Method for producing oligonucleotides using RNA ligase

Abstract

The present invention provides a method for producing oligonucleotides using an RNA ligase. The RNA ligase includes one or more enzymes from the RNA ligase family Rnl1, Rnl2, Rnl3, and Rnl5, and the oligonucleotides include natural or non-natural RNA. This method overcomes the conventional problem of the difficulty in efficiently synthesizing non-natural RNA strands, and is suitable for use in the field of RNA synthesis.

Description

[Technical Field]

[0001] The present invention relates to the field of RNA synthesis, and in particular to methods for producing oligonucleotides using RNA ligase.

[0002] This application is based on and claims priority from a Chinese application bearing application number 202211107027.1 and filed on September 8, 2022, the disclosure of which is incorporated herein in its entirety. [Background technology]

[0003] RNA drugs are a novel class of drugs that have attracted attention in recent years. Compared with traditional small molecule drugs and novel protein drugs, RNA drugs offer numerous advantages. For example, RNA drugs are highly targeted, binding only to specific sequences in target mRNA. Furthermore, compared with the research and development process for small molecule drugs, the research and development of RNA drugs is rapid. RNA drugs can be rapidly modified to meet individual needs. RNA drugs can generally be classified into four categories: RNA aptamers, antisense oligonucleotide drugs (ASOs), RNA interference (RNAi), and messenger RNA (mRNA). Of these, RNAi further includes microRNA (miRNA) and small interfering RNA (siRNA). mRNA drugs include mRNA drugs, mRNA-based cell therapy, and mRNA vaccines. Among these RNA drugs, the main components of ASOs and RNAi are oligonucleotides, typically 20–30 bases long, which are currently the most commonly used type of RNA drug.

[0004] Currently, solid-phase synthesis is the primary method for industrially synthesizing oligonucleotides. Synthesizing large quantities of oligonucleotides requires multiple rounds of accumulation, and the yield decreases with increasing oligonucleotide length. Furthermore, as the chain length increases, the impurities in the resulting product increase, making the purification process more complicated, all of which significantly increases costs. For RNAs with chain lengths of tens to hundreds of nt, primer synthesis is primarily achieved, but its synthesis scale is limited to the nmol to μmol range, making it difficult to scale up to production scale. Currently, large-volume RNA products can only be obtained through repeated solid-phase synthesis. For example, obtaining 1 mol of oligonucleotide requires approximately 200 rounds of accumulation. Furthermore, due to the characteristics of solid-phase synthesis, purity and yield decrease with increasing chain length. When primers are synthesized up to 80 nt in length, the crude product has a purity of only 40% and a yield of only approximately 55%.

[0005] Many new methods for synthesizing oligonucleotides are currently under investigation. Among these, the method of generating oligonucleotides using ligation reactions with RNA ligases is one of them, which has advantages such as high efficiency, low cost, and environmental friendliness. However, the RNA enzymes reported in the prior art cannot efficiently ligate non-natural RNA strands. Non-natural RNA drugs are an important new field of current medical research and development. Therefore, there is an urgent need to develop methods for rapid and efficient large-scale production of non-natural RNA strands. Summary of the Invention [Problem to be solved by the invention]

[0006] The main object of the present invention is to provide a method for producing oligonucleotides using RNA ligase to solve the problem in the prior art that it is difficult to efficiently synthesize non-natural RNA strands. [Means for solving the problem]

[0007] In order to achieve the above object, according to a first aspect of the present invention, A method for producing oligonucleotides using RNA ligase, comprising: The method includes ligating an RNA substrate with an RNA ligase to obtain an oligonucleotide, wherein the RNA ligase comprises any one or more of the enzymes of the RNA ligase family Rnl1, Rnl2, Rnl3, and Rnl5, and the oligonucleotide comprises natural RNA or non-natural RNA.

[0008] Furthermore, the RNA ligase may be any one or more of the enzymes set forth in SEQ ID NOs: 1 to 55, or an enzyme having 80% or more homology to the RNA ligase set forth in SEQ ID NOs: 1 to 55, preferably 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology thereto. The method for ligating a template strand and an RNA substrate, which are annealed and specifically bound to each other to form a nicked double-stranded nucleic acid structure, comprises the steps of: a) mixing a template strand and an RNA substrate, and annealing and specifically binding the RNA substrates to form a nicked double-stranded nucleic acid structure, wherein the number of the RNA substrates is 2 to 10; and b) ligating the nicks via a phosphodiester bond using the RNA ligase, wherein all of the ribonucleotides ligated via a phosphodiester bond are unnatural ribonucleotides, and preferably the number of the RNA substrates is 2 or 3.

[0009] Furthermore, each RNA substrate is an RNA fragment having a length of 2 to 100 nt, preferably 2 to 10 nt, more preferably 4 to 6 nt, preferably all ribonucleotides are unnatural ribonucleotides, and preferably the template strand comprises a single-stranded RNA template or a single-stranded DNA template.

[0010] To achieve the above object, according to a second aspect of the present invention, there is provided a method for producing single-stranded RNA, comprising: (a) mixing a single-stranded DNA template and an RNA substrate, annealing them to specifically bind to form a nicked DNA-RNA hybrid duplex, wherein the number of RNA substrates is 2 to 10; (b) using an RNA ligase to ligate the nicks via a phosphodiester bond to form a continuous DNA-RNA hybrid duplex; and (c) removing the DNA strand in the continuous DNA-RNA hybrid duplex to obtain single-stranded RNA, wherein the RNA ligase includes one or more enzymes from the RNA ligase family Rnl1, Rnl2, Rnl3, and Rnl5, and all of the ribonucleotides ligated via phosphodiester bonds are unnatural ribonucleotides. Preferably, the non-natural ribonucleotide comprises a ribonucleotide having one or more of a 2'-pentose ring modification, a phosphate group α-position modification, or a base modification; preferably, the 2'-pentose ring modification includes, but is not limited to, a 2'-methoxy modification (2'-OCH), a 2'-fluoro modification (2'-F), a 2'-trifluoromethoxy modification (2'-OF), a 2'-methoxyethyl modification (2'-OCH2CHOCH3), a 2'-allyl modification (2'-CH2CH=CH2), a 2'-amino modification (2'-NH2), or a 2'-azido (2'-N3) modification; preferably, the phosphate group α-position modification includes a phosphate group α-position thio modification (=S); and preferably, the base modification includes a methylation modification (-CH3) and / or an acetylation modification (-COCH3) at any one or more of the N1, N5, or N6 positions of the base.

