Method for enzymatic synthesis of 3'-o-phosphoric acid-modified nucleic acid

Abstract

Provided is a method for enzymatic synthesis of a 3'-O-phosphoric acid-modified nucleic acid, the method comprising using a phosphokinase to catalyze a nucleic acid monomer having a length of 1 nt or an oligonucleotide chain having a length of ≥ 2 nt, so as to obtain a 3'-O-phosphoric acid-modified nucleic acid monomer or oligonucleotide chain. The method can fill the technical blank that 3'-O-phosphoric acid-modified nucleic acids are difficult to synthesize via enzyme catalysis in the prior art, and the method is suitable for the field of synthesis of non-natural nucleotides.

Description

Enzymatic synthesis of nucleic acids with 3'-O-phosphate modification

[0001] This application is based on and claims priority to Chinese application CN application number 202410574630.3 filed on May 10, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the field of non-natural nucleotide synthesis, and more specifically, to a method for the enzymatic synthesis of nucleic acids with 3'-O-phosphate modification. Background Technology

[0003] Currently, oligonucleotide synthesis mainly relies on phosphorous amide solid-phase synthesis, but synthesizing nucleic acid chains longer than 200 nt remains highly challenging. Furthermore, the accuracy of this synthesis cannot meet current market demands for oligonucleotide products; the theoretical success rate for synthesizing nucleic acids longer than 200 nt is generally at most 37%. Existing oligonucleotide synthesis processes utilize large amounts of organic solvents, involving toxic reagents and posing significant PMI (Purpose Mass Index, the ratio of the total mass of all substances produced to the mass of the target product). For example, synthesizing a 20 nt oligonucleotide generates approximately 4000 kg of waste per kilogram of product. It also faces bottlenecks such as limitations in scale-up production. With the widespread application of oligonucleotide drugs, the demand for oligonucleotides is increasing daily; therefore, developing new methods for oligonucleotide synthesis is essential.

[0004] Enzymatic synthesis of oligonucleotides is a green synthetic process with advantages such as mild conditions, rapid coupling, and no hazardous waste generation. It can synthesize longer-chain oligonucleotides, thus showing promising application prospects. During enzymatic synthesis of oligonucleotides, reversible protection of the 3' end of the nucleotide or oligonucleotide is necessary to achieve specificity of the synthesized sequence and avoid the synthesis of erroneous sequences. The 3' end protecting group must be removable without leaving a trace after the enzymatic catalysis. In the template-free synthesis of oligonucleotides by nucleic acid polymerases, reversible 3' end capping of the nucleotide is required. After the coupling reaction, the 3' end cap needs to be removed to proceed to the next round of synthesis. Therefore, reversible 3' end capping of nucleic acid chains or nucleotides is crucial for the enzymatic synthesis of oligonucleotides. However, there are currently no reports on reversible enzymatic modification of the 3' end of oligonucleotides or nucleotides. Existing technologies mainly rely on chemical methods to synthesize nucleotides with reversible 3' end modifications, which are relatively cumbersome. Reversible modification of the 3' end of nucleic acid chains remains a technological gap.

[0005] Summary of the Invention

[0006] The main objective of this invention is to provide an enzymatic method for synthesizing nucleic acids with 3'-O-phosphate modification, thereby filling the technological gap in the prior art where it is difficult to synthesize nucleic acids with 3'-O-phosphate modification by enzyme catalysis.

[0007] To achieve the above objective, according to a first aspect of the present invention, a method for enzymatically synthesizing nucleic acids modified with 3'-O-phosphate is provided, the method comprising: using a phosphokinase to catalyze a nucleic acid monomer of length 1 nt or an oligonucleotide chain of length ≥2 nt to obtain a nucleic acid monomer or oligonucleotide chain with 3'-O-phosphate.

[0008] Furthermore, nucleic acid monomers include nucleosides or nucleotides; nucleosides are composed of a base and a pentose sugar; nucleotides are composed of a base, a pentose sugar, and a phosphate; the pentose sugar includes ribose or deoxyribose; the base is adenine, guanine, cytosine, or uracil; the number of phosphates in the nucleotide is 1, 2, or 3, and the corresponding nucleotides are nucleoside monophosphate, nucleoside diphosphate, or nucleoside triphosphate, respectively.

[0009] Furthermore, the oligonucleotide chain is an RNA or DNA chain with a length ≥2 nt.

[0010] Furthermore, the length of the oligonucleotide chain is 2-20 nt.

