Method for synthesizing double-stranded RNA and use of RNA ligase in synthesizing double-stranded RNA

Abstract

The present invention discloses a method for synthesizing double-stranded RNA and the use of RNA ligase in the synthesis of double-stranded RNA. The synthesis method includes step S1 of synthesizing single-stranded RNA fragments and step S2 of mixing the single-stranded RNA fragments and ligating the single-stranded RNA fragments with RNA ligase to form double-stranded RNA, where the RNA ligase is RNA ligase derived from Vibrio phage, Escherichia phage, or Klebsiella phage, for example. By applying the technical solution of the present invention, it is possible to specifically splice different nucleotide fragments, synthesize long fragment nucleotide chains, and catalyze the ligation of natural nucleotides and unnatural nucleotides, thereby solving the current technical difficulties in synthesizing nucleotide chains.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, and in particular to a method for synthesizing double-stranded RNA and the use of RNA ligase in synthesizing double-stranded RNA. [Background technology]

[0002] Ribonucleic acid (RNA) is a carrier of genetic information found in living cells and in some viruses and viroids. For a long time, RNA was thought to be merely a molecule that transports information between genes and proteins. In fact, at the very beginning of life, RNA was the only biological molecule capable of storing information and functioning as an enzyme. RNA not only functions as a messenger (mRNA) for protein synthesis but also has crucial regulatory functions. Nontranscribed RNAs include miRNA, siRNA, incRNA, and piwiRNA. There are over 400 miRNA molecules alone, which regulate at least one-third of human genes. Since the 1970s, gene vector technology, gene cloning technology, and gene editing technology have had a profound impact on modern gene therapy. Furthermore, RNA editing technology has made it possible to edit specific nucleotides in human cells at the genetic level. This technology can be used not only as a research tool but also as a temporary treatment for diseases caused by mutations.

[0003] The field of RNA medicine has developed rapidly in recent years. Small molecule nucleic acid drugs, such as siRNA and ASO, have made a name for themselves in areas such as rare diseases, while mRNA drugs, such as mRNA vaccines, have played an important role in the COVID-19 pandemic. Since the marketing approval of the first small molecule nucleic acid drug, fomivirsen, at the end of the last century, the development of the related industry has entered a "window of opportunity." Advances in delivery technology have led to a period of rapid growth in small molecule nucleic acid drugs in recent years. This period also saw the emergence of the first blockbuster small molecule nucleic acid drug, nusinersen. This drug, developed by Biogen, is an antisense oligonucleotide (ASO) drug for the treatment of spinal muscular atrophy (SMA) in adult and pediatric patients.

[0004] Currently, solid-phase synthesis is the most common method for synthesizing oligonucleotides (12-30 nucleotides). Solid-phase synthesis involves immobilizing nucleic acids on a solid support. The most commonly used solid support is controllable microporous glass beads (CPG), but their loading capacity is limited, typically 70-80 μmol / g. However, solid-phase synthesis of nucleotide chains has a major drawback: the length of the chain is limited, typically within 25 nucleotides. As the length of the chain increases, the yield decreases. When primers are synthesized up to approximately 80 nt, the crude product has a purity of only about 40%, significantly limiting the synthesis of nucleotide chains.

[0005] Enzyme-catalyzed synthesis is a promising catalytic method because it is an environmentally friendly and efficient method, requires relatively mild reaction conditions, and requires little or no organic reagents. Synthesizing nucleotide chains using ligases achieves environmentally friendly catalysis and overcomes the technical bottleneck of solid-phase synthesis by allowing for unlimited chain length synthesis. However, there are few literature or patent reports on the enzyme-catalyzed synthesis of natural or non-natural RNA double strands, and the synthesis of non-natural RNA double strands in particular has been extremely difficult. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides a method for synthesizing double-stranded RNA and the use of RNA ligase in synthesizing double-stranded RNA, in order to solve the technical problems that cannot be solved in the prior art when enzymatically catalyzing the synthesis of double-stranded natural or non-natural RNA. [Means for solving the problem]

[0007] To achieve the above object, one aspect of the present invention provides a method for synthesizing double-stranded RNA, comprising: step S1 synthesizing single-stranded RNA fragments; and step S2 mixing the single-stranded RNA fragments and ligating them with an RNA ligase to form double-stranded RNA, wherein the RNA ligase is derived from Vibrio phage, Escherichia phage, Klebsiella phage, Bacteriophage, thermophilic bacteriophage, Acidobacteria bacterium, Salmonella enterica, Yersinia phage vB_YepM_ZN18, Shigella phage pSs-1, or Buttiauxella phage vB_ButM_GuL6.

[0008] Furthermore, the single-stranded RNA fragment is 3 to 200 nucleotides in length, or an analog thereof.

