WO2023273366A1 - 核酸连接酶 - Google Patents

核酸连接酶 Download PDF

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WO2023273366A1
WO2023273366A1 PCT/CN2022/077538 CN2022077538W WO2023273366A1 WO 2023273366 A1 WO2023273366 A1 WO 2023273366A1 CN 2022077538 W CN2022077538 W CN 2022077538W WO 2023273366 A1 WO2023273366 A1 WO 2023273366A1
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nucleic acid
ligase
amino acid
mutation
rna
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PCT/CN2022/077538
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French (fr)
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丁春明
杨政权
金胜男
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温州医科大学
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Priority to EP22831198.1A priority Critical patent/EP4368708A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y605/00—Ligases forming phosphoric ester bonds (6.5)
    • C12Y605/01—Ligases forming phosphoric ester bonds (6.5) forming phosphoric ester bonds (6.5.1)
    • C12Y605/01001—DNA ligase (ATP) (6.5.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y605/00—Ligases forming phosphoric ester bonds (6.5)
    • C12Y605/01—Ligases forming phosphoric ester bonds (6.5) forming phosphoric ester bonds (6.5.1)
    • C12Y605/01002—DNA ligase (NAD+) (6.5.1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y605/00—Ligases forming phosphoric ester bonds (6.5)
    • C12Y605/01—Ligases forming phosphoric ester bonds (6.5) forming phosphoric ester bonds (6.5.1)
    • C12Y605/01003—RNA ligase (ATP) (6.5.1.3)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of biotechnology. Specifically, it relates to a new nucleic acid ligase obtained by mutation of a prior art nucleic acid ligase. The invention also relates to products comprising said enzymes.
  • Nucleic acid ligase is a metal ion-dependent enzyme that catalyzes the formation of phosphodiester bonds between the adjacent 3' and 5' ends of DNA or RNA to achieve intramolecular circularization or intermolecular linear ligation of substrate samples.
  • Nucleic acid ligases can be divided into DNA ligases and RNA ligases according to different catalytic substrates. Some ligases can catalyze the ligation reaction between DNA and RNA [1,2] .
  • natural nucleic acid ligases have a variety of unique properties, such as substrate specificity, sequence preference, thermostability, salt tolerance, and pH change resistance.
  • the complete nucleic acid ligation reaction is divided into three stages [1] .
  • the ligase binds to the adenosine group of ATP or NAD + , transfers the adenosine group in ATP or NAD + to the lysine residue in the conserved motif of the enzyme, releasing pyrophosphate, And form the intermediate of enzyme and adenosine.
  • This step is an equilibrium reaction that can proceed in both directions.
  • the intermediate of the enzyme and adenosine transfers its adenosine group to the 5' phosphate terminus of the nucleic acid substrate, forming a 5' adenylylated intermediate.
  • This step is also a counterbalance and can go both ways.
  • the lysine catalytic site of the enzyme binds to the 3' hydroxyl end of the nucleic acid, attacks the 5' adenylation end of the nucleic acid, catalyzes the formation of a phosphodiester bond between the two, and completes the ligation reaction.
  • Common ligases generally use 5' phosphorylated nucleic acid as a substrate to catalyze the three complete stages of the ligation reaction to realize the ligation reaction [3,4] .
  • Ligase is the basis of common molecular biology methods such as molecular cloning technology, high-throughput sequencing library preparation, gene synthesis and molecular diagnosis, and plays an important role in molecular biology research and molecular diagnosis technology [5,6] .
  • the ligase can be divided into two types: thermostable and non-thermostable.
  • thermostable there are many ligases for double-stranded DNA or RNA ligation reactions, but there are only a very limited selection of enzymes for single-stranded DNA ligation reactions, which can perform single-strand ligation at higher temperatures (such as >65°C) less thermostable ligases.
  • thermostable single-stranded nucleic acid ligases have been reported.
  • Thermostable TS2126 ligase (trade name CircLigase) can directly catalyze the ligation reaction between the 5' phosphate end and the 3' hydroxyl end at 65°C [7] , but the catalysis has a terminal sequence preference, resulting in selective ligation of substrates [7] , resulting in biased results.
  • the mutated Mth RNA thermostable ligase (trade name: Thermostable 5′AppDNA&RNA Ligase) can also catalyze the ligation reaction between single-stranded nucleic acids at higher temperatures, but its ligation efficiency is lower when it is used for single-stranded DNA ligation reactions. Low [8] .