[0011] Furthermore, phosphokinases include APSK enzymes or DPCK enzymes.

[0012] Further, the DPCK enzyme is any one of the proteins shown in SEQ ID NO: 1-SEQ ID NO: 10; the APSK enzyme is any one of the proteins shown in SEQ ID NO: 11-SEQ ID NO: 20.

[0013] Furthermore, nucleic acid monomers include natural nucleic acid monomers or non-natural nucleic acid monomers. The pentose sugar of natural nucleic acid monomers is ribose or deoxyribose, while the pentose sugar of non-natural nucleic acid monomers is modified ribose or deoxyribose, or a non-natural monosaccharide composed of 5 carbon atoms with a non-natural backbone.

[0014] Furthermore, oligonucleotide chains include natural oligonucleotide chains composed of natural nucleotides, or non-natural oligonucleotide chains containing non-natural nucleotides.

[0015] Furthermore, non-natural oligonucleotide chains are composed of non-natural nucleotides.

[0016] Furthermore, the method includes: using ATP or polyphosphate, and phosphokinase, to catalyze the use of nucleic acid monomers or oligonucleotide chains as substrates to obtain nucleic acid monomers or oligonucleotide chains with 3'-O-phosphate.

[0017] By applying the technical solution of this invention, phosphokinase can catalyze nucleic acid monomers or oligonucleotide chains to obtain nucleic acid monomers or oligonucleotide chains with 3'-O-phosphate, thereby realizing the enzymatic preparation of nucleic acids containing 3'-O-phosphate modification. Compared with the chemical synthesis in the prior art, this enzymatic preparation has many advantages such as production scale, yield, cost, environmental protection, and safety. Attached Figure Description

[0018] 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:

[0019] Figure 1 shows the liquid chromatography results of 3'-O-phosphoric acid modification using APSK-6 catalytic substrate according to Example 1 of the present invention.

[0020] Figure 2 shows the mass spectrometry results of 3'-O-phosphate modification using APSK-6 catalytic substrate according to Example 1 of the present invention.

[0021] Figure 3 shows the liquid chromatography results of AMP modification with APSK-3 catalysis according to Example 3 of the present invention, based on Example 3 of the present invention.

[0022] Figure 4 shows the mass spectrometry results of AMP modification with APSK-3 catalysis according to Example 3 of the present invention.

[0023] Figure 5 shows the liquid chromatography results of 3'-O-phosphoric acid modification using DPCK-7 catalyzed 5'-Pi-GA-OH-3' according to Example 2 of the present invention.

[0024] Figure 6 shows the liquid chromatography results of 3'-O-phosphoric acid modification using DPCK-3 catalysis for Pi-GmGfUmCf-OH according to Example 5 of the present invention.

[0025] Figure 7 shows the liquid chromatography results of 3'-O-phosphoric acid modification of UMP using APSK-6 catalysis according to Example 6 of the present invention. Detailed Implementation

[0026] 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.

[0027] 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, and the 5' position of the pentose sugar is connected to an -OH group instead of a phosphate group.

[0028] 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.

[0029] Using uridine and 5'-uridine monophosphate as examples, the structures of nucleosides and nucleotides containing uracil are shown:

[0030] Nucleic acid monomer: In this application, it refers to a single nucleoside or single nucleotide with a length of 1 nt.

[0031] As mentioned in the background section, in the prior art, the 3'-O-phosphate modification of oligonucleotides can only be obtained by chemical reaction. However, chemical synthesis is limited in terms of chain length, production capacity, and yield in industrial-scale production. Therefore, in this application, the inventors attempted to develop a method for synthesizing nucleotides with 3'-O modification through enzymatic catalysis, and proposed a series of protection schemes for this application.

[0032] In a first typical embodiment of this application, a method for enzymatically synthesizing nucleic acids modified with 3'-O-phosphate is provided. The method includes: using a phosphokinase to catalyze a nucleic acid monomer with a length of 1 nt or an oligonucleotide chain with a length of ≥2 nt to obtain a nucleic acid monomer or oligonucleotide chain with 3'-O-phosphate.

[0033] In the method described in this application, the inventors unexpectedly discovered that phosphokinases can catalyze the phosphorylation of the 3'-O portion of a 1-nt nucleic acid monomer or an oligonucleotide chain of 2 nt or longer, thereby obtaining a nucleic acid monomer with 3'-O-phosphate, or phosphorylating the 3'-O portion of a nucleotide located at the 3' end of an oligonucleotide chain, thereby obtaining an oligonucleotide chain containing 3'-O-phosphate at the 3' end. The above method enables the modification of oligonucleotide chains or nucleic acid monomers with 3'-O-phosphate.