[0011] Furthermore, the RNA ligase may comprise any one or more of the enzymes set forth in SEQ ID NOs: 1 to 55, or an enzyme having 80% or more homology to the RNA ligase set forth in SEQ ID NOs: 1 to 55, preferably 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology thereto; preferably, the DNA strand in the continuous DNA-RNA hybrid duplex is degraded and removed with a DNA enzyme; preferably, the DNA enzyme comprises DNase I, DNasel L1, or DNasel L2; preferably, the RNA is an RNA fragment having a length of 2 to 100 nt, preferably 2 to 10 nt, more preferably 4 to 6 nt; preferably, all ribonucleotides are unnatural ribonucleotides; and preferably, the number of RNA substrates is 2 to 3.

[0012] Furthermore, single-stranded RNA includes linear single-stranded RNA, semi-circular single-stranded RNA, and completely circular single-stranded RNA.

[0013] In order to achieve the above object, according to a third aspect of the present invention, there is provided a method for producing double-stranded RNA, comprising: (a) mixing a single-stranded RNA template and an RNA substrate, and annealing and specifically binding them to form a nicked double-stranded RNA, wherein the number of RNA substrates is 2 to 10; and (b) using an RNA ligase to ligate the nicks via a phosphodiester bond to form double-stranded RNA, wherein the RNA ligase includes one or more enzymes from the RNA ligase family Rnl1, Rnl2, Rnl3, and Rnl5, and all of the ribonucleotides ligated via phosphodiester bonds are unnatural ribonucleotides. Preferably, the non-natural ribonucleotide comprises a ribonucleotide having one or more of a 2'-pentose ring modification, a phosphate group α-position modification, or a base modification; preferably, the 2'-pentose ring modification includes, but is not limited to, a 2'-methoxy modification (2'-OCH), a 2'-fluoro modification (2'-F), a 2'-trifluoromethoxy modification (2'-OF), a 2'-methoxyethyl modification (2'-OCH2CHOCH3), a 2'-allyl modification (2'-CH2CH=CH2), a 2'-amino modification (2'-NH2), or a 2'-azido (2'-N3) modification; preferably, the phosphate group α-position modification includes a phosphate group α-position thio modification (=S); and preferably, the base modification includes a methylation modification (-CH3) and / or an acetylation modification (-COCH3) at any one or more of the N1, N5, or N6 positions of the base.

[0014] Furthermore, the RNA ligase includes any one or more of the enzymes shown in SEQ ID NOs: 1 to 55, or an enzyme having 80% or more homology to the RNA ligases shown in SEQ ID NOs: 1 to 55, preferably 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology; the single-stranded RNA template includes single-stranded RNA produced by the above-mentioned method for producing single-stranded RNA; and the double-stranded RNA includes linear double-stranded RNA, semi-circular double-stranded RNA, or fully circular double-stranded RNA.

[0015] Furthermore, the length of the RNA substrate is an RNA fragment of 2 to 100 nt, preferably 2 to 10 nt, more preferably 4 to 6 nt, and the number of RNA substrates is preferably 2 to 3.

[0016] Furthermore, all of the ribonucleotides in the RNA substrate are unnatural ribonucleotides. Preferably, the non-natural ribonucleotide comprises a ribonucleotide having one or more of a 2'-pentose ring modification, a phosphate group α-position modification, or a base modification; preferably, the 2'-pentose ring modification includes, but is not limited to, a 2'-methoxy modification (2'-OCH), a 2'-fluoro modification (2'-F), a 2'-trifluoromethoxy modification (2'-OF), a 2'-methoxyethyl modification (2'-OCH2CHOCH3), a 2'-allyl modification (2'-CH2CH=CH2), a 2'-amino modification (2'-NH2), or a 2'-azido (2'-N3) modification; preferably, the phosphate group α-position modification includes a phosphate group α-position thio modification (=S); and preferably, the base modification includes a methylation modification (-CH3) and / or an acetylation modification (-COCH3) at any one or more of the N1, N5, or N6 positions of the base.

[0017] According to the technical solution of the present invention, oligonucleotides can be produced using one or more enzymes from the RNA ligase family Rnl1, Rnl2, Rnl3, and Rnl5, which is more efficient and less costly than the conventional technique of producing oligonucleotides by solid-phase synthesis.