  • Another enzyme, Taq DNA ligase which can catalyze the ligation reaction between single-stranded DNA at higher temperature, has higher ligation efficiency, but it needs to use the complementary strand as a template as a guide, and cannot be used alone for ligation of single-stranded nucleic acids. reaction.
  • the transformation and modification of enzymes mainly include gene mutation, gene fusion, chemical modification, antibody modification and nucleic acid aptamer modification.
  • the genetic mutation of some ligases can change the characteristics of the ligase, improve the thermal stability or ligation efficiency of the ligase, and improve the salt tolerance or acid resistance of the ligase.
  • the mutation of the core lysine of Mth RNA ligase can make it directly use the pre-adenylated single-stranded DNA as the substrate for the ligation reaction, improving its ligation efficiency [8] .
  • HyperLigase Hyper-Thermostable Lysine-Mutatant ssDNA/RNA Ligase
  • HyperLigase WO2017160788A3
  • HyperLigase ssDNA/RNA Ligase has high thermal stability and can catalyze the ligation reaction between the 5' pre-adenylation substrate and the 3' hydroxyl terminal in a relatively high temperature range (such as 37-95°C). Nevertheless, the above genetically engineered ligase still has the defect that the connection efficiency is not high enough.
  • thermostable single-stranded nucleic acid ligases with higher ligation efficiency.
  • the present invention obtains the HyperLigase DNA/RNA ligase with new mutation sites on the basis of previous invention patents by screening potential gene mutation sites and carrying out gene mutation transformation. Compared with the original HyperLigase DNA/RNA Ligase ligase, these modified HyperLigase DNA/RNA Ligase ligases have higher enzymatic activity and improved their catalytic activity.
  • the present invention relates to the following aspects:
  • the present invention relates to a nucleic acid ligase comprising an amino acid sequence having mutations at one or more positions selected from positions 79, 281, 370 and 372 compared with the amino acid sequence of SEQ ID NO: 1.
  • the mutation at position 79 is that Arg is replaced by Ala
  • the mutation at position 281 is that Arg is replaced by Ala
  • the mutation at position 370 is that Lys is replaced by any natural amino acid except Lys
  • the mutation at position 372 is that Lys is replaced by any natural amino acid except Lys.
  • the mutation at position 372 is Lys replaced by Glu, Cys, Val, Ser, Gln, Ala, Leu, Thr or Phe.
  • the nucleic acid ligase of the present invention comprises an amino acid sequence selected from SEQ ID NOs: 2-7.
  • the nucleic acid ligase of the present invention comprises the amino acid sequence of SEQ ID NO: 2 or 3, wherein Xaa is a non-polar amino acid, preferably Ala. In one embodiment, the nucleic acid ligase of the present invention comprises the amino acid sequence of SEQ ID NO: 4 or 5.
  • the nucleic acid ligase of the present invention comprises the amino acid sequence of SEQ ID NO:6.
  • Xaa at positions 370 and 372 of SEQ ID NO: 6 is Ala.
  • the nucleic acid ligase of the present invention comprises the amino acid sequence of SEQ ID NO: 7, wherein Xaa at positions 79 and 281 are non-polar amino acids, preferably Ala. In one embodiment, Xaa at positions 370 and 372 of SEQ ID NO: 7 is a non-polar amino acid, preferably Ala.
  • the invention relates to nucleic acid molecules encoding the nucleic acid ligases of the invention.
  • the present invention relates to a vector comprising a nucleic acid molecule encoding a nucleic acid ligase of the present invention.
  • the invention relates to recombinant cells into which a nucleic acid of the invention or a vector of the invention has been introduced.
  • the present invention relates to a composition for ligating single-stranded DNA and/or RNA comprising the nucleic acid ligase of the present invention.
  • the present invention relates to a kit for ligating single-stranded DNA and/or RNA comprising the nucleic acid ligase of the present invention.
  • the present invention relates to the use of the nucleic acid ligase of the present invention in the preparation of products for ligating single-stranded DNA and/or RNA.
  • Figure 1 shows the electrophoresis identification of the mutant HyperLigase gene fragment amplified by inverse PCR (taking part of the results as an example).
  • Figure 2 shows the first-generation sequencing confirmation of wild-type and mutant HyperLigase bacterial fluids (taking the K370C site as an example).