[0034] In a preferred embodiment, the nucleic acid monomer includes a nucleoside or a nucleotide; the nucleoside is composed of a base and a pentose sugar; the nucleotide is composed of a base, a pentose sugar, and a phosphate; the pentose sugar includes ribose or deoxyribose; the base is adenine, guanine, cytosine, or uracil; the nucleotide contains one, two, or three phosphates, corresponding to nucleoside monophosphate, nucleoside diphosphate, or nucleoside triphosphate, respectively.

[0035] In a preferred embodiment, the oligonucleotide chain is an RNA chain or DNA chain with a length ≥2 nt.

[0036] In a preferred embodiment, the oligonucleotide chain is 2-20 nt in length, including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nt.

[0037] The above method can be used to perform 3'-O phosphorylation modification on nucleic acid monomers such as adenine ribonucleotide, guanine ribonucleotide, and cytosine ribonucleotide. It can also catalyze phosphorylation of single-stranded RNA of lengths including but not limited to 2-5 nt (2nt, 3nt, 4nt, or 5nt), catalyzing the 3'-O position of the nucleotide located at the 3' end of the single-stranded RNA to obtain nucleotides containing 3'-O-phosphate modification. This 3'-O-phosphate modification can be subsequently removed by alkaline phosphatase or polynucleotide kinase, achieving reversible modification of the original single nucleotide or oligonucleotide chain.

[0038] In a preferred embodiment, the oligonucleotide chain includes a first oligonucleotide chain or a second oligonucleotide chain, wherein the length of the first oligonucleotide chain is ≥2nt and the length of the second oligonucleotide chain is ≥3nt.

[0039] The first oligonucleotide chain is a first non-natural oligonucleotide chain containing non-natural nucleotides; preferably, the first non-natural oligonucleotide chain is composed of non-natural nucleotides.

[0040] Preferably, the second oligonucleotide chain comprises a natural oligonucleotide chain composed of natural nucleotides, or a second non-natural oligonucleotide chain containing non-natural nucleotides; more preferably, the second non-natural oligonucleotide chain comprises non-natural nucleotides.

[0041] In a preferred embodiment, the phosphokinase includes APSK enzyme or DPCK enzyme; preferably, the DPCK enzyme is any of the proteins shown in SEQ ID NO: 1-SEQ ID NO: 10; preferably, the APSK enzyme is any of the proteins shown in SEQ ID NO: 11-SEQ ID NO: 20.

[0042] In the prior art, APSK (adenosine-5′-phosphate sulfate kinase) enzymes catalyze phosphorylation using adenosine-5′-phosphate sulfate as a substrate and are one of the key enzymes involved in sulfur metabolism in plants and prokaryotes; DPCK (dephosphorylated coenzyme A kinase) enzymes catalyze phosphorylation using dephosphorylated coenzyme A as a substrate and are the final step in coenzyme A (CoA) biosynthesis. In this application, the inventors have discovered that phosphokinases, including but not limited to APSK or DPCK enzymes, can catalyze the catalysis of mononucleotide or oligonucleotide chains.

[0043] In a preferred embodiment, the nucleic acid monomer includes natural nucleic acid monomers or non-natural nucleic acid monomers. The pentose sugar of the natural nucleic acid monomer is ribose or deoxyribose, and the pentose sugar of the non-natural nucleic acid monomer is modified ribose or deoxyribose, or a non-natural monosaccharide composed of 5 C atoms with a non-natural backbone.

[0044] The aforementioned nucleic acid monomers include natural nucleosides or nucleotides, or non-natural nucleosides or nucleotides.

[0045] In the above method, the catalyzed nucleic acid monomers or oligonucleotide chains can be composed of natural nucleotides or contain non-natural nucleotides. This method can perform 3'-O-phosphorylation modification on the 3'-OH position of both natural and non-natural nucleotides.

[0046] Xeno-nucleic acid (XNA) is a class of nucleic acid molecules with a non-natural backbone or nucleic acid bases. Preferably, XNA 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. The structure of non-natural nucleosides differs from the above-mentioned XNA in the presence or absence of a phosphate group.