[0018] The drawings that form a part of this application are intended to provide a further understanding of the invention, and the illustrative embodiments of the invention and their descriptions are intended to illustrate the invention and are not intended to unduly limit the invention. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows the results of denaturing gel electrophoresis analysis according to Example 2 of the present invention. [Figure 2] 1 shows a UPLC chromatogram according to Example 2 of the present invention. [Figure 3] 1 shows the results of denaturing gel electrophoresis analysis of the enzymatic reaction between NgrRnl and JN02Rnl according to Example 3 of the present invention. [Figure 4] Electrophoresis results of the enzymatic ligation reaction using NgrRnl according to Example 4 of the present invention are shown. In FIG. 4, A shows the effect of substrate concentration on the reaction, B shows the effect of enzyme amount on the reaction, C shows the effect of reaction temperature on the reaction, D shows the effect of ATP concentration on the reaction, and E shows the effect of reaction time on the reaction. [Figure 5] 5 shows the electrophoresis results of the enzymatic ligation reaction using JN02Rnl according to Example 4 of the present invention. In FIG. 5, A shows the effect of substrate concentration on the reaction, B shows the effect of enzyme amount on the reaction, C shows the effect of reaction temperature on the reaction, D shows the effect of ATP concentration on the reaction, and E shows the effect of reaction time on the reaction. [Figure 6] 1 shows a UPLC chromatogram according to Example 4 of the present invention. [Figure 7] 1 shows a UPLC chromatogram according to Example 5 of the present invention. [Figure 8] FIG. 10 shows an electrophoresis diagram of catalytic synthesis of circular RNA by RnlARnl according to Example 6 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] It should be noted that, unless there is a contradiction, the examples and features of the examples of the present application can be combined with each other. The present invention will be described in more detail below with reference to examples.

[0021] As described in the background art, solid-phase synthesis is primarily used to synthesize oligonucleotides in the prior art, but solid-phase synthesis has various drawbacks. Many new methods for synthesizing oligonucleotides are currently under investigation. Among these, a method using ligation reactions with RNA ligases to produce oligonucleotides is one of them, boasting advantages such as high efficiency, low cost, and environmental friendliness. However, the RNA enzymes reported in the prior art cannot efficiently ligate non-natural RNA strands. Non-natural RNA drugs are currently an important new field in medical research and development. There is an urgent need to develop a method for rapid and efficient large-scale production of non-natural RNA strands. Therefore, in this application, the inventors attempted to explore an RNA ligation method using one or more enzymes from the RNA ligase family Rnl1, Rnl2, Rnl3, and Rnl5 to achieve ligation of non-natural ribonucleotides. Therefore, a series of protection schemes are proposed in this application.

[0022] In a first exemplary embodiment of the present application, a method for producing an oligonucleotide using an RNA ligase is provided, the method comprising the step of ligating an RNA substrate with an RNA ligase to obtain an oligonucleotide, wherein the RNA ligase comprises any one or more enzymes of the RNA ligase family Rnl1, Rnl2, Rnl3, and Rnl5, and the oligonucleotide comprises natural RNA or non-natural RNA.

[0023] Unnatural nucleic acids (XNAs) are nucleic acid molecules with unnatural backbones or nucleobases. Unnatural ribonucleotides are ribonucleotides with unnatural backbones or nucleobases. Common unnatural ribonucleotides include: (1) nucleotides chemically modified at the 2' position of the pentose ring: major chemical modifications include 2'-methoxy, 2'-fluoro, 2'-trifluoromethoxy, etc.; (2) nucleotides modified at the phosphate group: major thio modifications, etc.; (3) nucleotides chemically modified at the base: major modifications include methylation and acetylation at the N1, N5, and N6 positions. RNA containing such unnatural ribonucleotides is unnatural RNA. Unnatural ribonucleotides are ligated via phosphodiester bonds using one or more of the RNA ligases represented by SEQ ID NOs: 1 to 55 to form a chain of unnatural RNA. Nicked double-stranded RNA can be ligated using the above RNA ligases. Furthermore, ligation of extremely short non-natural RNAs can be achieved, with the minimum length of the non-natural RNA being 2 nt.

[0024] In a preferred embodiment, the RNA ligase comprises one or more of the enzymes set forth in SEQ ID NOs: 1 to 55, or an enzyme having 80% or more identity to the RNA ligase set forth in any of SEQ ID NOs: 1 to 55, preferably 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more identity thereto. Preferably, the method comprises: a) mixing a template strand and an RNA substrate, and annealing and specifically binding them to form a nicked double-stranded nucleic acid structure, wherein the number of RNA substrates is 2 to 10; and b) ligating the nicks using an RNA ligase via a phosphodiester bond, wherein all of the ribonucleotides ligated via a phosphodiester bond are unnatural ribonucleotides.

[0025] In the above-described RNA ligation method, a template strand capable of specifically binding to an RNA substrate (i.e., a short fragment of single-stranded RNA) is first mixed with two to ten or more RNA substrates, followed by annealing. Multiple RNA substrates are ligated to the template strand through specific complementary base pairing, forming a nicked double-stranded nucleic acid structure. Nicks are gaps between discontinuous RNA substrates that are not ligated by phosphodiester bonds. Using the above-described RNA ligase, discontinuous RNA substrates can be ligated by phosphodiester bonds, eliminating the nicks in the double-stranded nucleic acid structure and forming a continuous double-stranded nucleic acid structure. The ribonucleotides ligated via the above-mentioned phosphodiester bond are all unnatural ribonucleotides, i.e., the two ribonucleotides immediately on either side of the nick are all unnatural ribonucleotides, i.e., the bases at the 5'-end and 3'-end of the RNA substrate are all unnatural ribonucleotides (the 5'-end of the RNA substrate that complementarily pairs with the 3'-end of the template strand, or the 3'-end of the RNA substrate that complementarily pairs with the 3'-end of the template strand, may not be unnatural ribonucleotides). In the prior art, RNA ligases capable of ligating unnatural ribonucleotides are rare and have low activity.

[0026] According to the principle of RNA ligase ligation of nicked double-stranded RNA using the splint described above, two short RNA fragments typically form complementary strands with the splint RNA, and then RNA ligase ligates the 5'-phosphate group at the nick to the 3'-hydroxyl group to form a phosphodiester bond. When multiple short RNA fragments are complementary to the RNA splint, the same principle is followed: RNA ligase ligates the nicks one by one.

