WO2024119625A1 - 双链rna的合成方法及rna连接酶在双链rna合成中的应用 - Google Patents

双链rna的合成方法及rna连接酶在双链rna合成中的应用 Download PDF

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WO2024119625A1
WO2024119625A1 PCT/CN2023/077476 CN2023077476W WO2024119625A1 WO 2024119625 A1 WO2024119625 A1 WO 2024119625A1 CN 2023077476 W CN2023077476 W CN 2023077476W WO 2024119625 A1 WO2024119625 A1 WO 2024119625A1
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rna
amino acid
acid sequence
phage
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French (fr)
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洪浩
詹姆斯•盖吉
张娜
焦学成
刘芳
马翠萍
王召帅
耿宇菡
崔丽心
朱文轩
贾旭
杨益明
李娟�
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凯莱英生命科学技术(天津)有限公司
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  • the present invention relates to the field of biotechnology, and in particular to a method for synthesizing double-stranded RNA and application of RNA ligase in the synthesis of double-stranded RNA.
  • Ribonucleic acid is a genetic information carrier that exists in biological cells and some viruses and viroids.
  • RNA was considered to be just a molecule that transmits information between genes and proteins. In fact, at the beginning of the origin of life, RNA was the only life molecule that could store information and have enzyme functions.
  • mRNA messenger for protein synthesis
  • Non-transcribed RNA includes miRNA, siRNA, lncRNA, piwiRNA, etc. Among them, there are more than 400 miRNA molecules alone, which regulate at least one-third of human genes. Since the 1970s, gene carrier technology, gene cloning technology, gene editing technology, etc. have had a profound impact on modern gene therapy technology.
  • RNA editing technology has achieved the editing of specific nucleotides in human cells at the genetic level. This technology can not only be used as a research tool, but also as a temporary treatment for diseases caused by mutations.
  • RNA drugs In recent years, the field of RNA drugs has developed rapidly. On the one hand, small nucleic acid drugs represented by siRNA and ASO have shined in the fields of rare diseases, and on the other hand, mRNA drugs represented by mRNA vaccines have played an important role in the COVID-19 pandemic.
  • mRNA drugs represented by mRNA vaccines have played an important role in the COVID-19 pandemic.
  • fomivirsen was approved for marketing at the end of the last century, the development of related industries entered a "window period". With the advancement of delivery technology, small nucleic acid drugs have also ushered in a period of rapid growth in recent years. This stage also spawned the first blockbuster drug of small nucleic acid drugs, Nusinersen.
  • the drug is an antisense oligonucleotide (ASO) drug used to treat spinal muscular atrophy (SMA) in adults and children.
  • ASO antisense oligonucleotide
  • solid phase synthesis is to fix nucleic acids on a solid phase carrier to complete the synthesis reaction.
  • the most commonly used solid phase carrier is controlled pore glass beads (CPG), but the loading capacity is limited, generally 70-80 ⁇ mol/g.
  • CPG controlled pore glass beads
  • solid phase synthesis of nucleotide chains has great disadvantages.
  • the length of the synthetic chain is limited. It is generally used to synthesize chains within 25 nucleotides. As the length of the synthetic chain increases, the yield decreases. When the primer is synthesized to a chain length of ⁇ 80nt, the purity of the crude product is only ⁇ 40%, which greatly limits the synthesis of nucleotide chains.
  • Enzymatic synthesis is a green and efficient method with relatively mild reaction conditions and little or no organic reagents required, so it is a relatively promising catalytic method.
  • Using ligase to synthesize nucleotide chains can achieve green catalysis, and at the same time, the length of the synthetic chain is not limited, which can solve the technical bottleneck of solid-phase synthesis.
  • the present invention aims to provide a method for synthesizing double-stranded RNA and the application of RNA ligase in the synthesis of double-stranded RNA.
  • the invention solves the technical problem that it is difficult to complete the enzymatic synthesis of natural RNA or non-natural RNA double strands in the prior art.
  • a method for synthesizing double-stranded RNA comprises the following steps: S1, synthesizing single-stranded RNA fragments; S2, mixing the single-stranded RNA fragments and connecting the single-stranded RNA fragments to form double-stranded RNA using RNA ligase;
  • the RNA ligase is an RNA ligase derived from Vibrio phage, Escherichia phage, Klebsiella phage, Bacteriophage, thermophilic bacteriophage, Acidobacteriabacterium, Salmonella enterica, Yersiniaphage vB_YepM_ZN18, Shigella phage pSs-1 or Buttiauxella phage vB_ButM_GuL6.
  • the length of the single-stranded RNA fragment is 3 to 200 nucleotides or their analogs.
  • 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 Acidobacteriabacterium is shown in SEQ ID NO: 7; the amino acid sequence of the RNA ligase derived from Salmonellaenterica is shown in SEQ ID NO: 8; The amino acid sequence of the RNA ligase derived from YersiniaphagevB_YepM_ZNl8 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; the amino
  • the double-stranded RNA is natural RNA or non-natural RNA.
  • a ribonucleotide having one or more of a pentose ring 2' modification, a 3' modification, a phosphate ⁇ modification or a base modification; preferably, the pentose ring 2' 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; preferably, the phosphate ⁇ modification includes a phosphate ⁇ thio modification; 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.
  • the single-stranded RNA fragment is synthesized by solid phase synthesis.
  • the single-stranded RNA fragment includes single-stranded RNA fragment substrates 1 to substrate n and corresponding complementary single-stranded RNA fragments substrates n+1 to substrate n+n, wherein n ⁇ 2, and there is base complementary pairing of ⁇ 3 nucleotides between the complementary single-stranded RNA fragments; substrate 2 and substrate n+1, substrate n and substrate n+n-1 have base complementary pairing of ⁇ 2 nucleotides at the 5' end; optionally, the outer 5' and 3' ends of substrate 1 and substrate n+1 are connected by chemical bonds; optionally, the outer 5' and 3' ends of substrate n and substrate n+n are connected by chemical bonds.
  • 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.
  • RNA ligase in double-stranded RNA synthesis, wherein the RNA ligase is derived from Vibrio phage, Escherichia phage, Klebsiella phage, Bacteriophage, thermophilic bacteriophage, Acidobacteriabacterium, Salmonellaenterica, YersiniaphagevB_YepM_ZN18, Shigella phage pSs-1, or Buttiauxella phage vB_ButM_GuL6.
  • 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 Acidobacteriabacterium is shown in SEQ ID NO: 7; the amino acid sequence of the RNA ligase derived from Salmonellaenterica is shown in SEQ ID NO: 8; The amino acid sequence of the RNA ligase derived from YersiniaphagevB_YepM_ZNl8 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; the amino
  • the technical solution of the present invention can be used to specifically splice different nucleotide fragments, synthesize long nucleotide chains, and catalyze the connection between natural nucleotides and non-natural nucleotides, thus solving the current technical difficulties in synthesizing nucleotide chains.