[0047] Among them, locked nucleic acid modification is Hexitol nucleic acid modification 2'-methoxyethyl modification 2' methoxy group modified to 2' Fluorine modification 2'-FANA is ribuloNA is TNA is tPhoNA is dXNA is PS editing In the above structures, Base represents a base. For the above modifications of non-natural nucleotides, please refer to the following literature: Duffy K, Arangundy-Franklin S, Holliger P. Modified nucleic acids:replication,evolution,andnext-generation therapeutics[J].BMC Biology,2020,18(1):112.

[0048] In a preferred embodiment, the nucleic acid monomer includes natural nucleic acid monomers or non-natural nucleic acid monomers. The pentose sugar of the natural nucleic acid monomer is ribose or deoxyribose, and the pentose sugar of the non-natural nucleic acid monomer is modified ribose or deoxyribose, or a non-natural monosaccharide composed of 5 C atoms with a non-natural backbone.

[0049] The aforementioned nucleic acid monomers include natural nucleosides or nucleotides, or non-natural nucleosides or nucleotides. Non-natural nucleosides or nucleotides contain modifications at the 2' position of the ribose, modifications to the ribose backbone, base modifications, or modifications to the phosphate backbone (non-natural nucleosides lack phosphate groups).

[0050] In the above methods, the catalyzed nucleic acid monomers can be either natural nucleotides or natural nucleosides, or non-natural nucleotides or non-natural nucleosides. The catalyzed oligonucleotide chains can be composed of natural nucleotides or contain non-natural nucleotides.

[0051] The above method can not only perform 3'-O-phosphorylation modification on the 3'-OH of natural nucleosides or nucleotides, but also perform the same modification on the 3'-OH of non-natural nucleosides or nucleotides.

[0052] In a preferred embodiment, the non-natural oligonucleotide is composed of non-natural nucleotides.

[0053] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0054] Example 1

[0055] The phosphokinase 5′-phosphate-adenosine sulfate (APSK) or the phosphokinase DPCK of dephosphorylated coenzyme A (DPCK) acts directly on the 3′ end of the nucleic acid monomer or oligonucleotide chain to add a removable protecting group, 3′-O-phosphate.

[0056] The reaction system was added to a clean container to achieve a substrate concentration of 100 μM (5'-UmCfUmCf-3'). The final concentration of 5′-phosphate-sulfate-adenosine phosphokinase or dephosphorylated coenzyme A phosphokinase used was 0.2 mg / mL. 1 mM ATP, 10 mM MgCl2, and 100 mM Tris-HCl were added, and the pH was adjusted to 8.0. The reaction was carried out at 30 °C for 16 h. The conversion of the reaction system was detected by mass spectrometry.

[0057] In this application, m after A, C, G or U indicates a 2' methoxy modification of the ribonucleotide, and f indicates a 2' fluoro modification of the ribonucleotide.

[0058] The APSK or DPCK used are shown in Table 1.

[0059] Table 1

[0060] Where - indicates that a product was detected and the conversion rate is ≤0.1%, + indicates that a product was detected and the conversion rate is ≤1%, and ++ indicates that a product was detected and the conversion rate is ≤10%.

[0061] Figure 1 shows the liquid chromatography results catalyzed by the kinase shown in SEQ ID NO: 16, and Figure 2 shows the mass spectrometry results catalyzed by the kinase shown in SEQ ID NO: 16. The theoretical molecular weight of the substrate UmCf is 1192.20, the theoretical molecular weight of the product is 1271.17, and the product molecular weight (M-1: 1271.16) was detected. The product structure is shown in Formula I.

[0062] Simultaneously, a comparison was made using product standards, and the same peak elution time was detected, indicating that the reversible end capping of the 3' end was successfully performed.

[0063] Example 2

[0064] The reaction mixture was added to a clean container to achieve a substrate concentration of 100 μM. The final concentration of the 5′-phosphate-sulfate-adenosine phosphokinase or dephosphorylated coenzyme A phosphokinase used was 0.2 mg / mL. 1 mM ATP, 10 mM MgCl2, and 100 mM Tris-HCl were added, and the pH was adjusted to 8.0. The mixture was reacted at 30 °C for 16 h. The conversion was then analyzed by mass spectrometry. The results showed that the target product, 3′-O-phosphate, was generated in the phosphorylation reactions of various oligonucleotide substrates catalyzed by APSK-3 and DPCK-7. This phosphate group can be removed by alkaline phosphatase or polynucleotide kinase after phosphorylation.