[0027] In a preferred embodiment, each RNA substrate is an RNA fragment having a length of 2 to 100 nt, preferably 2 to 10 nt, more preferably 4 to 6 nt; preferably, all ribonucleotides are unnatural ribonucleotides; preferably, the template strand comprises a single-stranded RNA template or a single-stranded DNA template; and preferably, the number of RNA substrates is 2 to 3.

[0028] The RNA substrate may be RNA consisting of as few as two bases or as many as 100 bases. Parameters such as the length, number, and sequence of the RNA substrate can be flexibly adjusted based on factors such as RNA stability, ease of synthesis, specificity for the template strand, and the target non-natural RNA sequence. The RNA substrate may contain both non-natural and natural ribonucleotides, or may contain only non-natural ribonucleotides. The template strand that complementarily pairs with the RNA substrate and is used to guide the sequence of the RNA substrate may comprise a single-stranded RNA template or a single-stranded DNA template, and the resulting continuous double-stranded nucleic acid structure is a double-stranded RNA or a continuous DNA-RNA hybrid duplex, respectively.

[0029] A second exemplary embodiment of the present application provides a method for producing single-stranded RNA, comprising: (a) mixing a single-stranded DNA template and an RNA substrate, and annealing and specifically binding them to form a nicked DNA-RNA hybrid duplex, where the number of RNA substrates is 2 to 10; (b) using an RNA ligase to ligate the nicks via a phosphodiester bond to form a continuous DNA-RNA hybrid duplex; and (c) removing the continuous DNA-RNA hybrid duplex to obtain single-stranded RNA, where the RNA ligase includes one or more enzymes from the RNA ligase family Rnl1, Rnl2, Rnl3, and Rnl5, and all of the ribonucleotides ligated via phosphodiester bonds are unnatural ribonucleotides. Preferably, the unnatural ribonucleotide comprises a ribonucleotide having one or more of a 2'-pentose ring modification, a phosphate group α-position modification, or a base modification, and preferably, the 2'-pentose ring modification includes, but is not limited to, a 2'-methoxy modification (2'-OCH), a 2'-fluoro modification (2'-F), a 2'-trifluoromethoxy modification (2'-OF), a 2'-methoxyethyl modification (2'-OCHCHOCH), a 2'-allyl modification (2'-CHCH=CH), a 2'-amino modification (2'-NH), or a 2'-azido (2'-N) modification. Preferably, the phosphate group α-position modification includes a thio modification (=S) at the phosphate group α-position, and preferably, the base modification includes a methylation modification (-CH) and / or an acetylation modification (-COCH) at any one or more of the N1, N5, or N6 positions of the base.

[0030] In the above-mentioned single-stranded RNA production method, a continuous DNA-RNA hybrid duplex is produced using the above-mentioned RNA ligase with a single-stranded DNA template as the template strand. Next, one DNA strand of the continuous DNA-RNA hybrid duplex is degraded using a DNA enzyme or other method to obtain single-stranded RNA.

[0031] In a preferred embodiment, the RNA ligase comprises any one or more of the enzymes set forth in SEQ ID NOs: 1 to 55, or an enzyme having 80% or more homology to the RNA ligase set forth in SEQ ID NOs: 1 to 55, preferably 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology thereto; preferably, the DNA strand in the continuous DNA-RNA hybrid duplex is degraded and removed with the DNA enzyme; preferably, the DNA enzyme comprises DNase; preferably, the RNA substrate is an RNA fragment having a length of 2 to 100 nt, preferably 2 to 10 nt, more preferably 4 to 6 nt; preferably, all ribonucleotides are unnatural ribonucleotides; and preferably, the number of RNA substrates is 2 to 3.

[0032] In preferred embodiments, the single-stranded RNA comprises linear single-stranded RNA, semi-circular single-stranded RNA, or fully circular single-stranded RNA.

[0033] Semi-circular and fully circular RNA are single-stranded RNAs in which complementary bases form a double-stranded structure, while the non-complementary strand is a free single strand. If complementary sequences are present, they are likely to bind together in solution through hydrogen bonds, forming partially double-stranded RNA.

[0034] The length of the RNA substrate includes, but is not limited to, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 15 nt, 20 nt, 30 nt, 40 nt, 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, or 100 nt.

[0035] A third exemplary embodiment of the present application provides a method for producing double-stranded RNA, which includes: a) mixing a single-stranded RNA template and an RNA substrate, annealing them to specifically bind, and forming a nicked double-stranded RNA, where the number of RNA substrates is 2 to 10; and b) using an RNA ligase to ligate the nicks via a phosphodiester bond to form double-stranded RNA, where the RNA ligase includes one or more enzymes from the RNA ligase family Rnl1, Rnl2, Rnl3, and Rnl5, and all of the ribonucleotides ligated via a phosphodiester bond are unnatural ribonucleotides. Preferably, the unnatural ribonucleotide comprises a ribonucleotide having one or more of a 2'-pentose ring modification, a phosphate group α-position modification, or a base modification, and preferably, the 2'-pentose ring modification includes, but is not limited to, a 2'-methoxy modification (2'-OCH), a 2'-fluoro modification (2'-F), a 2'-trifluoromethoxy modification (2'-OF), a 2'-methoxyethyl modification (2'-OCHCHOCH), a 2'-allyl modification (2'-CHCH=CH), a 2'-amino modification (2'-NH), or a 2'-azido (2'-N) modification. Preferably, the phosphate group α-position modification includes a thio modification (=S) at the phosphate group α-position, and preferably, the base modification includes a methylation modification (-CH) and / or an acetylation modification (-COCH) at any one or more of the N1, N5, or N6 positions of the base.

[0036] In the method for producing double-stranded RNA, double-stranded RNA is produced using a single-stranded RNA template as a template strand and the RNA ligase.