  • Figure 3 shows the SDS-PAGE electrophoresis identification of the purified mutant HyperLigase (taking part of the results as an example).
  • Figure 4 shows the comparison of linear ligation reactivity between mutant HyperLigase and wild-type HyperLigase and CircLigase.
  • Each data is the mean of three independent repeated experiments; T-test; *: p ⁇ 0.05; **: p ⁇ 0.01, ***: p ⁇ 0.001.
  • Figure 5 shows the comparison of single-chain cyclization activity between mutant HyperLigase and wild-type HyperLigase.
  • Each data is the mean of three independent repeated experiments; T-test; *: p ⁇ 0.05; **: p ⁇ 0.01, ***: p ⁇ 0.001.
  • Figure 6 shows the comparison of linear ligation reactivity between mutant HyperLigase and wild-type HyperLigase.
  • Each data is the mean of three independent repeated experiments; T-test; *: p ⁇ 0.05; **: p ⁇ 0.01, ***: p ⁇ 0.001.
  • the diagram shows the summary of the cyclization efficiencies of all mutant Hyperligases sorted from high to low; each data is the average of three independent repeated experiments; T test; *: p ⁇ 0.05; **: p ⁇ 0.01, ** *: p ⁇ 0.001.
  • the figure shows the summary of the linear connection efficiency of all mutant Hyperligases sorted from high to low; each data is the average of three independent repeated experiments; T test; *: p ⁇ 0.05; **: p ⁇ 0.01, ** *: p ⁇ 0.001.
  • Figure 9 compares the linking activity between the wild-type Hyperligase and the K370P mutant on the RNA 3' hydroxyl end and the DNA 5' adenylation end, showing that the K370P mutation enhances Hyperligase's ability to bind the RNA 3' hydroxyl end to the DNA 5' adenylation ligation activity between the ends.
  • Figure 10 compares the ligation activity between wild-type Hyperligase and K370P mutants on DNA 3' hydroxyl end and RNA 5' adenylation end, showing that K370P mutation enhances Hyperligase's ability to bind DNA 3' hydroxyl end to RNA 5' adenylation ligation activity between the ends.
  • HyperLigase SEQ ID NO: i
  • the applicant screened out potential mutation sites through analysis and research, namely Arg79, Lys249, Lys370 and Lys372 of HyperLigase.
  • the NEBasechanger site-directed mutagenesis primer online design tool https://nebasechanger.neb.com
  • the wild-type HyperLigase protein SEQ ID NO: 1 expression plasmid as a template
  • high-fidelity DNA polymerase to carry out PCR reaction
  • the product is identified by electrophoresis to be consistent with the length of the amplicon, and then the fragments are purified (see Figure 1 ).
  • two sites K370 and K372 were selected for co-mutation to construct a double mutant K370A+K372A enzyme in which both sites were simultaneously mutated to alanine.
  • the detected mutant HyperLigase was stored in 50% glycerol solution at -20°C for a long time.
  • CircLigase is currently the single-stranded DNA ligase with the best activity known on the market.
  • a linear ligation reaction of intermolecular ligation was used to compare the activities of the two enzymes.
  • CircLigase linear connection reaction system is as follows:
  • HyperLigase linear ligation reaction system is as follows:
  • the HyperLigase reaction was carried out at 75°C, and the CingcLigase reaction was carried out at 60°C, and the reaction time was 6 hours. After the reaction, Urea-PAGE electrophoresis identification and gray scale analysis were performed to compare the activity difference of the enzymes (see Figure 4).
  • Embodiment 4 Mutant HyperLigase enzyme activity test
  • the reaction conditions were 60-75°C for 6 hours, and kept at 4°C.
  • Reaction product detection The cyclization product is identified by Urea-PAGE electrophoresis, the catalytic activity of the enzyme is characterized according to the change of the position of the electrophoretic band, and the ratio of the gray value of the cyclization product band to the substrate band in the catalytic reaction system of different mutant enzymes is compared Enzyme catalytic activity difference. The results are shown in Figure 5.
  • the reaction conditions were 60-75°C for 6 hours, and kept at 4°C.
  • the linear connection product is identified by Urea-PAGE electrophoresis, and the catalytic activity of the enzyme is characterized according to the change of the position of the electrophoretic band, according to the linear connection product band and the 3'-OH terminal substrate band in the catalytic reaction system of different mutant enzymes
  • the ratio of gray values compares the difference in catalytic activity of enzymes. The results are shown in Figure 6.