[0065] Examples of substrate sequences for this reaction are shown in Table 2.

[0066] Table 2

[0067] In this diagram, "+" indicates that the product was detected with a conversion rate ≤1%; "++" indicates that the product was detected with a conversion rate ≤10% and 1% < conversion rate; Pi represents a monophosphate group; m after A, C, G, or U indicates a 2' methoxy modification of the ribonucleotide, and f indicates a 2' fluoro modification of the ribonucleotide. s before A, C, G, or U indicates a thiomodification of the 5' phosphate of the ribonucleotide. d before A, C, G, or T indicates that the nucleotide is a deoxyribonucleotide; nucleotides without d are all ribonucleotides.

[0068] In the sequence shown in SEQ ID NO: 21, the ribonucleotides at positions 1, 2, 4, 6, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19 and 20 have a 2' methoxy modification, and the ribonucleotides at positions 3, 5, 7, 9 and 11 have a 2' fluorine modification.

[0069] In the sequence shown in SEQ ID NO: 22, the ribonucleotides at positions 1-10 have a 2' methoxy modification, the ribonucleotide at position 7 has a 2' fluorine modification, and the ribonucleotides at positions 2 and 3 have a 5' phosphate thio modification.

[0070] Figure 5 shows the LC-MS analysis spectrum of the reaction of 5'-Pi-GA-OH-3' with 3'-O-phosphoric acid modification catalyzed by DPCK-7. The theoretical molecular weight of the product is 772.08, and the detected molecular weight of the product (M-1: 771.0685) is 5'-Pi-GA-OH-Pi-3', where Pi represents the phosphate group.

[0071] The above-mentioned APSK or DPCK enzymes can also be used to catalyze the above-mentioned substrates.

[0072] Example 3

[0073] The reaction mixture was added to a clean container to achieve an AMP substrate concentration of 100 μM. The 5′-phosphate-sulfate-adenosine phosphokinase (APSK3) was used to achieve a final concentration of 0.2 mg / mL. 1 mM ATP, 10 mM MgCl2, and 100 mM Tris-HCl were added, and the pH was adjusted to 8.0. The mixture was reacted at 30 °C for 16 h. The conversion was analyzed by liquid chromatography and mass spectrometry, and the peak positions were determined using standards. The results showed a new peak at 6.384 min, the same position as the 3′Pi-AMP standard. Mass spectrometry confirmed a molecular weight of 428.1, compared to a theoretical molecular weight of 427.03, indicating product formation.

[0074] The LC-MS analysis spectra of AMP (adenosine monophosphate, 5' adenine nucleotide) modified with 3'-O-phosphate using APSK-3 catalysis are shown in Figures 3 and 4. Figure 3 is the liquid chromatography result of the reaction system, and Figure 4 is the mass spectrometry detection result of the phosphorylated product (peak at 6.384).

[0075] Example 4

[0076] The reaction mixture was added to a clean container to achieve a substrate concentration of 100 μM. The final concentration of the 5′-phosphate-sulfate-adenosine phosphokinase or dephosphorylated coenzyme A phosphokinase used was 0.2 mg / mL. 1 mM ATP, 10 mM MgCl2, and 100 mM Tris-HCl were added, and the pH was adjusted to 8.0. The reaction was carried out at 30 °C for 16 h. The reaction mixture was then analyzed by liquid chromatography and mass spectrometry. The results showed that product molecules were generated in the phosphorylation reactions of various nucleotides or nucleoside substrates catalyzed by APSK-3 and DPCK-7, indicating the formation of the target product, 3′-O-phosphate.

[0077] Table 3

[0078] Where "+" indicates that the product was detected and the conversion rate is ≤1%; "++" indicates that the product was detected and the conversion rate is 1% < 10%; "+++" indicates that the product was detected and the conversion rate is 10% < 20%; "+++++" indicates that the product was detected and the conversion rate is 20% < 30%; Pi represents a monophosphate group; 2Pi represents a diphosphate group; 3Pi represents a triphosphate group; m after A, C, G, or U indicates a 2' methoxy modification of the ribonucleotide, and f indicates a 2' fluorine modification of the ribonucleotide. d before A, C, G, or T indicates that the nucleotide is a deoxyribonucleotide; nucleotides without d are all ribonucleotides.