[0009] Furthermore, the amino acid sequence of an RNA ligase derived from a Vibrio phage is shown in SEQ ID NO: 1, 2, 5, 6, 13, 14, 17, or 19, the amino acid sequence of an RNA ligase derived from an Escherichia phage is shown in SEQ ID NO: 3 or 15, the amino acid sequence of an RNA ligase derived from a Klebsiella phage is shown in SEQ ID NO: 4 or 16, the amino acid sequence of an RNA ligase derived from an Acidobacteria bacterium is shown in SEQ ID NO: 7, the amino acid sequence of an RNA ligase derived from a Salmonella enterica is shown in SEQ ID NO: 8, the amino acid sequence of an RNA ligase derived from a Yersinia phage vB_YepM_ZN18 is shown in SEQ ID NO: 9, the amino acid sequence of an RNA ligase derived from a Shigella phage pSs-1 is shown in SEQ ID NO: 10, and the amino acid sequence of an RNA ligase derived from a Buttiauxella phage The amino acid sequence of the RNA ligase derived from vB_ButM_GuL6 is shown in SEQ ID NO: 11, the amino acid sequence of the RNA ligase derived from Bacteriophage is shown in SEQ ID NO: 12, and the amino acid sequence of the RNA ligase derived from thermophilic Bacteriophage is shown in SEQ ID NO: 18.

[0010] Furthermore, the double-stranded RNA may be natural RNA or non-natural RNA.

[0011] The ribonucleotide further comprises one or more of a pentose ring 2'-position modification, a 3'-position modification, a phosphate ion a-position modification, or a base modification, and preferably the pentose ring 2'-position modification includes a 2'-methoxy modification, a 2'-fluoro modification, a 2'-trifluoromethoxy modification, a 2'-methoxyethyl modification, a 2'-allyl modification, a 2'-amino modification, or a 2'-azido modification, and preferably the phosphate ion α-position modification includes a phosphate ion α-position thio modification, and preferably the base modification includes a methylation modification and / or an acetylation modification at any one or more of the N1, N5, or N6 positions of the base.

[0012] Furthermore, single-stranded RNA fragments are synthesized by employing solid-phase synthesis.

[0013] Furthermore, the single-stranded RNA fragments include single-stranded RNA fragments substrate 1 to substrate n and corresponding complementary single-stranded RNA fragments substrate n+1 to substrate n+n, where n is 2 or more, and between the complementary single-stranded RNA fragments, three or more nucleotide bases are complementarily paired, and at the 5'-ends of substrate 2 and substrate n+1, and substrate n and substrate n+n-1, two or more nucleotide bases are complementarily paired, and optionally, the outer 5'- and 3'-ends of substrate 1 and substrate n+1 are connected by a chemical bond, and optionally, the outer 5'- and 3'-ends of substrate n and substrate n+n are connected by a chemical bond.

[0014] Furthermore, the reaction temperature of S2 is 0°C to 60°C, preferably 4°C to 37°C, the reaction time is 0.5 hours to 24 hours, and the pH is 6 to 8.5.

[0015] According to another aspect of the present invention, there is provided use of an RNA ligase in synthesizing double-stranded RNA, wherein the RNA ligase is derived from Vibrio phage, Escherichia phage, Klebsiella phage, Bacteriophage, thermophilic bacteriophage, Acidobacteriabacterium, Salmonella enterica, Yersinia phage vB_YepM_ZN18, Shigella phage pSs-1, or Buttiauxella phage vB_ButM_GuL6.

[0016] Furthermore, the amino acid sequence of an RNA ligase derived from a Vibrio phage is shown in SEQ ID NO: 1, 2, 5, 6, 13, 14, 17, or 19, the amino acid sequence of an RNA ligase derived from an Escherichia phage is shown in SEQ ID NO: 3 or 15, the amino acid sequence of an RNA ligase derived from a Klebsiella phage is shown in SEQ ID NO: 4 or 16, the amino acid sequence of an RNA ligase derived from an Acidobacteria bacterium is shown in SEQ ID NO: 7, the amino acid sequence of an RNA ligase derived from a Salmonella enterica is shown in SEQ ID NO: 8, the amino acid sequence of an RNA ligase derived from a Yersinia phage vB_YepM_ZN18 is shown in SEQ ID NO: 9, the amino acid sequence of an RNA ligase derived from a Shigella phage pSs-1 is shown in SEQ ID NO: 10, and the amino acid sequence of an RNA ligase derived from a Buttiauxella phage The amino acid sequence of the RNA ligase derived from vB_ButM_GuL6 is shown in SEQ ID NO: 11, the amino acid sequence of the RNA ligase derived from Bacteriophage is shown in SEQ ID NO: 12, and the amino acid sequence of the RNA ligase derived from thermophilic Bacteriophage is shown in SEQ ID NO: 18. [Effects of the Invention]

[0017] By applying the technical solutions of the present invention, different nucleotide fragments can be specifically spliced, long fragment nucleotide chains can be synthesized, and the ligation of natural nucleotides and non-natural nucleotides can be catalyzed, thereby solving the current technical difficulties in synthesizing nucleotide chains. [Brief explanation of the drawings]