[0037] In a preferred embodiment, the RNA ligase comprises any one or more of the enzymes set forth in SEQ ID NOs: 1 to 55, or an enzyme having 80% or more homology to the RNA ligase set forth in SEQ ID NOs: 1 to 55, preferably 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology thereto; the single-stranded RNA template preferably comprises a single-stranded RNA produced by the above-mentioned method for producing single-stranded RNA; the double-stranded RNA preferably comprises a linear double-stranded RNA, a semi-circular double-stranded RNA, or a completely circular double-stranded RNA; the length of the RNA substrate preferably is an RNA fragment of 2 to 100 nt, preferably 2 to 10 nt, more preferably 4 to 6 nt; preferably, all ribonucleotides in the RNA substrate are unnatural ribonucleotides; and preferably, the number of RNA substrates is 2 to 3. Preferably, the non-natural ribonucleotide comprises a ribonucleotide having one or more of a 2'-pentose ring modification, a phosphate group α-position modification, or a base modification; preferably, the 2'-pentose ring modification includes, but is not limited to, a 2'-methoxy modification (2'-OCH), a 2'-fluoro modification (2'-F), a 2'-trifluoromethoxy modification (2'-OF), a 2'-methoxyethyl modification (2'-OCH2CHOCH3), a 2'-allyl modification (2'-CH2CH=CH2), a 2'-amino modification (2'-NH2), or a 2'-azido (2'-N3) modification; preferably, the phosphate group α-position modification includes a phosphate group α-position thio modification (=S); and preferably, the base modification includes a methylation modification (-CH3) and / or an acetylation modification (-COCH3) at any one or more of the N1, N5, or N6 positions of the base.

[0038] The above-mentioned methods for producing single-stranded RNA and double-stranded RNA may be used in combination. For example, first, single-stranded RNA is obtained using a single-stranded DNA template as the template strand by the method for producing single-stranded RNA. Next, double-stranded RNA is produced using the obtained single-stranded RNA as the template strand. The double-stranded RNA is then heated to unwind it, forming two single-stranded RNAs, which are then bound to an RNA substrate. The above-mentioned production method is repeated to obtain large amounts of single-stranded RNA or double-stranded RNA. The above-mentioned production method is low-cost, highly efficient, and has great application value in industrial production.

[0039] The beneficial effects of the present invention will be described in detail below with reference to specific examples.

[0040] Example 1 Expression and purification of RNA ligase Plasmids containing the expression sequences of 55 target proteins (sequences are as follows) were transformed into E. coli BL21(DE3) competent cells. After expression in E. coli, the expressed proteins were purified in two steps using an affinity column (Ni-NTA) and an ion column (QFF or SPFF), yielding 55 proteins with relatively high purity.

[0041] SEQ ID NO: 1 (DpRnl, from Diplonema papillatum, Rnl2 family) [ka]

[0042] SEQ ID NO:2 (AcNPVRnl, derived from Autographa californica nucleopolyhedrovirus, Rnl2 family) [ka]

[0043] SEQ ID NO:3 (SfRnl, from Shigella flexneri, Rnl2 family) [ka]

[0044] SEQ ID NO:4 (MthRnl, from Methanothermobacter thermautotrophicus str. Delta H, Rnl3 family) [ka]

[0045] SEQ ID NO:5 (Pab1020Rnl, from Pyrococcus abyssi, Rnl3 family) [ka]

[0046] SEQ ID NO:6 (NgrRnl, from Naegleria gruberi, Rnl5 family): [ka]

[0047] SEQ ID NO:7 (DraRnl, from Deinococcus radiodurans, Rnl5 family) [ka]

[0048] SEQ ID NO:8 (DdRnl, from Dictyostelium discoideum AX4, Rnl5 family) [ka]

[0049] SEQ ID NO:9 (GzRnl, from Gibberella zeae, Rnl5 family) [ka]

[0050] SEQ ID NO:10 (NcRnl, from Neurospora crassa, Rnl5 family) [ka]

[0051] SEQ ID NO:11 (PoRnl, from Pyricularla oryzae 70-15, Rnl5 family) [ka]

[0052] SEQ ID NO:12 (SppRnl, derived from Shigella phage pSs-1, Rnl2 family): [ka] SEQ ID NO: 13 (BpRnl, derived from Buttiauxella phage vB_ButM_GuL6, Rnl2 family) [ka]

[0053] SEQ ID NO:14 (TbgRnl, from Trypanosoma brucei gambiense DML972, Rnl2 family) [ka]

[0054] SEQ ID NO: 15 (F48Rnl, derived from Klebsiella phage vB_Kpn_F48, Rnl1 family) [ka]

[0055] SEQ ID NO:16 (PhRnl, from Pyrococcus horikoshii OT3, Rnl3 family) [ka]

[0056] SEQ ID NO: 17 (RB69Rnl, from Enterobacteria phage RB69, Rnl1 family) [ka]

[0057] SEQ ID NO: 18 (RnlB-BRnl, from Aeromonas virus, Rnl2 family) [ka]

[0058] SEQ ID NO:19 (UAlRnl, derived from Escherichia phage UFV-AREG1, Rnl1 family) [ka]

[0059] SEQ ID NO: 20 (KP27Rnl, from Klebsiella phage KP21, Rnl2 family): [ka]

[0060] SEQ ID NO:21 (FpRnl, from Fusarium proliferatum, Rnl5 family) [ka]

[0061] SEQ ID NO:22 (TbRnl, from Thermococcus barophilus, Rnl1 family) [ka]

[0062] SEQ ID NO:23 (TbREL1Rnl, from Trypanosoma brucei, Rnl2 family) [ka]

[0063] SEQ ID NO:24 (TbREL2Rnl, from Trypanosoma brucei gambiense DAL972, Rnl2 family) [ka]

[0064] SEQ ID NO: 25 (JS98Rnl, derived from Escherichia phage JS98, Rnl2 family) [ka]