  • FIG1 shows a denaturing polyacrylamide gel electrophoresis result diagram in Example 1
  • FIG2 shows a UPLC detection spectrum of a control without enzyme reaction in Example 1
  • FIG3 shows a UPLC detection spectrum of the reaction catalyzed by lig20 in Example 1;
  • FIG4 shows the denaturing polyacrylamide gel electrophoresis result in Example 2.
  • FIG5 shows the denaturing polyacrylamide gel electrophoresis result in Example 2.
  • FIG6 shows a UPLC detection spectrum of a control without enzyme reaction in Example 2.
  • FIG7 shows a UPLC detection spectrum of the reaction catalyzed by lig24 in Example 2.
  • FIG8 shows the peak time of the positive chain product in Example 2.
  • FIG9 shows the peak time of the antisense strand product in Example 2.
  • FIG10 shows the peak time of the double-stranded product in Example 2.
  • FIG11 shows the SDS-PAGE gel image detection reaction results in Example 3.
  • FIG12 shows the SDS-PAGE gel image detection reaction results in Example 4.
  • FIG13 shows the UPLC detection spectrum in Example 4.
  • FIG. 14 shows the results of mass spectrometry detection in Example 4.
  • Natural RNA refers to naturally occurring nucleotides, an RNA chain formed by connecting them through phosphate bonds, with a hydroxyl group at the 2’ position and a phosphorus-oxygen bond as the phosphate backbone.
  • Non-natural RNA refers to an RNA chain formed by non-natural nucleotides connected by phosphate bonds, the 2’ position of which can be modified, such as 2’F, 2’methoxy, 2’MOE, LNA, etc., and can also be modified on the base or on the phosphate group, such as phosphorus-sulfur modification.
  • the present invention uses ligase to synthesize natural or non-natural RNA chains, filling the technical gap.
  • a method for synthesizing double-stranded RNA comprises the following steps: S1, synthesizing single-stranded RNA fragments; S2, mixing the single-stranded RNA fragments and connecting the single-stranded RNA fragments using RNA ligase to form double-stranded RNA;
  • the RNA ligase is an RNA ligase derived from Vibrio phage, Escherichia phage, Klebsiella phage, Bacteriophage, thermophilic bacteriophage, Acidobacteriabacterium, Salmonella enterica, YersiniaphagevB_YepM_ZNl8, Shigella phage pSs-1 or Buttiauxella phage vB_ButM GuL6.
  • the technical solution of the present invention can be used to specifically splice different nucleotide fragments, synthesize long nucleotide chains, and catalyze the connection between natural nucleotides and non-natural nucleotides, thus solving the current technical difficulties in synthesizing nucleotide chains.
  • the length of the single-stranded RNA fragment is 3 to 200 nucleotides or their analogs, preferably 3 to 20 nucleotides or their analogs.
  • 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 Acidobacteriabacterium is shown in SEQ ID NO: 7; the amino acid sequence of the RNA ligase derived from Salmonellaenterica is shown in SEQ ID NO: 8; the amino acid sequence of the RNA ligase derived from YersiniaphagevB_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;
  • the double-stranded RNA is natural RNA or non-natural RNA.
  • the non-natural RNA may contain 2' methoxy, 2' fluorine, 2' MOE, 2' amino, 2' methyl, FANA, LNA or PS, etc.
  • 2' methoxy, 2' fluorine and PS have better effects.
  • the single-stranded RNA fragment can be synthesized by solid phase synthesis, that is, the synthesis method in the prior art.
  • the single-stranded RNA fragment includes single-stranded RNA fragment substrates 1 to n and corresponding complementary single-stranded RNA fragment substrates n+1 to n+n, wherein n ⁇ 2, and there are ⁇ 3 nucleotides of base complementary pairing between the complementary single-stranded RNA fragments; substrate 2 and substrate n+1, substrate n and substrate n+n-1 have ⁇ 2 nucleotides of base complementary pairing at the 5'end; optionally, the 5' and 3' ends of the outer side (outer side, i.e., the unconnected side) of substrate 1 and substrate n+1 are connected by chemical bonds; optionally, the 5' and 3' ends of the outer side (outer side, i.e., the unconnected side) of substrate n and substrate n+n are connected by chemical bonds.
  • reaction formula it can be understood in conjunction with the following reaction formula:
  • the reaction temperature of S2 is 0°C to 60°C, more preferably 4°C to 37°C, the reaction time is 0.5h to 24h, and the pH is 6 to 8.5.
  • RNA ligase in double-stranded RNA synthesis
  • the RNA ligase is an RNA ligase derived from Vibrio phage, Escherichia phage, Klebsiella phage, Bacteriophage, thermophilic bacteriophage, Acidobacteriabacterium, Salmonellaenterica, YersiniaphagevB_YepM_ZN18, Shigella phage pSs-1 or Buttiauxella phage vB_ButM_GuL6.
  • These ligases can be used to directly react and connect without annealing reaction, thereby providing synthesis efficiency.
  • 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 Acidobacteriabacterium is shown in SEQ ID NO: 7; the amino acid sequence of the RNA ligase derived from Salmonellaenterica is shown in SEQ ID NO: 8; the amino acid sequence of the RNA ligase derived from YersiniaphagevB_YepM_ZNl8 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
  • the inventors mined genes from various species in the gene library, cloned them into Escherichia coli, and used Pet28A as the expression vector.
  • the constructed strain containing the target gene was cultured after IPTG induction, and the cells were collected by centrifugation, ultrasonically broken, and the supernatant was obtained after centrifugation.
  • the supernatant was purified by nickel column, combined with ion column purification or molecular sieve purification to obtain pure enzyme.
  • DsRNA activity-1 is the activity of the following reaction:
  • DsRNA activity-2 is the activity of the following reaction:
  • amino acid sequence of some enzymes is as follows:
  • lig-20 (SEQ ID NO. 6, Vibriophage):
  • lig-21 (SEQ ID NO.1, Vibriophage):
  • lig-22 (SEQ ID NO. 2, Vibriophage):
  • lig-25 (SEQ ID NO.5, Vibriophage):
  • lig-26 (SEQ ID NO.7, AbRn1, from Acidobacteriabacterium, Rnl1 family):
  • lig-27 (SEQ ID NO.8, SeRn1, from Salmonella enterica, Rn12 family):
  • lig-28 (SEQ ID NO.9, YpRn1, source YersiniaphagevB_Yepi_ZN18, Rn12 family):
  • lig-29 (SEQ ID NO.10, SppRn1, from Shigella phage pSs-1, Rn12 family):
  • lig-30 (SEQ ID NO.11, BpRn1, source Buttiauxella phage vB_ButM_GuL6, Rn12 family):
  • lig-31 (SEQ ID NO.12, BpARn1, from Bacteriophage AR1, Rnl1 family):
  • lig-32 (SEQ ID NO.13, VPhRn1, from Vibrio phage, Rnl1 family):
  • lig-33 (SEQ ID NO.14, VPpRnl, source Vibrio phage phi-ST2, Rnl2 family):
  • lig-34 (SEQ ID NO.15, EPRn1, from Escherichia phage JN02, Rnl1 family):
  • lig-35 (SEQ ID NO.16, KPRn1, from Klebsiella phage KP15, Rn12 family):
  • lig-36 (SEQ ID NO.17, VPJRn1, source Vibrio phage JS98, Rn12 family):
  • lig-37 (SEQ ID NO.18, TBRn1, from thermophilic bacteriophage RM378, Rnl1 family):
  • lig-38 (SEQ ID NO.19, VPphRnl, from Vibrio phage phi-ST2, Rnl1 family):
  • the ligase gene was constructed on 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 Escherichia coli BL21 (DE3) expression strain.
