

16. The use of the nucleoside monomer analog of any one of claims 1 to 2, or the nucleotide monomer analog of any one of claims 3 to 4, or the method for synthesizing the oligonucleotide having a reversibly modified 3' end of any one of claims 5 to 10, or the method for preparing the oligonucleotide of any one of claims 11 to 15, in the preparation of oligonucleotides.

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