  • a 21-base RNA sequence is selected as the acceptor, and a 35-base 5'-end pre-adenylated and 3'-end blocked modified DNA is used as the donor, and the 3' hydroxyl end of the RNA and the 5' adenylyl of the DNA are carried out
  • the ligation test was carried out at the ends.
  • the reaction system is as follows:
  • reaction conditions 65°C, 30min/1h; 4°C, keep.
  • the reaction product was identified by Urea-PAGE electrophoresis
  • RNA sequence was selected as the donor, and a 55-base single-stranded DNA with a hydroxyl group at the 3' end was used as the acceptor, and the 5' adenylation end of the RNA was tested for connection with the 3' hydroxyl end of the DNA.
  • the reaction system is as follows:
  • Reaction conditions 65°C, 30min; 16°C, keep.
  • thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties[J]. 1): 135-42.

Abstract

提供了一种核酸连接酶，其包含与现有的Hyperligase的氨基酸序列（如SEQ ID NO：1所示）相比，在选自第79位、281位、370位和372位的一个或多个位置具有突变的氨基酸序列。又提供了编码该酶的核酸分子、包含该核酸分子的载体以及包含该核酸分子或该载体的重组细胞。还提供了包含该酶的组合物以及该酶的用途。

Description

核酸连接酶 技术领域
本发明涉及生物技术领域。具体涉及基于现有技术的核酸连接酶进行突变而得到的新的核酸连接酶。本发明还涉及包含所述酶的产品。
背景技术
核酸连接酶是一种金属离子依赖性酶，催化DNA或RNA相邻的3’末端与5’末端之间形成磷酸二酯键，实现底物样品的分子内环化或分子间线性连接反应。核酸连接酶根据催化底物的不同可分为DNA连接酶和RNA连接酶。部分连接酶可催化DNA与RNA之间的连接反应 [1，2]。根据物种来源的不同，天然的核酸连接酶具有多种独特的特性，如底物特异性、序列偏好性、热稳定性、耐盐、耐pH变化等。
一般认为，完整的核酸连接反应分为三个阶段进行 [1]。第一阶段，连接酶与ATP或NAD +的腺苷基团结合，将ATP或NAD +中的腺苷基团转移至酶的保守基序中的赖氨酸残基，释放出焦磷酸盐，并形成酶和腺苷的中间体。该步骤是一种平衡反应，能够双向进行。在第二阶段，酶和腺苷的中间体将其腺苷基团转移至核酸底物的5’磷酸末端，形成5’腺苷酰化的中间产物。该步骤也是一种平衡反向，能够双向进行。第三阶段，酶的赖氨酸催化位点与核酸的3’羟基末端结合，攻击核酸的5’腺苷酰化末端，催化二者之间形成磷酸二酯键，完成连接反应。常见的连接酶一般以5’磷酸化的核酸为底物，催化连接反应完整的三个阶段，实现连接反应 [3，4]。
连接酶是分子克隆技术、高通量测序文库制备、基因合成和分子诊断等常用分子生物学方法的基础，在分子生物学研究和分子诊断技术中占据重要地位 [5，6]。按照酶的反应温度的不同，可将连接酶分为热稳定型和非热稳定型两类。目前，用于双链DNA或RNA连接反应的连接酶较多，但用于单链DNA连接反应的酶仅有极其有限的选择，能够在较高温度下(如＞65℃)进行单链连接的热稳定连接酶更少。