[0065] SEQ ID NO:26 (VgRnl, from Variovorax gossypii, Rnl5 family) [ka]

[0066] SEQ ID NO:27 (UpRnl, from Undibacterium pigrum, Rnl5 family) [ka]

[0067] SEQ ID NO: 28 (PpPRnl, derived from Panteoa phage Phynn, Rnl5 family, Rnl5 family) [ka]

[0068] SEQ ID NO:29 (SpMRnl, from Stenotrophomonas phage Marzo, Rnl1 family) [ka]

[0069] SEQ ID NO: 30 (WaRnl, from Waterburya agarophytonicola, Rnl1 family): [ka]

[0070] SEQ ID NO:31 (SavRnl, from Streptomyces avermitilis, Rnl2 family) [ka]

[0071] SEQ ID NO: 32 (P000VRnl, derived from Escherichia phage p000v, Rnl2 family) [ka]

[0072] SEQ ID NO: 33 (FsRnl, from Fusarium subglutinans, Rnl5 family) [ka]

[0073] SEQ ID NO: 34 (JN02Rnl, derived from Vibrio phage JN02, Rnl2 family) [ka]

[0074] SEQ ID NO:35 (SsRnl, from Shigella sonnel, Rnl2 family) [ka]

[0075] SEQ ID NO:36 (ToRnl, from Thelonectria olida, Rnl5 family) [ka]

[0076] SEQ ID NO: 37 (CpMRnl, derived from Citrobacter phage Merlin, Rnl2 family) [ka]

[0077] SEQ ID NO:38 (FaRnl, from Fusarium albosuccineum, Rnl5 family) [ka]

[0078] SEQ ID NO: 39 (RnlARnl, derived from Vibrio phagent-1, Rnl1 family) [ka]

[0079] SEQ ID NO:40 (SH7Rnl, derived from Shigella phage SH7, Rn2 family) [ka]

[0080] SEQ ID NO: 41 (ST2Rnl, from Vibrio phage phi-ST2, Rnl1 family): [ka]

[0081] SEQ ID NO: 42 (KP15Rnl, derived from Klebsiella phage KP15, Rnl2 family) [ka]

[0082] SEQ ID NO: 43 (JN02Rnl, from Escherichia phage JN02, Rnl2 family): [ka]

[0083] SEQ ID NO: 44 (YpRnl, from Yersinia phage JC221, Rnl2 family) [ka]

[0084] SEQ ID NO:45 (CpRnl, derived from Cronobacter phage vB_CsaM_GAP161, Rnl2 family) [ka]

[0085] SEQ ID NO: 46 (AmeRnl, from Amsacta moorei entomopox virus, Rnl5 family) [ka]

[0086] SEQ ID NO: 47 (CsRnl, from Chitinophaga sp. S165, Rnl5 family) [ka]

[0087] SEQ ID NO:48 (CAbRnl, from Candidatus Aminicenantes bacterium, Rnl5 family) [ka]

[0088] SEQ ID NO: 49 (MrRnl, from Massila rubra, Rnl5 family) [ka]

[0089] SEQ ID NO:50 (IbRnl, from Ignavibacteriaceae bacterium, Rnl5 family) [ka]

[0090] SEQ ID NO:51 (TaRnl, from Thermoprotei archaeon, Rnl3 family) [ka]

[0091] SEQ ID NO:52 (KpRnl, from Klebsiella pneumoniae, Rnl1 family) [ka]

[0092] SEQ ID NO:53 (AbRnl, from Acidobacteria bacterium, Rnl1 family) [ka]

[0093] SEQ ID NO:54 (SeRnl, from Salmonella enterica, Rnl2 family) [ka]

[0094] SEQ ID NO: 55 (YpRnl, derived from Yersinia phage vB_YepM_ZN18, Rnl2 family) [ka]

[0095] Example 2 In the present invention, several non-natural RNA substrates were designed: P8 (4nt):mA-fC-mG-fG, P9 (5nt):mG-fG-mU-fC-mA, P10 (6nt):fC-mU-fG-mA-fG-mU, The expected target product P13 (SEQ ID NO: 56) is 15 nt in length (molecular weight 5037), The length of the splint RNA (single-stranded RNA template) P14 (SEQ ID NO: 57) is 15 nt (molecular weight 4917). P13 (15nt):mA-fC-mG-fG-mG-fG-mU-fC-mA-fC-mU-fG-mA-fG-mU (SEQ ID NO:56). P14 (15nt):mA-fC-mU-mC-mA-fG-fU-mG-mA-fC-fC-mC-fC-mG-mU (SEQ ID NO:57). m represents a 2'-OCH3 modification, and f represents a 2'-F modification.

[0096] First, we developed analytical methods for the target product. These methods included denaturing gel electrophoresis (DEN) and ultra-performance liquid chromatography (UPLC). The results of the denaturing gel electrophoresis analysis, shown in Figure 1, indicated that the target product P13 and the single-stranded RNA template P14 formed a tightly bound double-stranded product. The UPLC analysis results, shown in Figure 2, also confirmed the presence of the double-stranded product.