  • the expression strain was inoculated into 5mL LB medium (containing 50 ⁇ g/mL) at an inoculum of 0.1% and cultured at 37°C for 16h.
  • the obtained induced culture solution was centrifuged at 10000rpm for 20min at 4°C to obtain bacterial sludge.
  • the bacterial sludge was resuspended with lysis buffer (50mM Tris-Cl pH8.0, 0-500mM NaCl, 10% glycerol) to make the final concentration of the bacteria 10%, and the crude enzyme solution was obtained after ultrasonic disruption and centrifugation.
  • the crude enzyme solution was filtered with a 0.22 ⁇ m filter membrane to obtain an enzyme solution that was purified with a nickel column. After desalting and replacing the solution, it was purified again with a strong anion column.
  • the obtained enzyme solution was stored in 30% glycerol after desalting and replacing the solution.
  • FIG1 shows the gel detection result of lig-20 purified by a nickel column
  • FIG2 shows the gel detection result of lig-20 purified by an ion column.
  • the final ligase purification effect is shown in FIG3, indicating that the purification method adopted by the present invention has purified pure enzymes for catalytic reactions.
  • Ligation catalyzes the synthesis of double-stranded RNA
  • 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 method, that is, traditional chemical synthesis method.
  • control group containing 400 ⁇ M substrate, no enzyme, and the corresponding volume of 50 mM Tris-Cl buffer was added to replace the enzyme.
  • the results of UPLC detection of samples are shown in Figure 6 (control group, containing 400 ⁇ M substrate, no enzyme, adding the corresponding volume of 50 mM Tris-Cl buffer to replace the enzyme, reacting at 25°C for 3 hours, heating at 98°C for 2 minutes, keeping warm at 80°C for 20 minutes, and obtaining the supernatant after centrifugation for UPLC analysis), Figure 7 (reaction catalyzed by ligase lig24. The peak at 21.662 min is the double-stranded product peak), Figure 8.
  • the peak time of the sense chain product is 15.022 min
  • Figure 9 the peak time of the antisense chain product is 15.213 min
  • Figure 10 the peak time of the mixed solution of the sense chain product and the antisense chain product, that is, the peak time of the double-stranded product is 21.683 min, and the peak at 15.192 min is the excess antisense chain product.
  • the results showed that the peak position of the product was consistent with that of the standard, and there was basically no substrate remaining.
  • MS identification the result of the positive chain was 7926.24, and the theoretical value of the positive chain was 7925.98 ⁇ 7.
  • the MS identification result of the antisense chain was 8150.25, and the theoretical value of the antisense chain was 8152.07 ⁇ 8.
  • the results were consistent with the standard, indicating that lig-20, 21, 22, 23, 24, 25, 35, 31, 34, 37, and 32 catalyzed the ligation reaction to generate ligated double-stranded RNA.
  • Ligation catalyzes the synthesis of double-stranded RNA
  • Ligase catalyzes the joining of natural RNA double strands
  • the substrates 26 to 29 were mixed in equal proportions, 10ul of 1mM ATP was added, 10*ligation buffer (containing 50mMtris-Cl, 10mM MgCl2 , 1mM DTT) was added, 10 ⁇ L (2mg/ml) of pure enzymes of lig20 and 24 were added to different reaction tubes respectively, water was added to make the substrate concentration 50uM, the reaction was carried out at 25°C for 3h, heated at 98°C for 2min, and kept at 80°C for 20min to inactivate the enzyme. The supernatant was obtained after centrifugation.
  • the results showed that the positive chain product (formed by the connection of SEQ ID NO.26 and SEQ ID NO.27) was 4488.15, and the antisense chain product (formed by the connection of SEQ ID NO.28 and SEQ ID NO.29) was 6304.92.
  • the theoretical values of the positive chain were 4485.73 ⁇ 4 and the antisense chain were 6302.79 ⁇ 6.
  • the generation of the products was confirmed, indicating that lig-20 and 24 catalyzed the connection reaction to generate the connected double-stranded RNA.
  • Ligation catalyzes the synthesis of double-stranded RNA
  • Ligase catalyzes the ligation of unnatural RNA duplexes
  • lane 1 nucleic acid molecular weight standard
  • lane 2 sense chain product
  • lane 3 antisense chain 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 ⁇ M substrate, connection buffer, ATP, only without enzyme, using an equal volume of 50 mM tris-Cl to replace the enzyme, reacting at 25°C for 3h, heating at 98°C for 2min, keeping at 80°C for 20min, and obtaining the supernatant after centrifugation for gel running
  • lane 11 sense chain product
  • lane 12 antisense chain 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-
  • FIG14 shows the result of mass spectrometry detection: the lig-20 reaction system after reaction was subjected to mass spectrometry detection, and the result of molecular weight was consistent with the product molecular weight, indicating that the connected product was generated.
  • + indicates an activity of 50% to 60%
  • ++ indicates an activity of 60% to 80%
  • +++ indicates an activity of >80%.
  • Ligation catalyzes the synthesis of double-stranded RNA
  • the molecular weight of the sense chain product (formed by connecting SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36, and SEQ ID NO.37) was identified by MS as 11227.4, and the molecular weight of the antisense chain product (formed by connecting SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, and SEQ ID NO.41) was 10583.7.
  • the theoretical values of the sense chain product were 11225 ⁇ 8, and the theoretical values of the antisense chain product were 10580 ⁇ 8.
  • the formation of the products was confirmed, indicating that lig-20, 21, 22, 23, 24, and 25 catalyzed the connection reaction to generate the connected double-stranded RNA.
  • the theoretical value of the sense chain was 47781.4 ⁇ 10, and the theoretical value of the antisense chain was 50121 ⁇ 10.
  • the generation of the product was confirmed, indicating that lig-20, 21, 22, 23, 24, 25 catalyzed the ligation reaction to generate the ligated double-stranded RNA.
  • the above-mentioned embodiments of the present invention achieve the following technical effects: green and environmentally friendly, can specifically splice different nucleotide fragments, can synthesize long fragment nucleotide chains, and can catalyze the connection between natural nucleotides and non-natural nucleotides, solving the current technical difficulties in synthesizing nucleotide chains.