[0018] The drawings in the specification that form a part of this application are used to provide a further understanding of the present invention, and the exemplary embodiments of the present invention and the description thereof are used to interpret the present invention and are not intended to unduly limit the present invention. [Figure 1]FIG. 1 shows the results of denaturing polyacrylamide gel electrophoresis in Example 1. [Figure 2] 1 shows the UPLC detection spectrum of a control reaction in Example 1 in which no enzyme was added. [Figure 3] 1 shows the UPLC detection spectrum of the reaction catalyzed by lig20 in Example 1. [Figure 4] FIG. 1 shows the results of denaturing polyacrylamide gel electrophoresis in Example 2. [Figure 5] FIG. 1 shows the results of denaturing polyacrylamide gel electrophoresis in Example 2. [Figure 6] 1 shows the UPLC detection spectrum of a control reaction in Example 2 in which no enzyme was added. [Figure 7] 1 shows the UPLC detection spectrum of the reaction catalyzed by lig24 in Example 2. [Figure 8] 1 shows the peak times of sense strand products in Example 2. [Figure 9] 1 shows the peak times of antisense strand products in Example 2. [Figure 10] 1 shows the peak times of double-stranded products in Example 2. [Figure 11] 1 shows the reaction results detected by SDS-PAGE gel image in Example 3. [Figure 12] 1 shows the reaction results detected by SDS-PAGE gel image in Example 4. [Figure 13] 1 shows the UPLC detection spectrum in Example 4. [Figure 14] 1 shows the results of mass spectrometry detection in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0019] It should be noted that, unless there is a contradiction, the embodiments and features of the embodiments of the present application can be combined with each other. The present invention will be described in detail below by combining embodiments with reference to the drawings.

[0020] Terminology Native RNA: refers to an RNA chain in which naturally occurring nucleotides are joined via phosphate ester bonds, the 2' position of which is hydroxy, and the phosphate backbone is a phosphorus-oxygen bond.

[0021] Non-natural RNA: refers to an RNA strand in which non-natural nucleotides are connected via phosphate ester bonds, the 2' position of which may be modified, such as 2'F, 2'methoxy, 2'MOE, LNA, etc., and the modification may be at the base or at the phosphate group, such as a phosphorothioate modification.

[0022] Currently, there are few literature or patent reports on the enzymatic synthesis of double-stranded natural or non-natural RNA, and the synthesis of double-stranded non-natural RNA is particularly difficult. The present invention fills this technical gap by using a ligase to synthesize natural or non-natural RNA strands.

[0023] According to a representative embodiment of the present invention, there is provided a method for synthesizing double-stranded RNA, comprising: step S1 synthesizing single-stranded RNA fragments; and step S2 mixing the single-stranded RNA fragments and ligating the single-stranded RNA fragments with an RNA ligase to form double-stranded RNA, wherein the RNA ligase is derived from Vibrio phage, Escherichia phage, Klebsiella phage, Bacteriophage, thermophilic bacteriophage, Acidobacteria bacterium, Salmonella enterica, Yersinia phage vB_YepM_ZN18, Shigella phage pSs-1, or Buttiauxella phage vB_ButM_GuL6.

[0024] By applying the technical solutions of the present invention, different nucleotide fragments can be specifically spliced, long fragment nucleotide chains can be synthesized, and the ligation of natural nucleotides and non-natural nucleotides can be catalyzed, thereby solving the current technical difficulties in synthesizing nucleotide chains.

[0025] In one embodiment of the present invention, the length of the single-stranded RNA fragment is 3 to 200 nucleotides or an analog thereof, preferably 3 to 20 nucleotides or an analog thereof.

[0026] Preferably, the amino acid sequence of the RNA ligase derived from Vibrio phage is shown in SEQ ID NO: 1, 2, 5, 6, 13, 14, 17, or 19, the amino acid sequence of the RNA ligase derived from Escherichia phage is shown in SEQ ID NO: 3 or 15, the amino acid sequence of the RNA ligase derived from Klebsiella phage is shown in SEQ ID NO: 4 or 16, the amino acid sequence of the RNA ligase derived from Acidobacteria bacterium is shown in SEQ ID NO: 7, the amino acid sequence of the RNA ligase derived from Salmonella enterica is shown in SEQ ID NO: 8, the amino acid sequence of the RNA ligase derived from Yersinia phage vB_YepM_ZN18 is shown in SEQ ID NO: 9, the amino acid sequence of the RNA ligase derived from Shigella phage pSs-1 is shown in SEQ ID NO: 10, and the amino acid sequence of the RNA ligase derived from Buttiauxella phage The amino acid sequence of the RNA ligase derived from vB_ButM_GuL6 is shown in SEQ ID NO: 11, the amino acid sequence of the RNA ligase derived from Bacteriophage is shown in SEQ ID NO: 12, and the amino acid sequence of the RNA ligase derived from thermophilic Bacteriophage is shown in SEQ ID NO: 18.

[0027] According to one exemplary embodiment of the present invention, the double-stranded RNA is natural RNA or non-natural RNA, where the non-natural RNA may include 2' methoxy, 2' fluoro, 2' MOE, 2' amino, 2' methyl, FANA, LNA, or PS, etc., where the effects of 2' methoxy, 2' fluoro, and PS are relatively good.

[0028] In the present invention, the single-stranded RNA fragment may be synthesized by employing solid-phase synthesis, that is, by a conventional synthesis method.