[0097] Example 3 Multi-fragment non-natural RNAs were specifically ligated using non-natural single-stranded RNA templates. In the experiment, P8, P9, P10, and P14 were first annealed and joined, and then two nicks were ligated using an expressed and purified RNA ligase. The initial reaction volume was 10 μL. The reaction conditions were: substrate concentrations (P8, P9, P10, and P14) of 20 μM each, enzyme concentration of 0.2 mg / mL, ATP concentration of 1 mM, and ligase reaction buffer (T4 DNA Ligase Reaction Buffer (10x), NEB, product number B0202S). The reaction temperature was 16°C and the reaction time was 16 hours. After the reaction was completed, the reaction was heated and centrifuged, and the supernatant was collected and subjected to denaturing gel electrophoresis analysis. The results are shown in Table 1. All 55 RNA ligases demonstrated ligation activity. To verify the reaction, 50 μL of NgrRnl and JN02Rnl, which have higher catalytic activity, were selected for the reaction. Denaturing gel electrophoresis analysis showed that the enzymatic reaction between NgrRnl and JN02Rnl produced the desired product with high purity (see Figure 3). In Figure 3, lane 1 is the ssRNA ladder, lane 2 is P11, lane 3 is P12, lane 4 is P13, lane 5 is P14, lane 6 is P13+P14, lane 7 is the MthRnI reaction, lane 8 is the NgrRnl reaction, lane 9 is the JN02Rnl reaction, and lane 10 is the reaction without ligase. Furthermore, mass spectrometry analysis showed that the molecular weight of the product observed in the denaturing gel electrophoresis analysis was consistent with the predicted molecular weight of the desired product, again fully verifying the production of the desired product. [Table 1] TIFF2025536870000058.tif40160 Note: "+", "++", and "+++" in the table represent RNA ligase catalytic activity from low to high.

[0098] Example 4 The enzymatic ligation reaction of NgrRnl and JN02Rnl was optimized. Experiments were performed to optimize single variables, including substrate concentration, enzyme amount, reaction time, reaction temperature, and ATP concentration. As shown in Figures 4 and 5, M represents the ssRNA ladder, P11 represents the P11 substrate, P12 represents the P12 substrate, P13 represents the P13 standard, P13+P14 represents the double-stranded nucleic acid formed by the combination of P13 and P14, and the blank represents the reaction system without added enzyme.

[0099] Figure 4 shows gel images of enzymatic ligation reactions using NgrRnI, in which A shows the effect of substrate concentration on the reaction, B shows the effect of enzyme amount on the reaction, C shows the effect of reaction temperature on the reaction, D shows the effect of ATP concentration on the reaction, and E shows the effect of reaction time on the reaction.

[0100] Figure 5 shows gel images of enzymatic ligation reactions using JN02Rnl, in which A shows the effect of substrate concentration on the reaction, B shows the effect of enzyme amount on the reaction, D shows the effect of reaction temperature on the reaction, D shows the effect of ATP concentration on the reaction, and E shows the effect of reaction time on the reaction.

[0101] The optimization results show that a substrate concentration of 400 μM produces the most desired product and achieves a relatively high conversion rate. The optimization results for each enzyme amount show that an enzyme amount of 0.2 mg / mL results in the highest conversion rate. The experimental results for each reaction time show that the reaction reaches equilibrium after 2 hours and the conversion rate does not increase further. The optimization results for each temperature show that increasing the temperature can accelerate the reaction, but also results in the production of more impurities, so the optimal reaction temperature is 16°C. The optimization results for the amount of ATP in the reaction show that 0.5 mM is sufficient for an enzyme amount of 0.2 mg / mL.

[0102] The ligation reaction was performed under the optimized conditions described above: NgrRnl, 0.2 mg / mL enzyme, 400 μM substrate, 16°C reaction temperature, 16 h reaction time, and 0.5 mM ATP. After the reaction was completed, the target product was analyzed by UPLC. The UPLC analysis results shown in Figure 6 indicated that P9 and P10 reacted almost completely, with a high yield of approximately 90% of the target product. After ion column purification, the target product was purified to a purity of over 90%. The main impurities were unreacted P8, P11 formed by ligation of P8 and P9, and P12 formed by ligation of P9 and P10. P11 (9nt):mA-fC-mG-fG-mG-fG-mU-fC-mA P12 (lint):mG-fG-mU-fC-mA-fC-mU-fG-mA-fG-mU (SEQ ID NO:58)

[0103] Example 5 The present invention attempted to use DNA splint (single-stranded DNA template) P16 (SEQ ID NO:59) instead of RNA splint P14 to carry out the reaction. P16: AACTCAGTGACCCCGTA (SEQ ID NO:59)

[0104] The primary purpose of using DNA splints is to digest the DNA splints in the system with DNase I after the reaction is complete, thereby obtaining a more pure single-stranded RNA product. UPLC analysis showed that 18 proteins exhibited high RNA ligation activity. Among them, RnlB-BRnl, NgrRnl, SppRnl, CpMRnl, RnlARnl, KP15Rnl, KP27Rnl, WaRnl, and ST2Rnl not only produced the desired product but also very few impurities, resulting in a system purity of approximately 75%. After the reaction was complete, DNase I was added to the reaction system, which was then incubated at 37°C for 1 hour to terminate the reaction. After the reaction was completed, the reaction mixture was subjected to UPLC analysis. Spectra of RnlARnl and ST2Rnl before and after splint digestion are shown in Figure 7. The analytical results demonstrated that DNase I successfully digested the DNA splints.

[0105] Example 6 The present inventors also attempted to ligate a perfectly circular RNA using these enzymes. The perfectly circular RNA substrate was a non-natural RNA strand 38 nt in length, with the following sequence: SEQIDNO: 60:(H1, 38nt, m:2'-OCH3 modification) [ka]

[0106] Reaction conditions: substrate H1 concentration 20 μM, enzyme amount 0.2 mg / mL, ATP 1 mM, ligase reaction buffer, reaction temperature 16°C, reaction time 16 hours. After the reaction was completed, mass spectrometry confirmed that the desired product had been produced. Using RnlARnl ligase as an example, the results of denaturing gel electrophoresis analysis shown in Figure 8 showed that RnlARnl had the highest activity for ligating complete circular RNA, reaching over 50%.