目前已经有几种热稳定单链核酸连接酶报道。热稳定TS2126连接酶(商品名为CircLigase)能够直接在65℃催化5’磷酸末端与3’羟基末端之间的连接反应 [7]，但催化具有末端序列偏好性，导致底物的选择 性连接 [7]，造成结果的偏差。基因突变后的Mth RNA热稳定连接酶(商品名为Thermostable 5′AppDNA&RNA Ligase)同样能够在较高温度下催化单链核酸之间的连接反应，但其用于单链DNA连接反应时连接效率较低 [8]。另一种能够在较高温度下催化单链DNA之间连接反应的酶Taq DNA连接酶虽然具有较高的连接效率，但需要以互补链为模板作为引导，无法单独用于单链核酸的连接反应。
酶的改造和修饰主要有基因突变、基因融合、化学修饰、抗体修饰和核酸适配体修饰等方法。对部分连接酶进行基因突变改造能够改变连接酶的特性，提高连接酶的热稳定性或连接效率，改善连接酶的耐盐性或耐酸性等。对Mth RNA连接酶核心赖氨酸的突变能够使其直接以预腺苷酰化的单链DNA为底物进行连接反应，提高其连接效率 [8]。对超级耐热丁酸栖高温菌Hyperthermus butylicus来源的Hbut_1550基因编码的蛋白质核心赖氨酸进行基因突变赋予了该蛋白质单链DNA/RNA连接的能力，研究人员因此将其改造为热稳定DNA/RNA连接酶(被命名为Hyper-Thermostable Lysine-Mutatant ssDNA/RNA Ligase，以下简称HyperLigase，WO2017160788A3)。HyperLigase ssDNA/RNA Ligase具有很高的热稳定性，能够在较高的温度范围内(如37-95℃)催化5’预腺苷酰化底物与3’羟基末端之间的连接反应。尽管如此，上这一基因工程改造后的连接酶仍然存在连接效率不够高的缺陷。
因此，本领域仍然需要连接效率更高的热稳定单链核酸连接酶。
发明内容
本发明通过筛选潜在的基因突变位点并进行基因突变改造，在前人发明专利的基础上，获得了带有新的突变位点的HyperLigase DNA/RNA连接酶。相比于原有的HyperLigase DNA/RNA Ligase连接酶，这些经过改造的HyperLigase DNA/RNA Ligase连接酶具有更高的酶活性，提高了其催化活性。
具体地，本发明涉及以下方面：
一方面，本发明涉及核酸连接酶，其包含与SEQ ID NO：1的氨基酸序列相比，在选自79位、281位、370位和372位的一个或多个位置具有突变的氨基酸序列。在一个实施方案中，所述79位的突变是Arg替换为Ala，所述281位的突变是Arg替换为Ala，所述370位的 突变是Lys替换为除Lys外的任何天然氨基酸，并且所述372位的突变是Lys替换为除Lys外的任何天然氨基酸。在一个实施方案中，所述372位的突变是Lys替换为Glu、Cys、Val、Ser、Gln、Ala、Leu、Thr或Phe。
在一个实施方案中，本发明的核酸连接酶包含选自SEQ ID NOs：2-7的氨基酸序列。
在一个实施方案中，本发明的核酸连接酶包含SEQ ID NO：2或3的氨基酸序列，其中Xaa是非极性氨基酸，优选是Ala。在一个实施方案中，本发明的核酸连接酶包含SEQ ID NO：4或5的氨基酸序列。
在一个实施方案中，本发明的核酸连接酶包含SEQ ID NO：6的氨基酸序列。在一个实施方案中，SEQ ID NO：6的第370和372位的Xaa是Ala。
在一个实施方案中，本发明的核酸连接酶包含SEQ ID NO：7的氨基酸序列，其中第79和281位的Xaa是非极性氨基酸，优选是Ala。在一个实施方案中，SEQ ID NO：7的第370和372位的Xaa是非极性氨基酸，优选是Ala。
另一方面，本发明涉及编码本发明的核酸连接酶的核酸分子。
另一方面，本发明涉及载体，其包含编码本发明的核酸连接酶的核酸分子。
另一方面，本发明涉及重组细胞，其中引入了本发明的核酸或本发明的载体。
另一方面，本发明涉及用于连接单链DNA和/或RNA的组合物，其包含本发明的核酸连接酶。
另一方面，本发明涉及用于连接单链DNA和/或RNA的试剂盒，其包含本发明的核酸连接酶。
另一方面，本发明涉及本发明的核酸连接酶在制备用于连接单链DNA和/或RNA的产品中的用途。
附图说明
图1显示反向PCR扩增突变型HyperLigase基因片段电泳鉴定(以部分结果为例)。
图2显示野生型与突变型HyperLigase菌液一代测序确认(以 K370C位点为例)。