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Abstract

本发明公开了一种双链RNA的合成方法及RNA连接酶在双链RNA合成中的应用。其中，该合成方法包括以下步骤：S1，合成单链RNA片段；S2，将单链RNA片段混合采用RNA连接酶将单链RNA片段连接形成双链RNA；RNA连接酶为来源为Vibrio phage、Escherichia phage或Klebsiella phage的RNA连接酶等。应用本发明的技术方案，能特异性拼接不同的核苷酸片段，能合成长片段核苷酸链，同时能催化天然核苷酸和非天然核苷酸的连接，解决了目前合成核苷酸链的技术难题。

Description

双链RNA的合成方法及RNA连接酶在双链RNA合成中的应用 技术领域
本发明涉及生物技术领域，具体而言，涉及一种双链RNA的合成方法及RNA连接酶在双链RNA合成中的应用。
背景技术
核糖核酸是存在于生物细胞以及部分病毒、类病毒中的遗传信息载体。在很长的一段时间内，RNA被认为只是在基因与蛋白质之间进行信息传递的分子。其实，在生命起源之初，RNA是唯一的生命分子，既可储存信息，也具有酶的功能。RNA除了充当蛋白质合成的信使之外(mRNA)，还具有着非常重要的调控功能，非转录RNA包括miRNA，siRNA，lncRNA，piwiRNA等等。其中，仅miRNA分子就有400多种，调控至少三分之一的人类基因。从20世纪70年代起，基因载体技术、基因克隆技术、基因编辑技术等给现代基因疗法技术带来了深刻的影响。同时RNA编辑技术实现了从遗传水平对人体细胞中特定的核苷酸进行编辑，这一技术不仅可以用作研究工具，而且可作为由突变引发的疾病的临时治疗方法。
近年来，RNA药物领域飞速发展，一方面是以siRNA和ASO为代表的小核酸药物在罕见病等领域大放异彩，另一方面是以mRNA疫苗为代表的mRNA药物在新冠疫情中发挥了重要的作用。上个世纪末首个小核酸药物fomivirsen获批上市以后，相关产业发展进入了一段“空窗期”。随着递送技术的进步，小核酸药物同样在近几年迎来了快速增长期。而这一阶段也酝酿出了小核酸药物的首个重磅炸弹药物Nusinersen。该药由Biogen开发，是一款反义寡核苷酸(ASO)药物，用于治疗成人和儿童患者的脊髓性肌萎缩(SMA)。
目前寡核苷酸(12-30个核苷酸)通用的合成方法多为固相合成法，而固相合成是将核酸固定在固相载体上完成合成反应的，最常用的固相载体为可控微孔玻璃珠(CPG)，但是载量有限，一般为70-80μmol/g。但是固相合成核苷酸链存在很大的缺点，合成链长受限，一般用于合成25个核苷酸以内的链长，随着合成链长的增加，产率降低，引物合成至～80nt链长时粗品纯度只有～40％，因此极大限制了核苷酸链的合成。
酶法催化合成是一种绿色高效的方式，反应条件比较温和，对有机试剂需求小甚至不需要有机试剂，因此是一种比较有前景的催化方式。利用连接酶合成核苷酸链，可以实现绿色催化，同时合成链长不受限制，可以解决固相合成的技术瓶颈。但文献或专利中对于酶法催化合成天然RNA或非天然RNA双链的报道几乎没有，尤其是对于非天然RNA的双链合成更是很难做到。
发明内容
本发明旨在提供一种双链RNA的合成方法及RNA连接酶在双链RNA合成中的应用，以 解决现有技术中酶法催化合成天然RNA或非天然RNA双链难以完成的技术问题。
为了实现上述目的，根据本发明的一个方面，提供了一种双链RNA的合成方法。该合成方法包括以下步骤：S1，合成单链RNA片段；S2，将单链RNA片段混合采用RNA连接酶将单链RNA片段连接形成双链RNA；RNA连接酶为来源为Vibrio phage、Escherichia phage、Klebsiella phage、Bacteriophage、thermophilic bacteriophage、Acidobacteriabacterium、Salmonellaenterica、YersiniaphagevB_YepM_ZN18、Shigella phage pSs-1或Buttiauxella phage vB_ButM_GuL6的RNA连接酶。
进一步地，单链RNA片段的长度为3～200个核苷酸或其类似物。
进一步地，来源为Vibrio phage的RNA连接酶的氨基酸序列如SEQ ID NO：1、2、5、6、13、14、17或19所示；来源为Escherichia phage的RNA连接酶的氨基酸序列如SEQ ID NO：3或15所示；来源为Klebsiella phage的RNA连接酶的氨基酸序列如SEQ ID NO：4或16所示；来源为Acidobacteriabacterium的RNA连接酶的氨基酸序列如SEQ ID NO：7所示；来源为Salmonellaenterica的RNA连接酶的氨基酸序列如SEQ ID NO：8所示；来源为YersiniaphagevB_YepM_ZNl8的RNA连接酶的氨基酸序列如SEQ ID NO：9所示；来源为Shigella phage pSs-1的RNA连接酶的氨基酸序列如SEQ ID NO：10所示；来源为Buttiauxella phage vB_ButM_GuL6的RNA连接酶的氨基酸序列如SEQ ID NO：11所示，来源为Bacteriophage的RNA连接酶的氨基酸序列如SEQ ID NO：12所示；来源为thermophilic Bacteriophage的RNA连接酶的氨基酸序列如SEQ ID NO：18所示。
进一步地，双链RNA为天然RNA或非天然RNA。
进一步地，具有戊糖环2’位修饰、3’位修饰、磷酸根α位修饰或碱基修饰中的一种或多种的核糖核苷酸；优选地，戊糖环2’位修饰包括2’-甲氧基修饰、2’-氟修饰、2’-三氟甲氧基修饰、2’-甲氧乙基修饰、2’-烯丙基修饰、2’-氨基修饰或2’-叠氮基修饰；优选地，磷酸根α位修饰包括磷酸根α位硫代修饰；优选地，碱基修饰包括在碱基的N1、N5或N6位中任意一处或多处进行的甲基化修饰和/或乙酰化修饰。
进一步地，单链RNA片段为采用固相合成法合成。
进一步地，单链RNA片段包括单链RNA片段底物1～底物n及对应互补的单链RNA片段底物n+1～底物n+n，其中，n≥2，互补的单链RNA片段之间有≥3个核苷酸的碱基互补配对；底物2和底物n+1、底物n和底物n+n-1的5’端有≥2个核苷酸的碱基互补配对；可选的，底物1和底物n+1外侧的5’和3’端由化学键相连；可选的，底物n和底物n+n外侧的5’和3’端由化学键相连。
进一步地，S2的反应温度为0℃～60℃，优选为4℃～37℃，反应时间为0.5h～24h，pH为6～8.5。
根据本发明的另一方面，提供了一种RNA连接酶在双链RNA合成中的应用，其中，RNA连接酶为来源为Vibrio phage、Escherichia phage、Klebsiella phage、Bacteriophage、thermophilic  bacteriophage、Acidobacteriabacterium、Salmonellaenterica、YersiniaphagevB_YepM_ZN18、Shigella phage pSs-1或Buttiauxella phage vB_ButM_GuL6的RNA连接酶。