[0079] Example 5

[0080] The reaction mixture was added to a clean container to achieve a substrate concentration of 100 μM for Pi-GmGfUmCf-OH and a final DPCK-3 concentration of 0.2 mg / mL. 1 mM ATP, 10 mM MgCl2, and 100 mM Tris-HCl were added, and the pH was adjusted to 8.0. The mixture was reacted at 30 °C for 16 h. The reaction mixture was then analyzed by liquid chromatography and mass spectrometry. The results showed that product peaks were generated in all reaction systems, as shown in Figure 6. The molecular weight of the product was detected by mass spectrometry, indicating the formation of the target product, 3'-O-phosphate.

[0081] Example 6

[0082] The reaction mixture was added to a clean container to achieve a UMP substrate concentration of 100 μM and a final APSK-6 concentration of 0.2 mg / mL. 1 mM ATP, 10 mM MgCl2, and 100 mM Tris-HCl were added, and the pH was adjusted to 8.0. The mixture was reacted at 30 °C for 16 h. The reaction mixture was then analyzed by liquid chromatography and mass spectrometry. The results showed the formation of a new peak in the reaction mixture. Mass spectrometry confirmed the molecular weight of the new peak to be M-1: 403.0, while the theoretical molecular weight of the product is 404.0, as shown in Figure 7. The peak elution time was the same as that of the product standard, indicating the formation of the target product, 3'-O-phosphate.

[0083] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: Utilizing the two enzyme-catalyzed methods described above, phosphokinases such as APSK or DPCK can catalyze nucleic acid monomers or oligonucleotide chains to obtain nucleic acid monomers or oligonucleotide chains with 3'-O-phosphate, thus realizing the enzymatic preparation of nucleotides containing 3'-O-phosphate modification. This method enables the enzymatic synthesis of nucleotides modified with 3'-O-phosphate. Compared to chemical methods for synthesizing nucleic acid chains with protecting groups, the above method can directly add 3'-O-phosphate modification, which is a reversible modification group, to the 3' end of the nucleic acid chain, and the protection can be easily removed, thereby achieving a green, environmentally friendly, and efficient purpose.

[0084] 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 method for enzymatic synthesis of nucleic acids modified with 3'-O-phosphate, characterized in that, The method includes: Nucleic acid monomers or oligonucleotide chains with 3'-O-phosphate are obtained by using phosphokinase to catalyze nucleic acid monomers with a length of 1 nt or oligonucleotide chains with a length of ≥2 nt.

2. The method according to claim 1, characterized in that, The nucleic acid monomer includes nucleosides or nucleotides; The nucleoside is composed of a base and a pentose sugar; The nucleotide is composed of the base, the pentose sugar, and a phosphate group; The pentose sugar includes ribose or deoxyribose; The base is adenine, guanine, cytosine, or uracil; The nucleotide contains one, two, or three phosphates, and the corresponding nucleotides are nucleoside monophosphate, nucleoside diphosphate, or nucleoside triphosphate, respectively.

3. The method according to claim 1, characterized in that, The oligonucleotide chain is an RNA or DNA chain with a length ≥2 nt.

4. The method according to claim 3, characterized in that, The length of the oligonucleotide chain is 2-20 nt.

5. The method according to claim 1, characterized in that, The phosphokinases include APSK enzymes or DPCK enzymes.

6. The method according to claim 5, characterized in that, The DPCK enzyme is any one of the proteins shown in SEQ ID NO: 1-SEQ ID NO: 10; The APSK enzyme is any one of the proteins shown in SEQ ID NO: 11-SEQ ID NO:

20.

7. The method according to claim 2, characterized in that, The nucleic acid monomers include natural nucleic acid monomers or non-natural nucleic acid monomers. The pentose sugar of the natural nucleic acid monomers is ribose or deoxyribose, and the pentose sugar of the non-natural nucleic acid monomers is modified ribose or deoxyribose, or a non-natural monosaccharide composed of 5 carbon atoms with a non-natural backbone.

8. The method according to claim 1, characterized in that, The oligonucleotide chain includes a natural oligonucleotide chain composed of natural nucleotides, or a non-natural oligonucleotide chain containing non-natural nucleotides.

9. The method according to claim 8, characterized in that, The non-natural oligonucleotide chain is composed of the non-natural nucleotides.

10. The method according to any one of claims 1-9, characterized in that, The method includes: using ATP or polyphosphate, and the phosphokinase, to catalyze the nucleic acid monomer or the oligonucleotide chain as a substrate to obtain the nucleic acid monomer or oligonucleotide chain of the 3'-O-phosphate.

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