[0029] According to one exemplary embodiment of the present invention, the single-stranded RNA fragments include single-stranded RNA fragment Substrate 1 to Substrate n and corresponding complementary single-stranded RNA fragment Substrate n+1 to Substrate n+n, where n is 2 or greater, and three or more nucleotide bases are complementary paired between the complementary single-stranded RNA fragments, and two or more nucleotide bases are complementary paired at the 5' ends of Substrate 2 and Substrate n+1, and Substrate n and Substrate n+n-1, and optionally, the 5' and 3' ends on the outside (the outside is the unligated side) of Substrate 1 and Substrate n+1 are connected by a chemical bond, and optionally, the 5' and 3' ends on the outside (the outside is the unligated side) of Substrate n and Substrate n+n are connected by a chemical bond. Specifically, this can be understood with reference to the following reaction formula:

[0030] JPEG2025540301000002.jpg54162 Preferably, the reaction temperature of S2 is 0°C to 60°C, more preferably 4°C to 37°C, the reaction time is 0.5 hours to 24 hours, and the pH is 6 to 8.5.

[0031] According to one exemplary embodiment of the present invention, there is provided the use of an RNA ligase in the synthesis of double-stranded RNA, the RNA ligase being derived from Vibrio phage, Escherichia phage, Klebsiella phage, Bacteriophage, thermophilic bacteriophage, Acidobacteria bacterium, Salmonella enterica, Yersinia phage vB_YepM_ZN18, Shigella phage pSs-1, or Buttiauxella phage vB_ButM_GuL6. Use of these ligases allows direct ligation without an annealing reaction, improving synthesis efficiency. Preferably, the amino acid sequence of the RNA ligase derived from Vibrio phage is shown in SEQ ID NO: 1, 2, 5, 6, 13, 14, 17, or 19, the amino acid sequence of the RNA ligase derived from Escherichia phage is shown in SEQ ID NO: 3 or 15, the amino acid sequence of the RNA ligase derived from Klebsiella phage is shown in SEQ ID NO: 4 or 16, the amino acid sequence of the RNA ligase derived from Acidobacteria bacterium is shown in SEQ ID NO: 7, the amino acid sequence of the RNA ligase derived from Salmonella enterica is shown in SEQ ID NO: 8, the amino acid sequence of the RNA ligase derived from Yersinia phage vB_YepM_ZN18 is shown in SEQ ID NO: 9, the amino acid sequence of the RNA ligase derived from Shigella phage pSs-1 is shown in SEQ ID NO: 10, and the amino acid sequence of the RNA ligase derived from Buttiauxella phage The amino acid sequence of the RNA ligase derived from vB_ButM_GuL6 is shown in SEQ ID NO: 11, the amino acid sequence of the RNA ligase derived from Bacteriophage is shown in SEQ ID NO: 12, and the amino acid sequence of the RNA ligase derived from thermophilic Bacteriophage is shown in SEQ ID NO: 18.

[0032] The beneficial effects of the present invention will be further illustrated below with reference to examples.

[0033] To catalyze the double-stranded ligation of short-stranded or non-natural short-stranded RNAs, the inventors extracted various genes from different species from a gene library and cloned them into E. coli. Using Pet28A as an expression vector, the strain containing the constructed target gene was induced with IPTG, cultured, centrifuged to harvest the cells, sonicated, and centrifuged again to obtain the supernatant. The supernatant was purified using a nickel column and then combined with ion column or molecular sieve purification to obtain a pure enzyme.

[0034] A clean reaction vessel was charged with 0.1 μmol of each sequence fragment (final concentration 200 μM), 0.1 mg of ligase, 0.4 μmol of ATP, 0.5 μmol of DTT, and 25 μmol of MgCl. The volume was then supplemented with Tris-Cl buffer to a total volume of 500 μL. The reaction mixture was mixed to a final Tris-Cl concentration of 50 mM and incubated at 25°C for 1 to 20 hours. The resulting reaction mixture was subjected to polyacrylamide gel electrophoresis or UPLC detection, and the results shown in Table 3 were obtained. The results indicate that most enzymes lacked ligation activity, and only a few enzymes possessed catalytic activity.

[0035] DsRNA activity-1 is the activity of the following reaction:

[0036] JPEG2025540301000003.jpg35166, where the specific sequence is shown in Table 1.

[0037] [Table 1] DsRNA activity-2 is the activity of the following reaction:

[0038] JPEG2025540301000005.jpg29166, where the specific sequence is shown in Table 2.

[0039] [Table 2] [Table 3] TIFF2025540301000008.tif216164Note: "-" indicates that no product was produced, "++" indicates that the conversion was greater than 10% but less than 50%, "+++" indicates that the conversion was greater than 50% but less than 70%, and "++++" indicates that the conversion was greater than 70%.

[0040] Here, the amino acid sequence of a portion of the enzyme is as follows:

[0041] lig-20 (SEQ ID NO: 6, Vibrio phage): TIFF2025540301000009.tif35164lig-21 (SEQ ID NO: 1, Vibrio phage): TIFF2025540301000010.tif36164lig-22 (SEQ ID NO: 2, Vibrio phage): TIFF2025540301000011.tif40164lig-23 (SEQ ID NO: 3, Escherichia phage): TIFF2025540301000012.tif36164lig-24 (SEQ ID NO: 4, Klebsiella phage): TIFF2025540301000013.tif35164lig-25 (SEQ ID NO: 5, Vibrio phage): TIFF2025540301000014.tif35164lig-26 (SEQ ID NO: 7, AbRnl, from the Rnl1 family of Acidobacteriabacterium): TIFF2025540301000015.tif40166lig-27 (SEQ ID NO: 8, SeRnl, from the Rnl2 family of Salmonella enterica): TIFF2025540301000016.tif35166lig-28 (SEQ ID NO: 9, YpRnl, from the Rnl2 family of YersiniaphagevB_YepM_ZN18): TIFF2025540301000017.tif36166lig-29 (SEQ ID NO: 10, SppRnl, from the Rnl2 family of Shigella phage pSs-1): TIFF2025540301000018.tif35163lig-30 (SEQ ID NO: 11, BpRnl, from the Rnl2 family of Buttiauxella phage vB_ButM_GuL6): TIFF2025540301000019.tif35163lig-31 (SEQ ID NO: 12, BpARnl, from the Rnl1 family of Bacteriophage AR1): TIFF2025540301000020.tif41163lig-32 (SEQ ID NO: 13, VPhRnl, from the Rnl1 family of Vibrio phage): TIFF2025540301000021.tif40165lig-33 (SEQ ID NO: 14, VPpRnl, from the Rnl2 family of Vibrio phage phi-ST2): TIFF2025540301000022.tif40165lig-34 (SEQ ID NO: 15, EPRnl, from the Rnl1 family of Escherichia phage JN02): TIFF2025540301000023.tif40165lig-35 (SEQ ID NO: 16, KPRnl, from the Rnl2 family of Klebsiella phage KP15): TIFF2025540301000024.tif34164lig-36 (SEQ ID NO: 17, VPJRnl, from the Rnl2 family of Vibrio phage JS98): TIFF2025540301000025.tif35164lig-37 (SEQ ID NO: 18, TBRnl, from the Rnl1 family of thermophilic bacteriophage RM378): TIFF2025540301000026.tif45164lig-38 (SEQ ID NO: 19, VPphRnl, from the Rnl1 family of Vibrio phage Phi-ST2): TIFF2025540301000027.tif34163

[0042] Example 1 Ligase production The ligase gene was constructed in the pET28a vector, and a histidine tag was added to the N-terminus of the ligase to facilitate affinity chromatography purification. The constructed recombinant plasmid was transformed into the E. coli BL21(DE3) expression strain. The expression strain was inoculated into 5 mL of LB medium (containing 50 μg / mL) at a 0.1% inoculum size and cultured at 37°C for 16 h. The resulting culture was transferred to 500 mL of LB medium at a 1% inoculum size and cultured at 37°C until an OD of 0.6 was reached. 0.1 mM IPTG was added and the culture was induced at 20°C for 16 h. The resulting induced culture was centrifuged at 10,000 rpm at 4°C for 20 min to obtain a bacterial sludge. The bacterial sludge was resuspended in lysis buffer (50 mM Tris-Cl pH 8.0, 0-500 mM NaCl, 10% glycerin) to a final bacterial cell concentration of 10%, disrupted by sonication, and centrifuged to obtain a crude enzyme solution. The crude enzyme solution was filtered through a 0.22 μm filter membrane, and the resulting enzyme solution was purified using a nickel column. After desalting and liquid exchange, a secondary purification was performed using a strong anion column. The resulting enzyme solution was desalted, liquid exchange, and stored in 30% glycerin. Figure 1 shows the gel detection results of Lig-20 purified using the nickel column, and Figure 2 shows the gel detection results of Lig-20 purified using the ion column. The final ligase purification efficiency is shown in Figure 3. This demonstrates that the purification methods used in this invention were able to produce pure enzymes suitable for catalytic reactions.

[0043] Example 2 Ligation of double-stranded RNA by catalytic synthesis JPEG2025540301000028.jpg30163 [Table 4] Note: "m" indicates a 2' methoxy modification and "f" indicates a 2' fluoro modification.

[0044] The substrates in Table 4 (SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25) were prepared by solid phase synthesis, i.e., conventional chemical synthesis methods.