图3显示纯化后突变型HyperLigase的SDS-PAGE电泳鉴定(以部分结果为例)。
图4显示突变型HyperLigase与野生型HyperLigase以及CircLigase的线性连接反应活性比较。每个数据均为三次独立重复实验的平均值；T检验；*：p＜0.05；**：p＜0.01，***：p＜0.001。
图5显示突变型HyperLigase与野生型HyperLigase单链环化反应活性比较。每个数据均为三次独立重复实验的平均值；T检验；*：p＜0.05；**：p＜0.01，***：p＜0.001。
图6显示突变型HyperLigase与野生型HyperLigase线性连接反应活性比较。每个数据均为三次独立重复实验的平均值；T检验；*：p＜0.05；**：p＜0.01，***：p＜0.001。
图7显示所有突变型Hyperligase环化效率汇总(N＝3)。图示所有突变型Hyperligase的环化效率按照从高到低排序的汇总；每个数据均为三次独立重复实验的平均值；T检验；*：p＜0.05；**：p＜0.01，***：p＜0.001。
图8显示所有突变型Hyperligase线性连接效率汇总(N＝3)。图示所有突变型Hyperligase的线性连接效率按照从高到低排序的汇总；每个数据均为三次独立重复实验的平均值；T检验；*：p＜0.05；**：p＜0.01，***：p＜0.001。
图9比较了野生型Hyperligase与K370P突变体对RNA 3’羟基末端与DNA 5’腺苷酰化末端之间的连接活性，显示K370P突变增强Hyperligase对RNA 3’羟基末端与DNA 5’腺苷酰化末端之间的连接活性。
图10比较了野生型Hyperligase与K370P突变体对DNA 3’羟基末端与RNA 5’腺苷酰化末端之间的连接活性，显示K370P突变增强Hyperligase对DNA 3’羟基末端与RNA 5’腺苷酰化末端之间的连接活性。
具体实施方式
提供以下实施例以展示优选实施方案。本领域的技术人员应认识到下文实施例中公开的技术代表本发明人发现的在本文公开方法的实 施中功能良好的技术，因此可认为其构成实施的优选模式。然而，按照本公开，本领域的技术人员将认识到在公开的具体实施方案中能进行不脱离本文公开方法的实质和范围的许多改变，并且仍然获得同样的或类似的结果。
实施例1.定点突变技术构建突变型HyperLigase蛋白表达质粒
从现有技术的HyperLigase(SEQ ID NO：i)出发，申请人经过分析研究，筛选出潜在突变位点，即HyperLigase的Arg79、Lys249、Lys370和Lys372。
根据已筛选的突变位点及拟改造成的对应氨基酸，使用NEBasechanger定点突变引物在线设计工具(https：//nebasechanger.neb.com)进行背靠背点突变引物设计。合成相应引物后，以野生型HyperLigase蛋白(SEQ ID NO：1)表达质粒为模板，使用高保真DNA聚合酶进行PCR反应，产物经电泳鉴定与扩增子长度一致后进行片段纯化(见图1)。纯化后DNA经末端磷酸化和自身连接环化，产物转化至BL21(DE3)感受态细胞，涂布在含有抗生素的平板上，挑选抗生素筛选得到的菌落并测序确认序列正确(见图2)，获得突变型HyperLigase蛋白表达质粒。上述过程均使用商品化试剂并按照试剂盒说明书操作。HyperLigase蛋白质与标签蛋白His标签共同表达，标签位于目的蛋白的N端或C端。
除上述单独突变外，选择K370与K372两位点进行共突变构建两位点同时突变为丙氨酸的双突变K370A+K372A酶。
实施例2.蛋白诱导表达与纯化
自平板上挑取阳性菌落接种至LB培养基内进行复苏，复苏后菌液10～50倍继续扩大培养，转至1L LB培养基培养至OD600吸光度0.6～0.8，使菌液达对数生长期，加入IPTG诱导剂至终浓度0.1～1mmol/L，14～37℃条件下诱导培养6～24h。收集菌体沉淀后经超声处理破碎菌体，对超声破碎产物进行纯化。纯化使用手工法或仪器法，按照仪器设备指南进行，洗脱后蛋白测定浓度后转化为摩尔浓度，并使用SDS-PAGE进行纯度鉴定(见图3)。检测后的突变型HyperLigase于50％甘油溶液中-20℃长期保存。
实施例3.