进一步地，来源为Vibrio phage的RNA连接酶的氨基酸序列如SEQ ID NO：1、2、5、6、13、14、17或19所示；来源为Escherichia phage的RNA连接酶的氨基酸序列如SEQ ID NO：3或15所示；来源为Klebsiella phage的RNA连接酶的氨基酸序列如SEQ ID NO：4或16所示；来源为Acidobacteriabacterium的RNA连接酶的氨基酸序列如SEQ ID NO：7所示；来源为Salmonellaenterica的RNA连接酶的氨基酸序列如SEQ ID NO：8所示；来源为YersiniaphagevB_YepM_ZNl8的RNA连接酶的氨基酸序列如SEQ ID NO：9所示；来源为Shigella phage pSs-1的RNA连接酶的氨基酸序列如SEQ ID NO：10所示；来源为Buttiauxella phage vB_ButM_GuL6的RNA连接酶的氨基酸序列如SEQ ID NO：11所示，来源为Bacteriophage的RNA连接酶的氨基酸序列如SEQ ID NO：12所示；来源为thermophilic Bacteriophage的RNA连接酶的氨基酸序列如SEQ ID NO：18所示。
应用本发明的技术方案，能特异性拼接不同的核苷酸片段，能合成长片段核苷酸链，同时能催化天然核苷酸和非天然核苷酸的连接，解决了目前合成核苷酸链的技术难题。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示出了实施例1中变性聚丙烯酰胺凝胶电泳结果图；
图2示出了实施例1中未加酶反应对照UPLC检测图谱；
图3示出了实施例1中lig20催化的反应UPLC检测图谱；
图4示出了实施例2中变性聚丙烯酰胺凝胶电泳结果图；
图5示出了实施例2中变性聚丙烯酰胺凝胶电泳结果图；
图6示出了实施例2中未加酶反应对照UPLC检测图谱；
图7示出了实施例2中lig24催化的反应UPLC检测图谱；
图8示出了实施例2中正义链产品的出峰时间；
图9示出了实施例2中反义链产品的出峰时间；
图10示出了实施例2中双链产品的出峰时间；
图11示出了实施例3中SDS-PAGE胶图检测反应结果；
图12示出了实施例4中SDS-PAGE胶图检测反应结果；
图13示出了实施例4中UPLC检测图谱；
图14示出了实施例4中质谱检测的结果。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
名词解释
天然RNA：指天然存在的核苷酸，通过磷酸酯键相连形成的RNA链，其2’位为羟基，磷酸骨架为磷氧键。
非天然RNA：指由非天然的核苷酸通过磷酸酯键相连形成的RNA链，其2’位可以为修饰后的，如2’F、2’甲氧基、2’MOE、LNA等，也可在碱基上进行修饰、也可在磷酸基团上进行修饰，如磷硫修饰。
目前，文献或专利中对于酶法催化合成天然RNA或非天然RNA双链的报道几乎没有，尤其是对于非天然RNA的双链合成更是很难做到。本发明利用连接酶合成天然或非天然RNA链，填补了技术空白。
根据本发明一种典型的实施方式，提供一种双链RNA的合成方法。该合成方法包括以下步骤：S1，合成单链RNA片段；S2，将单链RNA片段混合采用RNA连接酶将单链RNA片段连接形成双链RNA；RNA连接酶为来源为Vibrio phage、Escherichia phage、Klebsiella phage、Bacteriophage、thermophilic bacteriophage、Acidobacteriabacterium、Salmonellaenterica、YersiniaphagevB_YepM_ZNl8、Shigella phage pSs-1或Buttiauxella phage vB_ButM GuL6的RNA连接酶。
应用本发明的技术方案，能特异性拼接不同的核苷酸片段，能合成长片段核苷酸链，同时能催化天然核苷酸和非天然核苷酸的连接，解决了目前合成核苷酸链的技术难题。
在本发明一实施方式中，单链RNA片段的长度为3～200个核苷酸或其类似物，优选为3～20个核苷酸或其类似物。
优选的，来源为Vibrio phage的RNA连接酶的氨基酸序列如SEQ ID NO：1、2、5、6、13、14、17或19所示；来源为Escherichia phage的RNA连接酶的氨基酸序列如SEQ ID NO：3或15所示；来源为Klebsiella phage的RNA连接酶的氨基酸序列如SEQ ID NO：4或16所示；来源为Acidobacteriabacterium的RNA连接酶的氨基酸序列如SEQ ID NO：7所示；来源为Salmonellaenterica的RNA连接酶的氨基酸序列如SEQ ID NO：8所示；来源为YersiniaphagevB_YepM_ZN18的RNA连接酶的氨基酸序列如SEQ ID NO：9所示；来源为Shigella phage pSs-1的RNA连接酶的氨基酸序列如SEQ ID NO：10所示；来源为Buttiauxella phage vB_ButM_GuL6的RNA连接酶的氨基酸序列如SEQ ID NO：11所示，来源为 Bacteriophage的RNA连接酶的氨基酸序列如SEQ ID NO：12所示；来源为thermophilic Bacteriophage的RNA连接酶的氨基酸序列如SEQ ID NO：18所示。
根据本发明一种典型的实施方式，双链RNA为天然RNA或非天然RNA。其中，非天然RNA中可以含有2’甲氧基、2’氟、2’MOE、2’氨基、2’甲基、FANA、LNA或PS等。其中，2’甲氧基、2’氟、PS效果较好。
在本发明中，单链RNA片段可以采用固相合成法合成，即现有技术中的合成方式合成。
根据本发明一种典型的实施方式，单链RNA片段包括单链RNA片段底物1～底物n及对应互补的单链RNA片段底物n+1～底物n+n，其中，n≥2，互补的单链RNA片段之间有≥3个核苷酸的碱基互补配对；底物2和底物n+1、底物n和底物n+n-1的5’端有≥2个核苷酸的碱基互补配对；可选的，底物1和底物n+1外侧(外侧即未连接的一侧)的5’和3’端由化学键相连；可选的，底物n和底物n+n外侧(外侧即未连接的一侧)的5’和3’端由化学键相连。具体的，可结合下列反应式理解：
优选的，S2的反应温度为0℃～60℃，更优选为4℃～37℃，反应时间为0.5h～24h，pH为6～8.5。