[0107] From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: The ligation of unnatural ribonucleotides can be achieved by using any one or more of the Rnl1, Rnl2, Rnl3, and Rnl5 families of enzymes, including but not limited to one or more of the enzymes set forth in SEQ ID NOs: 1-55. Compared with the prior art method of producing oligonucleotides by solid-phase synthesis, this method is more efficient and less costly. Furthermore, all of the above-described RNA ligases can simultaneously ligate multiple nicked double-stranded nucleic acid structures, with high reaction efficiency and high conversion rates. Most reactions are usually completed within two hours, with high conversion rates.

[0108] The above is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will recognize that the present invention can be modified and changed in various ways. Any modifications, equivalent replacements, improvements, etc. made without departing from the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. 1. A method for producing oligonucleotides using RNA ligase, comprising: ligating an RNA substrate with said RNA ligase to obtain said oligonucleotide; the RNA ligase comprises any one or more of the enzymes of the RNA ligase family Rnl1, Rnl2, Rnl3, and Rnl5; The method, wherein the oligonucleotide comprises natural RNA or non-natural RNA.

2. The RNA ligase comprises one or more of the enzymes represented by SEQ ID NOs: 1 to 55; Preferably, the method comprises: a) mixing the template strand with the RNA substrates, the number of which is 2-10, preferably 2-3, and annealing them to specifically bind to form a nicked double-stranded nucleic acid structure; b) ligating the nicks with a phosphodiester bond using the RNA ligase; all of the ribonucleotides ligated by the phosphodiester bond are non-natural ribonucleotides; Preferably, the non-natural ribonucleotide comprises a ribonucleotide having one or more of a pentose ring 2'-position modification, a phosphate group α-position modification, or a base modification; 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 modification at the α-position of the phosphate group comprises a thio modification at the α-position of the phosphate group; 2. The method of claim 1, wherein the base modifications preferably comprise methylation and / or acetylation modifications made at any one or more of the N1, N5, or N6 positions of the base.

3. each of said RNA substrates is an RNA fragment of 2 to 100 nt, preferably 2 to 10 nt, more preferably 4 to 6 nt; Preferably, all of the ribonucleotides are non-natural ribonucleotides; 3. The method of claim 2, wherein the template strand preferably comprises a single-stranded RNA template or a single-stranded DNA template.

4. A method for producing single-stranded RNA, comprising: a) mixing a single-stranded DNA template with 2-10 RNA substrates, and annealing and specifically binding to form a nicked DNA-RNA hybrid duplex; b) ligating the nicks with a phosphodiester bond using an RNA ligase to form a contiguous DNA-RNA hybrid duplex; c) removing the DNA strand in the continuous DNA-RNA hybrid duplex to obtain the single-stranded RNA; the RNA ligase comprises any one or more of the enzymes of the RNA ligase family Rnl1, Rnl2, Rnl3, and Rnl5; A method for producing single-stranded RNA, wherein all of the ribonucleotides ligated via phosphodiester bonds are non-natural ribonucleotides.

5. The RNA ligase comprises one or more of the enzymes represented by SEQ ID NOs: 1 to 55; Preferably, the DNA strand in the continuous DNA-RNA hybrid double strand is degraded and removed with a DNA enzyme; Preferably, the DNA enzyme comprises DNase1, DNase1L1, or DNase1L2; Preferably, the length of the RNA substrate is an RNA fragment of 2 to 100 nt, preferably 2 to 10 nt, more preferably 4 to 6 nt; Preferably, all of the ribonucleotides are non-natural ribonucleotides; Preferably, the non-natural ribonucleotide comprises a ribonucleotide having one or more of a pentose ring 2'-position modification, a phosphate group α-position modification, or a base modification; 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 modification at the α-position of the phosphate group comprises a thio modification at the α-position of the phosphate group; Preferably, the base modifications comprise methylation and / or acetylation modifications made at any one or more of the N1, N5, or N6 positions of the base; 5. The method for producing single-stranded RNA according to claim 4, wherein the number of the RNA substrates is preferably 2 to 3.

6. 5. The method for producing single-stranded RNA according to claim 4, wherein the single-stranded RNA includes linear single-stranded RNA, semi-circular single-stranded RNA, or completely circular single-stranded RNA.

7. A method for producing double-stranded RNA, comprising: a) mixing a single-stranded RNA template with 2 to 10 RNA substrates, and annealing and specifically binding them to form nicked double-stranded RNA; b) ligating the nicks with a phosphodiester bond using an RNA ligase to form the double-stranded RNA; the RNA ligase comprises any one or more of the enzymes of the RNA ligase family Rnl1, Rnl2, Rnl3, Rnl5; A method for producing double-stranded RNA, wherein all of the ribonucleotides ligated by phosphodiester bonds are non-natural ribonucleotides.

8. The RNA ligase comprises any one or more of SEQ ID NOs: 1 to 55; Preferably, the single-stranded RNA template is a single-stranded RNA produced by the method for producing a single-stranded RNA according to any one of claims 4 to 6, 8. The method for producing double-stranded RNA according to claim 7, wherein the double-stranded RNA includes linear double-stranded RNA, semi-circular double-stranded RNA, or completely circular double-stranded RNA.

9. the RNA substrate is an RNA fragment of 2 to 100 nt, preferably 2 to 10 nt, more preferably 4 to 6 nt; The method for producing double-stranded RNA according to claim 7, wherein the number of the RNA substrates is preferably 2 to 3.

10. all ribonucleotides of the RNA substrate are the unnatural ribonucleotides; Preferably, the non-natural ribonucleotide comprises a ribonucleotide having one or more of a pentose ring 2'-position modification, a phosphate group α-position modification, or a base modification; 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 modification at the α-position of the phosphate group comprises a thio modification at the α-position of the phosphate group; 8. The method for producing double-stranded RNA according to claim 7, wherein the base modifications preferably include methylation and / or acetylation modifications performed at any one or more of the N1, N5, or N6 positions of the base.

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