[0045] 0.2 μmol of each of the six substrates in Table 4 (final concentration 400 μM) was added to 0.4 μmol of ATP and diluted with 10× ligation buffer (50 mM Tris-Cl, 10 mM MgCl, 1 mM Add DTT) to make the final volume 500μL, add 0.1mg of pure enzymes of lig20, 21, 22, 23, 24, 25 into different reaction tubes, react at 25℃ for 3h, heat at 98℃ for 2min, and keep at 80℃ for 20min to inactivate the enzymes, and then centrifuge to obtain the supernatant, which was analyzed and detected by denaturing polyacrylamide gel electrophoresis. As a result, a very bright product band was obtained, as shown in Figure 4 (1. Sense strand product standard (formed by ligating SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22); 2. Antisense strand product standard (formed by ligating SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25); 3. Sense strand + antisense strand; 4. Nucleic acid molecular weight marker; 5. Trypanosoma brucei brucei ligase; 6. Reaction catalyzed by Lig21; 7. Reaction catalyzed by Lig22; 8. Reaction catalyzed by Lig24; 9. Reaction catalyzed by Lig23; 10. Reaction catalyzed by Lig20; 11. Reaction catalyzed by Lig25; 12. Control group, containing 400 μM substrate, no enzyme, and an equivalent volume of 50 mM See Figure 5 (1. sense strand; 2. antisense strand; 3. nucleic acid molecular weight marker; 4. sense strand + antisense strand; 5. reaction catalyzed by lig35; 6. reaction catalyzed by lig31; 7. reaction catalyzed by lig34; 8. reaction catalyzed by lig1; 9. reaction catalyzed by lig15; 10. reaction catalyzed by lig37; 11. reaction catalyzed by lig16; 12. reaction catalyzed by lig32; 13. control, containing 400 µM substrate, no enzyme, and an equivalent volume of 50 mM Tris-Cl buffer added instead of enzyme).The results of UPLC detection of the samples are shown in Figure 6 (control group, containing 400 μM substrate but no enzyme; an equivalent volume of 50 mM Tris-Cl buffer was added instead of the enzyme; the reaction was carried out at 25°C for 3 hours, heated at 98°C for 2 minutes, and then incubated at 80°C for 20 minutes. After centrifugation, the supernatant was collected and subjected to UPLC analysis), Figure 7 (reaction catalyzed by ligase lig24; the peak at 21.662 min is the peak of the double-stranded product), Figure 8 (the peak time of the sense strand product is 15.022 min), Figure 9 (the peak time of the antisense strand product is 15.213 min), and Figure 10 (the peak time of the mixed solution of the sense strand product and the antisense strand product; i.e., the peak time of the double-stranded product is 21.683 min; the peak at 15.192 min is the excess antisense strand product). The results showed that the peak position of the product matched the standard and that almost no substrate remained. MS analysis revealed that the sense strand was 7926.24, with a theoretical value of 7925.98±7. MS analysis revealed that the antisense strand was 8150.25, with a theoretical value of 8152.07±8. These results matched the standard, indicating that the ligation reaction was catalyzed by lig-20, 21, 22, 23, 24, 25, 35, 31, 34, 37, and 32, resulting in the production of ligated double-stranded RNA.

[0046] Example 3 Ligation of double-stranded RNA by catalytic synthesis JPEG2025540301000030.jpg33163Ligation of natural RNA double strands is catalyzed by ligase.

[0047] [Table 5] Substrates 26 to 29 were mixed in equal proportions, 10 μL of 1 mM ATP was added, and 10× ligation buffer (containing 50 mM Tris-Cl, 10 mM MgCl2, and 1 mM DTT) was added. 10 μL of pure enzymes (2 mg / ml) of lig20 and lig24 were added to separate reaction tubes. Water was added to adjust the substrate concentration to 50 μM. The reaction was allowed to proceed at 25°C for 3 hours, heated at 98°C for 2 minutes, and then incubated at 80°C for 20 minutes to inactivate the enzymes. The supernatant was centrifuged and analyzed by MS. The sense strand product (formed by ligating SEQ ID NO:26 and SEQ ID NO:27) was 4488.15, and the antisense strand product (formed by ligating SEQ ID NO:28 and SEQ ID NO:29) was 6304.92. The theoretical values ​​for the sense strand and antisense strand were 4485.73±4 and 6302.79±6, respectively. This confirmed the production of the product, indicating that Lig-20 and 24 catalyzed the ligation reaction and produced ligated double-stranded RNA. The results are shown in Figure 11 (SDS-PAGE gel image of the detection reaction. Lane 1 shows the molecular weight marker, lane 2 shows the negative control (the system contains substrate, reaction buffer, and ATP, but no enzyme; an equivalent volume of 50 mM Tris-Cl was used instead of the enzyme), and lanes 3 and 4 show the reaction system catalyzed by ligase).

[0048] Example 4 Ligation of double-stranded RNA by catalytic synthesis JPEG2025540301000032.jpg35164Ligation of non-natural RNA duplexes is catalyzed by ligase.

[0049] [Table 6] Note: "m" indicates a 2' methoxy modification, "f" indicates a 2' fluoro modification, and "s" indicates a phosphorothioate modification.

[0050] Substrates 30 to 33 were mixed in equal proportions, and 10 μl of 1 mM ATP was added. 10× ligation buffer (containing 50 mM Tris-Cl, 10 mM MgCl2, and 1 mM DTT) was added. 10 μL of pure enzyme (2 mg / ml) for lig20, 21, 22, 23, 24, 25, 31, 32, 33, 34, 35, 36, and 37 was added to each reaction tube. Water was added to bring the substrate concentration to 200 μM. A control reaction without enzyme was also set up at the same time. 10 μl of 10× ligation buffer (containing 50 mM Tris-Cl, 10 mM MgCl2, and 1 mM DTT) was added. The supernatant was collected and analyzed by MS. The detected molecular weight of the sense strand product (formed by ligation of SEQ ID NO:30 and SEQ ID NO:31) was 8726.9, and the detected molecular weight of the antisense strand product (formed by ligation of SEQ ID NO:32 and SEQ ID NO:33) was 7765.9. The theoretical molecular weights of the sense strand and antisense strand were 8727.2±3 and 7766.0±3, respectively. This indicates that ligases lig-20, lig-21, lig-22, lig-23, lig-24, lig-25, lig-31, lig-32, lig-33, lig-34, lig-35, lig-36, and lig-37 catalyzed the ligation reaction, resulting in the production of ligated double-stranded RNA. However, lig-16, lig-17, lig-18, and lig-19 did not produce any products. The SDS-PAGE gel Figure 12 shows essentially complete conversion of the substrate and the formation of a product band.