根据本发明一种典型的实施方式，提供RNA连接酶在双链RNA合成中的应用，其中，RNA连接酶为来源为Vibrio phage、Escherichia phage、Klebsiella phage、Bacteriophage、thermophilic bacteriophage、Acidobacteriabacterium、Salmonellaenterica、YersiniaphagevB_YepM_ZNl8、Shigella phage pSs-1或Buttiauxella phage vB_ButM_GuL6的RNA连接酶。采用这些连接酶可以不经过退火反应直接进行反应连接，提供合成效率。优选的，来源为Vibrio phage的RNA连接酶的氨基酸序列如SEQ ID NO：1、2、5、6、13、14、17或19所示；来源为Escherichia phage的RNA连接酶的氨基酸序列如SEQ ID NO：3或15所示；来源为Klebsiella phage的RNA连接酶的氨基酸序列如SEQ ID NO：4或16所示；来源为Acidobacteriabacterium的RNA连接酶的氨基酸序列如SEQ ID NO：7所示；来源为Salmonellaenterica的RNA连接酶的氨基酸序列如SEQ ID NO：8所示；来源为YersiniaphagevB_YepM_ZNl8的RNA连接酶的氨基酸序列如SEQ ID NO：9所示；来源为Shigella phage pSs-1的RNA连接酶的氨基酸序列如SEQ ID NO：10所示；来源为Buttiauxella phage vBButM_GuL6的RNA连接酶的氨基酸序列如SEQ ID NO：11所示，来源为Bacteriophage的RNA连接酶的氨基酸序列如SEQ ID NO：12所示；来源为thermophilic  Bacteriophage的RNA连接酶的氨基酸序列如SEQ ID NO：18所示。
下面将结合实施例进一步说明本发明的有益效果。
为了能催化短链RNA或非天然的短链RNA的双链连接，发明人在基因库中挖掘了多种不同种属来源的基因，克隆至大肠杆菌中，表达载体是Pet28A，构建的含目的基因的菌种经IPTG诱导表达后进行培养，离心收集菌体，进行超声破碎，离心后获得上清液。上清液经镍柱纯化，结合离子柱纯化或结合分子筛纯化后得到纯酶。
在干净的反应器中，加入各个序列片段各0.1μmol(200μM终浓度)，加入连接酶0.1mg，ATP 0.4μmol，0.5μmolDTT，5μmolMgCl2，用Tris-Cl缓冲液补充至500μL，使Tris-Cl终浓度50mM反应体系经混合后，在25℃进行温育1～20h，得到的反应液经聚丙烯酰胺凝胶电泳或者UPLC检测，得到如下表3所示结果，结果显示大多数酶没有连接活性，只有少部分酶具有催化活性。
DsRNA活性-1为以下反应的活性：
其中，具体序列见表1。
表1
DsRNA活性-2为以下反应的活性：
其中，具体序列见表2。
表2

表3


注：“-”代表：无产品生成，“++”代表：转化率大于10％小于50％，“+++”代表：转化率大于50％小于70％，“++++”代表：转化率大于70％。
其中，部分酶的氨基酸序列如下：
lig-20(SEQ ID NO.6，Vibriophage)：
lig-21(SEQ ID NO.1，Vibriophage)：
lig-22(SEQ ID NO.2，Vibriophage)：
lig-23(SEQ ID NO.3，Escherichia phage)：
lig-24(SEQ ID NO.4，Klebsiella phage)：
lig-25(SEQ ID NO.5，Vibriophage)：
lig-26(SEQ ID NO.7，AbRnl，来源Acidobacteriabacterium，Rnl1家族)：
lig-27(SEQ ID NO.8，SeRnl，来源Salmonellaenterica，Rnl2家族)：
lig-28(SEQ ID NO.9，YpRnl，来源YersiniaphagevB_Yepi_ZN18，Rnl2家族)：

lig-29(SEQ ID NO.10，SppRnl，来源Shigella phage pSs-1，Rnl2家族)：
lig-30(SEQ ID NO.11，BpRnl，来源Buttiauxella phage vB_ButM_GuL6，Rnl2家族)：
lig-31(SEQ ID NO.12，BpARnl，来源Bacteriophage AR1，Rnl1家族)：
lig-32(SEQ ID NO.13，VPhRnl，来源Vibrio phage，Rnl1家族)：
lig-33(SEQ ID NO.14，VPpRnl，来源Vibrio phage phi-ST2，Rnl2家族)：

lig-34(SEQ ID NO.15，EPRnl，来源Escherichia phage JN02，Rnl1家族)：
lig-35(SEQ ID NO.16，KPRnl，来源Klebsiella phage KP15，Rnl2家族)：
lig-36(SEQ ID NO.17，VPJRnl，来源Vibrio phage JS98，Rnl2家族)：
lig-37(SEQ ID NO.18，TBRnl，来源thermophilic bacteriophage RM378，Rnl1家族)：
lig-38(SEQ ID NO.19，VPphRnl，来源Vibrio phage phi-ST2，Rnl1家族)：
实施例1
连接酶的制备
将连接酶基因构建与pET28a载体上，在连接酶的N端增加组氨酸标签方便亲和层析纯化，构建的重组质粒转化入大肠杆菌BL21(DE3)表达菌株。表达菌株以0.1％的接种量接种至5mLLB培养基中(含50μg/mL)，与37℃培养16h。得到的培养液以1％的接种量转接到500mL LB培养基中，与37℃培养至OD＝0.6时，加入0.1mM的IPTG，在20℃诱导16h。得到的诱导培养液在4℃，经10000rpm离心20min获得菌泥。菌泥用裂解缓冲液(50mM Tris-Cl pH8.0，0～500mM NaCl，10％甘油)重悬，使得菌体终浓度为10％，经超声破碎，离心后得到粗酶液。粗酶液用0.22，μm滤膜过滤后得到的酶液用镍柱进行纯化，经过脱盐换液后，用强阴离子柱进行二次纯化，得到的酶液经脱盐换液后储存与30％甘油中。其中，图1示出了镍柱纯化lig-20的凝胶检测结果图，图2示出了离子柱纯化lig-20的凝胶检测结果图。最终连接酶纯化效果如图3所示，表明经过本发明采用的纯化方式，均纯化出了纯净的酶用于催化反应。
实施例2
连接催化合成双链RNA
表4

注：“m”代表：2’甲氧基修饰，“f”代表：2’氟修饰。
表4中的底物(SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25)通过固相合成方法制备的，即传统的化学合成方法。
将表4中的6种底物各取0.2μmol(终浓度400μM)，加入ATP 0.4μmol，加入10*连接缓冲液(含50mMtris-Cl，10mM MgCl2，1mM DTT)使得终体积为500μL，不同的反应管中分别加入lig20、21、22、23、24、25各个纯酶0.1mg，在25℃反应3h，98℃加热2min，80℃保温20min使得酶失活，离心后得上清液，用变性聚丙烯酰胺凝胶电泳进行分析检测，结果生成了亮度非常高的产物条带，具体见图4(1.正义链产品标准品(SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22连接形成)；2.反义链产品标准品(SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25连接形成)；3.正义链+反义链；4.核酸分子量标记；5.由来源于Trypanosoma brucei brucei的连接酶催化的反应；6.由lig21催化的反应；7.由lig22催化的反应；8.由lig24催化的反应；9.由lig23催化的反应；10.由lig20催化的反应；11.由lig25催化的反应；12.对照组，含400μM底物，不含酶，添加对应体积的50mM Tris-Cl缓冲液替代酶)和图5(1.正义链；2.反义链；3.核酸分子量标记；4.正义链+反义链；5.由lig35催化的反应；6.由lig31催化的反应；7.由lig34催化的反应；8.由lig1催化的反应；9.由lig15催化的反应；10.由lig37催化的反应；11.由lig16催化的反应；12.由lig32催化的反应；13.对照组，含400μM底物，不含酶，添加对应体积的50mM Tris-Cl缓冲液替代酶)。将样品进行UPLC检测结果见图6(对照组，含400μM底物，不含酶，添加对应体积的50mM Tris-Cl缓冲液替代酶，在25℃反应3h，98℃加热2min，80℃保温20min，离心后得上清液进行UPLC分析)、图7(连接酶lig24催化后的反应。21.662min峰为双链产品峰)、图8.正义链产品的出峰时间15.022min、图9(反义链产品的出峰时间15.213min)和图10(正义链产品和反义链产品的混合溶液的出峰时间，即双链产品的出峰时间21.683min，15.192min峰为超量的反义链产品)。结果发现，产品的出峰位置与标准品一致，且基本无底物剩余，经MS鉴定后结果正义链为7926.24，正义链理论值7925.98±7，反义链MS鉴定结果为8150.25，反义链理论值8152.07±8，结果与标准品一致，表明lig-20、21、22、23、24、25、35、31、34、37、32催化了连接反应，生成了连接后的双链RNA。
实施例3
连接催化合成双链RNA
连接酶催化连接天然RNA双链连接
表5
将26～29底物等比例混合，加入1mM ATP 10ul，加入10*连接缓冲液(含50mMtris-Cl，10mM MgCl2，1mM DTT)，不同反应管中分别加入lig20、24纯酶10μL(2mg/ml)，加入水使得底物浓度为50uM，在25℃反应3h，98℃加热2min，80℃保温20min使得酶失活，离心后得上清液，用MS鉴定后结果正义链产品(SEQ ID NO.26和SEQ ID NO.27连接形成)4488.15，反义链产品(SEQ ID NO.28和SEQ ID NO.29连接形成)6304.92，理论值正义链4485.73±4，反义链6302.79±6，确定产品的生成，表明lig-20、24催化了连接反应，生成了连接后的双链RNA。结果见图11(SDS-PAGE胶图检测反应结果，泳道1表示分子量标记，泳道2表示阴性对照(体系中含有底物、反应缓冲液、ATP，不含酶，使用相应体积的50mMtris-Cl替代酶)，泳道3和4分别为连接酶催化后的反应体系)。
实施例4
连接催化合成双链RNA
连接酶催化连接非天然RNA双链连接
表6
注：“m”代表：2’甲氧基修饰，“f”代表：2’氟修饰。“s”表示：硫代磷酸酯修饰
将30～33底物等比例混合，加入1mM ATP 10ul，加入10*连接缓冲液(含50mMtris-Cl，10mM MgCl2，1mM DTT)，不同反应管中分别加入lig20、21、22、23、24、25、31、32、33、34、35、36、37纯酶10μL(2mg/ml)，加入水使得底物浓度为200μM，同时设置不加酶的对照反应，补加10μL50mMtris-Cl，在25℃反应3h，98℃加热2min，80℃保温20min使得酶失活，离心后得上清液，用MS鉴定后结果正义链产品(SEQ ID NO.30和SEQ ID NO.31连接形成)检测分子量为8726.9，反义链产品(SEQ ID NO.32和SEQ ID NO.33连接形成)检测分子量为7765.9，理论正义链分子量为8727.2±3，理论反义链分子量为7766.0±3，表明连接酶lig-20、21、22、23、24、25、31、32、33、34、35、36、37催化了连接反应，生成了连接后的双链RNA。而lig-16、17、18、19没有产品的生成。SDS-PAGE胶图12显示，底物基本已经转化完全，生成了产品条带。
图12，其中，泳道1：核酸分子量标准；泳道2：正义链产品；泳道3：反义链产品；泳道4：lig-20；泳道5：lig-21；泳道6：lig-22；泳道7：lig-23；泳道8：lig-24；泳道9：lig-25；泳道10：阴性对照，含有底物200μM、连接缓冲液、ATP、仅没有加酶，使用相等体积的50mMtris-Cl替代酶，经25℃反应3h，98℃加热2min，80℃保温20min，离心后得上清液进行跑胶；泳道11：正义链产品；泳道12：反义链产品；泳道13：lig-37；泳道14：lig-33；泳道15：lig-36；泳道16：lig-32；泳道17：lig-31；泳道18：lig-35；泳道19：lig-34。泳道20：核酸分子量标准，泳道21：正义链和反义链产品的混合体系，泳道22：lig-16，泳道23：lig-17，泳道24：lig-1，泳道25：lig-19。
进行UPLC检测，结果已经催化完全，结果见图13(连接酶催化的连接反应，1表示正义链和反义链混合后的标准品，在UPLC中是两条链分开的，与胶图的结果也是一致的。2～5表示由连接酶催化后的反应体系，分别为lig-20、21、22、23)。
图14示出了质谱检测的结果：经反应后的lig-20反应体系进行质谱检测，结果分子量是符合产品分子量的，表明生成了连接后的产品。
对反应进行优化，对反应pH和反应温度进行优化，结果在Ph＝6～8℃均能生成目的产品，最优pH是7.5，在0℃～40℃均能催化生成产品，最优反应温度是16℃。结果如下表7。
表7
注：+表示活性50％～60％；++表示活性60％～80％；+++表示活性＞80％。
实施例5
连接催化合成双链RNA
表8
注：“m”代表：2’甲氧基修饰，“f”代表：2’氟修饰。
将表8中的底物各取0.1μmol(终浓度为200μM)，加入10mM ATP 20μl，不同反应管中分别加入lig20、21、22、23、24、25纯酶0.1mg，加入10*连接缓冲液(含50mMtris-Cl，10 mM MgCl2，1mM DTT)，使得终体积为0.5mL，在25℃反应3h，98℃加热2min，80℃保温20min使得酶失活，离心后得上清液，用MS鉴定正义链产品(SEQ ID NO.34、SEQ ID NO.35SEQ ID NO.36、SEQ ID NO.37连接形成)分子量为11227.4，反义链产品(SEQ ID NO.38、SEQ ID NO.39SEQ ID NO.40、SEQ ID NO.41连接形成)分子量为10583.7，正义链产品理论值为11225±8，反义链产品理论值为10580±8，确定产品的生成，表明lig-20、21、22、23、24、25催化了连接反应，生成了连接后的双链RNA。
实施例6
连接催化合成长链RNA
表9
注：“m”代表：2’甲氧基修饰，“f”代表：2’氟修饰。