[0051] In Figure 12, lane 1: nucleic acid molecular weight standard; lane 2: sense strand product; lane 3: antisense strand product; lane 4: lig-20; lane 5: lig-21; lane 6: lig-22; lane 7: lig-23; lane 8: lig-24; lane 9: lig-25; lane 10: negative control, containing 200 μM substrate, ligation buffer, and ATP, but no enzyme added, and an equal volume of 50 mM Tris-Cl was used for the enzyme. The reaction was carried out at 25°C for 3 hours, heated at 98°C for 2 minutes, and incubated at 80°C for 20 minutes. The mixture was then centrifuged to obtain the supernatant, which was then subjected to gel electrophoresis. Lane 11: sense strand product; lane 12: antisense strand product; lane 13: lig-37; lane 14: lig-33; lane 15: lig-36; lane 16: lig-32; lane 17: lig-31; lane 18: lig-35; lane 19: lig-34. Lane 20: nucleic acid molecular weight standard; lane 21: a mixture of sense and antisense strand products; lane 22: lig-16; lane 23: lig-17; lane 24: lig-1; lane 25: lig-19.

[0052] UPLC detection revealed complete catalysis. The results are shown in Figure 13 (ligation reaction catalyzed by ligase. 1 shows a standard mixture of sense and antisense strands. In UPLC, the two strands were separated, which is consistent with the gel results. 2 to 5 represent reaction systems catalyzed by ligases (lig-20, 21, 22, and 23), respectively). Figure 14 shows the results of mass spectrometry. Mass spectrometry analysis of the lig-20 reaction system after the reaction showed that the molecular weight matched the molecular weight of the product, indicating that a ligation product was produced.

[0053] The reaction was optimized by optimizing the reaction pH and reaction temperature. As a result, the target product could be produced at pH = 6 to 8 °C, with the optimal pH being 7.5. The product could be catalyzed at pH = 0 to 40 °C, with the optimal reaction temperature being 16 °C. The results are shown in Table 7.

[0054] [Table 7] Note: + indicates 50%-60% activity, ++ indicates 60%-80% activity, +++ indicates >80% activity.

[0055] Example 5 Ligation of double-stranded RNA by catalytic synthesis JPEG2025540301000035.jpg33161 [Table 8] Note: "m" indicates a 2' methoxy modification and "f" indicates a 2' fluoro modification.

[0056] 0.1 μmol of each substrate in Table 8 (final concentration 200 μM) was added to 20 μl of 10 mM ATP, and 0.1 mg of pure enzymes lig20, 21, 22, 23, 24, and 25 were added to different reaction tubes, respectively, and 10× ligation buffer (50 mM Tris-Cl, 10 mM MgCl, 1 mM The mixture was incubated at 25°C for 3 hours, heated at 98°C for 2 minutes, and then incubated at 80°C for 20 minutes to inactivate the enzyme. The supernatant was centrifuged and analyzed by MS. The molecular weight of the sense strand product (formed by ligating SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37) was 11,227.4, and the molecular weight of the antisense strand product (formed by ligating SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41) was 10,583.7. The theoretical values ​​for the sense strand product and the antisense strand product were 11,225±8 and 10,580±8, respectively. This confirmed the production of the products, indicating that lig-20, 21, 22, 23, 24, and 25 catalyzed the ligation reaction and produced ligated double-stranded RNA.

[0057] Example 6 Catalytic synthesis of long RNA ligation [Table 9] Note: "m" indicates a 2' methoxy modification and "f" indicates a 2' fluoro modification.

[0058] 0.1 μmol of each substrate in Table 9 was added to 20 μl of 10 mM ATP. 0.1 mg of pure enzymes (lig-21, 22, 23, 24, and 25) were added to separate reaction tubes. 10× ligation buffer (containing 50 mM Tris-Cl, 10 mM MgCl2, and 1 mM DTT) was added to a final volume of 0.5 mL. The mixture was incubated at 25°C for 3 hours, heated at 98°C for 2 minutes, and then incubated at 80°C for 20 minutes to inactivate the enzymes. The supernatant was centrifuged and analyzed by MS. The molecular weight of the sense strand was 47789.4, and the molecular weight of the antisense strand was 50130.2. The theoretical values ​​for the sense strand and antisense strand were 47781.4±10 and 50121±10, respectively. This confirmed the production of the product, indicating that lig-20, 21, 22, 23, 24, and 25 catalyzed the ligation reaction to produce ligated double-stranded RNA.

[0059] From the above description, it can be seen that the above embodiments of the present invention have achieved the following technical effects: they are environmentally friendly, can specifically splice different nucleotide fragments, can synthesize long fragment nucleotide chains, and can catalyze the ligation of natural nucleotides and non-natural nucleotides, thereby solving the current technical challenges in synthesizing nucleotide chains.

[0060] The above description is merely a preferred embodiment of the present invention, and is not intended to limit the present invention. Those skilled in the art can make various modifications and variations to the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for synthesizing double-stranded RNA, comprising: Step S1 of synthesizing single-stranded RNA fragments; and step S2 of mixing the single-stranded RNA fragments and ligating the single-stranded RNA fragments with an RNA ligase to form the double-stranded RNA, wherein the RNA ligase is selected from the group consisting of Vibrio phage, Escherichia phage, Klebsiella phage, Bacteriophage, thermophilic bacteriophage, Acidobacteria bacterium, Salmonella enterica, Yersinia phage vB_YepM_ZN18, Shigella phage pSs-1, and Buttiauxella phage. A method for synthesizing double-stranded RNA, characterized in that the RNA ligase is derived from vB_ButM_GuL6.