将表9中的底物各取0.1μmol，加入10mMATP 20μl，不同反应管中分别加入lig-21、22、23、24、25纯酶0.1mg，加入10*连接缓冲液(含50mMtris-Cl，10mM MgCl2，1mM DTT)，使得终体积为0.5mL，在25℃反应3h，98℃加热2min，80℃保温20min使得酶失活，离心后得上清液，用MS鉴定正义链分子量为47789.4，反义链分子量为50130.2，正义链理论值为47781.4±10，反义链理论值为50121±10，确定产品的生成，表明lig-20、21、22、23、24、25催化了连接反应，生成了连接后的双链RNA。
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：绿色环保，能特异性拼接不同的核苷酸片段，能合成长片段核苷酸链，同时能催化天然核苷酸和非天然核苷酸的连接，解决了目前合成核苷酸链的技术难题。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种双链RNA的合成方法，其特征在于，包括以下步骤：
    S1，合成单链RNA片段；
    S2，将所述单链RNA片段混合，采用RNA连接酶将所述单链RNA片段连接形成所述双链RNA；所述RNA连接酶为来源为Vibrio phage、Escherichia phage、Klebsiella phage、Bacteriophage、thermophilic bacteriophage、Acidobacteriabacterium、Salmonellaenterica、YersiniaphagevB_YepM_ZN18、Shigella phage pSs-1或Buttiauxella phage vB_ButM_GuL6的RNA连接酶。
  2. 根据权利要求1所述的合成方法，其特征在于，所述单链RNA片段具有3～200个核苷酸或其类似物。
  3. 根据权利要求2所述的合成方法，其特征在于，所述来源为Vibrio phage的RNA连接酶的氨基酸序列如SEQ ID NO：1、2、5、6、13、14、17或19所示；所述来源为Escherichia phage的RNA连接酶的氨基酸序列如SEQ ID NO：3或15所示；所述来源为Klebsiella phage的RNA连接酶的氨基酸序列如SEQ ID NO：4或16所示；所述来源为Acidobacteriabacterium的RNA连接酶的氨基酸序列如SEQ ID NO：7所示；所述来源为Salmonellaenterica的RNA连接酶的氨基酸序列如SEQ ID NO：8所示；所述来源为YersiniaphagevB_YepM_ZN18的RNA连接酶的氨基酸序列如SEQ ID NO：9所示；所述来源为Shigella phage pSs-1的RNA连接酶的氨基酸序列如SEQ ID NO：10所示；所述来源为Buttiiauxella phage vB_ButM_GuL6的RNA连接酶的氨基酸序列如SEQ ID NO：11所示，所述来源为Bacteriophage的RNA连接酶的氨基酸序列如SEQ ID NO：12所示；所述来源为thermophilic Bacteriophage的RNA连接酶的氨基酸序列如SEQ ID NO：18所示。
  4. 根据权利要求1所述的合成方法，其特征在于，所述双链RNA为天然RNA或非天然RNA。
  5. 根据权利要求4所述的合成方法，其特征在于，所述非天然RNA具有戊糖环2’位修饰、3’位修饰、磷酸根α位修饰或碱基修饰中的一种或多种的核糖核苷酸；
    优选地，所述戊糖环2’位修饰包括2’-甲氧基修饰、2’-氟修饰、2’-三氟甲氧基修饰、2’-甲氧乙基修饰、2’-烯丙基修饰、2’-氨基修饰或2’-叠氮基修饰；
    优选地，所述磷酸根α位修饰包括磷酸根α位硫代修饰；
    优选地，所述碱基修饰包括在碱基的N1、N5或N6位中任意一处或多处进行的甲基化修饰和/或乙酰化修饰。
  6. 根据权利要求1所述的合成方法，其特征在于，所述单链RNA片段采用固相合成法合成。
  7. 根据权利要求1所述的合成方法，其特征在于，所述单链RNA片段包括单链RNA片段底物1～底物n及对应互补的单链RNA片段底物n+1～底物n+n，其中，n≥2，
    互补的单链RNA片段之间有≥3个核苷酸的碱基互补配对；
    底物2和底物n+1、底物n和底物n+n-1的5’端有≥2个核苷酸的碱基互补配对；
    可选的，底物1和底物n+1外侧的5’和3’端由化学键相连；
    可选的，底物n和底物n+n外侧的5’和3’端由化学键相连。
  8. 根据权利要求1所述的合成方法，其特征在于，所述S2的反应温度为0℃～60℃，优选为4℃～37℃，反应时间为0.5h～24h，pH为6～8.5。
  9. RNA连接酶在双链RNA合成中的应用，其中，所述RNA连接酶为来源为Vibrio phage、Escherichia phage、Klebsiella phage、Bacteriophage、thermophilic bacteriophage、Acidobacteriabacterium、Salmonellaenterica、YersiniaphagevB_YepM_ZN18、Shigella phage pSs-1或Buttiiauxella phage vB_ButM_GuL6的RNA连接酶。
  10. 根据权利要求9所述的应用，其特征在于，所述来源为Vibrio phage的RNA连接酶的氨基酸序列如SEQ ID NO：1、2、5、6、13、14、17或19所示；所述来源为Escherichia phage的RNA连接酶的氨基酸序列如SEQ ID NO：3或15所示；所述来源为Klebsiella phage的RNA连接酶的氨基酸序列如SEQ ID NO：4或16所示；所述来源为Acidobacteriabacterium的RNA连接酶的氨基酸序列如SEQ ID NO：7所示；所述来源为Salmonellaenterica的RNA连接酶的氨基酸序列如SEQ ID NO：8所示；所述来源为YersiniaphagevB_YepM_ZN18的RNA连接酶的氨基酸序列如SEQ ID NO：9所示；所述来源为Shigella phage pSs-1的RNA连接酶的氨基酸序列如SEQ ID NO：10所示；所述来源为Buttiiauxella phage vB_ButM_GuL6的RNA连接酶的氨基酸序列如SEQ ID NO：11所示，所述来源为Bacteriophage的RNA连接酶的氨基酸序列如SEQ ID NO：12所示；所述来源为thermophilic Bacteriophage的RNA连接酶的氨基酸序列如SEQ ID NO：18所示。
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CN111971396A (zh) * 2018-03-30 2020-11-20 住友化学株式会社 单链rna的制造方法
CN113444698A (zh) * 2020-03-25 2021-09-28 味之素株式会社 连接酶突变体

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CN111971396A (zh) * 2018-03-30 2020-11-20 住友化学株式会社 单链rna的制造方法
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