2. 2. The method of claim 1, wherein the single-stranded RNA fragment has 3 to 200 nucleotides, or analogs thereof.

3. The amino acid sequence of the RNA ligase derived from Vibrio phage is represented by SEQ ID NO: 1, 2, 5, 6, 13, 14, 17, or 19, the amino acid sequence of the RNA ligase derived from Escherichia phage is represented by SEQ ID NO: 3 or 15, the amino acid sequence of the RNA ligase derived from Klebsiella phage is represented by SEQ ID NO: 4 or 16, the amino acid sequence of the RNA ligase derived from Acidobacteria bacterium is represented by SEQ ID NO: 7, the amino acid sequence of the RNA ligase derived from Salmonella enterica is represented by SEQ ID NO: 8, the amino acid sequence of the RNA ligase derived from Yersinia phagevB_YepM_ZN18 is represented by SEQ ID NO: 9, and the amino acid sequence of the RNA ligase derived from Shigella 3. The method of claim 2, wherein the amino acid sequence of the RNA ligase derived from Buttiauxella phage pSs-1 is shown in SEQ ID NO: 10, the amino acid sequence of the RNA ligase derived from Buttiauxella phage vB_ButM_GuL6 is shown in SEQ ID NO: 11, the amino acid sequence of the RNA ligase derived from Bacteriophage is shown in SEQ ID NO: 12, and the amino acid sequence of the RNA ligase derived from thermophilic Bacteriophage is shown in SEQ ID NO:

18.

4. 2. The synthesis method according to claim 1, wherein the double-stranded RNA is natural RNA or non-natural RNA.

5. The non-natural RNA has one or more ribonucleotides modified at the 2' position of the pentose ring, modified at the 3' position, modified at the α position of the phosphate ion, or modified at the base; Preferably, the modification at the 2'-position of the pentose ring comprises a 2'-methoxy modification, a 2'-fluoro modification, a 2'-trifluoromethoxy modification, a 2'-methoxyethyl modification, a 2'-allyl modification, a 2'-amino modification, or a 2'-azido modification; Preferably, the α-position modification of the phosphate ion comprises a thio-modification of the α-position of the phosphate ion, 5. The method of claim 4, wherein the base modifications preferably include methylation and / or acetylation modifications at any one or more of the N1, N5, or N6 positions of the base.

6. 2. The method according to claim 1, wherein the single-stranded RNA fragment is synthesized by solid-phase synthesis.

7. the single-stranded RNA fragments include single-stranded RNA fragment substrate 1 to substrate n, and corresponding complementary single-stranded RNA fragment substrate n+1 to substrate n+n, where n is 2 or more; Between complementary single-stranded RNA fragments, three or more nucleotide bases are complementary paired, At the 5' ends of substrate 2 and substrate n+1, and substrate n and substrate n+n-1, the bases of two or more nucleotides are complementary paired, Optionally, the outer 5' and 3' ends of Substrate 1 and Substrate n+1 are connected by a chemical bond; 2. The method of claim 1, wherein optionally the outer 5' and 3' ends of substrate n and substrate n+n are connected by a chemical bond.

8. The method of claim 1, wherein the reaction temperature of S2 is 0°C to 60°C, preferably 4°C to 37°C, the reaction time is 0.5h to 24h, and the pH is 6 to 8.

5.

9. 1. Use of an RNA ligase in the synthesis of double-stranded RNA, wherein the RNA ligase is derived from Vibrio phage, Escherichia phage, Klebsiella phage, Bacteriophage, thermophilic bacteriophage, Acidobacteriabacterium, Salmonella enterica, Yersinia phage vB_YepM_ZN18, Shigella phage pSs-1, or Buttiauxella phage vB_ButM_GuL6.

10. The amino acid sequence of the RNA ligase derived from Vibrio phage is represented by SEQ ID NO: 1, 2, 5, 6, 13, 14, 17, or 19, the amino acid sequence of the RNA ligase derived from Escherichia phage is represented by SEQ ID NO: 3 or 15, the amino acid sequence of the RNA ligase derived from Klebsiella phage is represented by SEQ ID NO: 4 or 16, the amino acid sequence of the RNA ligase derived from Acidobacteria bacterium is represented by SEQ ID NO: 7, the amino acid sequence of the RNA ligase derived from Salmonella enterica is represented by SEQ ID NO: 8, the amino acid sequence of the RNA ligase derived from Yersinia phagevB_YepM_ZN18 is represented by SEQ ID NO: 9, and the amino acid sequence of the RNA ligase derived from Shigella The use according to claim 9, wherein the amino acid sequence of the RNA ligase derived from Buttiauxella phage pSs-1 is shown in SEQ ID NO: 10, the amino acid sequence of the RNA ligase derived from Buttiauxella phage vB_ButM_GuL6 is shown in SEQ ID NO: 11, the amino acid sequence of the RNA ligase derived from Bacteriophage is shown in SEQ ID NO: 12, and the amino acid sequence of the RNA ligase derived from thermophilic Bacteriophage is shown in SEQ ID NO: 18.

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