突变后HyperLigase酶与原Hyperligase和CircLigase酶活性比较
CircLigase是市场上目前已知活性最佳的单链DNA连接酶，为测试突变前后HyperLigase与CircLigase活性的差异，使用分子间连接的线性连接反应进行两酶活性比较。
CircLigase线性连接反应体系如下：
Figure PCTCN2022077538-appb-000001
HyperLigase线性连接反应体系如下：
Figure PCTCN2022077538-appb-000002
HyperLigase反应在75℃条件下进行，CingcLigase反应在60℃条件下进行，反应时间均为6h，反应结束Urea-PAGE电泳鉴定和灰度分析比较酶的活性差异(见图4)。
测试结果显示，K370P突变、K372E突变、K370C突变均明显提升了HyperLigase的催化活性，且突变后的HyperLigase活性明显高于CircLigase。
实施例4.突变型HyperLigase酶活性测试
(1)环化效率测试
使用合成的预腺苷酰化底物，按下表制备分子内连接的环化反应 体系：
Figure PCTCN2022077538-appb-000003
反应条件为60～75℃反应6小时，4℃保持。
反应产物检测：环化产物经Urea-PAGE电泳鉴定，按照电泳条带位置的变化表征酶的催化活性，根据不同突变酶催化反应体系中环化产物条带与底物条带灰度值的比例比较酶的催化活性差异。结果见图5。
单链环化反应结果显示，R79、R281、K370、K372四位点氨基酸突变为丙氨酸后明显提高酶的活性，K370和K372联合突变为丙氨酸提高酶的催化活性。同时，对于K370和K372位点，赖氨酸突变为其他氨基酸均可提高酶的催化活性。
(2)线性连接效率测试
使用合成的5’端腺苷酰化、3’末端含有阻断修饰的底物，与3’末端为羟基的底物，按下表制备线性连接反应体系：
Figure PCTCN2022077538-appb-000004
反应条件为60～75℃反应6小时，4℃保持。
反应产物检测：线性连接产物经Urea-PAGE电泳鉴定，按照电泳条带位置的变化表征酶的催化活性，根据不同突变酶催化反应体系中线性连接产物条带与3’-OH末端底物条带灰度值的比例比较酶的催化 活性差异。结果见图6。
线性连接反应测试结果显示，R79、R281、K370、K372四位点氨基酸突变为丙氨酸或K370和K372联合突变为丙氨酸后明显提高酶的活性。同时，对于K370和K372位点，赖氨酸突变为其他氨基酸均可提高酶的催化活性。
进一步，对K370和K372位点突变为其他氨基酸，结果显示该两位点突变为任意氨基酸后，活性均较野生型HyperLigase明显提高(图5、6)。
对于上述突变位点的活性测试，线性连接反应结果与单链环化反应结果一致。
综上所述，R79、R281、K370、K372四位点氨基酸突变或K370和K372联合突变明显提高酶的活性。同时，对于K370和K372位点，赖氨酸突变为其他氨基酸均可提高酶的催化活性(参见图7、图8)。
实施例5
K370P突变核酸连接酶对RNA的3’-羟基末端与DNA的5’腺苷酰化末端的连接活性测试
选择21碱基的RNA序列作为受体，35碱基5’末端预腺苷酰化且3’末端阻断修饰的DNA作为供体，进行RNA的3’羟基末端与DNA的5’腺苷酰化末端进行连接测试。反应体系如下：
Figure PCTCN2022077538-appb-000005
反应条件：65℃，30min/1h；4℃，保持。反应产物进行Urea-PAGE电泳鉴定
结果如图9所示，其中显示K370P突变增强Hyperligase对RNA 3’ 羟基末端与DNA 5’腺苷酰化末端之间的连接活性。从图中可以看出K370P的连接产物条带深度有少量增加，且AppDNA的底物条带相较于野生型更弱，说明更多的底物被用于连接，由此说明酶的连接活性有所提升。
实施例7
K370P突变核酸连接酶对DNA的3’羟基末端与RNA的5’腺苷酰化末端的连接活性测试
选择28碱基的RNA序列作为供体，3’末端为羟基的55碱基单链DNA作为受体，进行RNA的5’腺苷酰化末端与DNA的3’羟基末端进行连接测试。反应体系如下：
Figure PCTCN2022077538-appb-000006
反应条件：65℃，30min；16℃，保持。
结果如图10所示，其中显示K370P突变增强Hyperligase对DNA3’羟基末端与RNA 5’腺苷酰化末端之间的连接活性。可以明显看出K370P的OH-DNA底物量减少，AppRNA条带几乎全部用尽，说明更多的底物被用于连接。但是连接产物条带没有明显更亮。这是因为连接产物中RNA被降解，K370P的连接产物的降解条带比野生型更亮。
参考文献：
[1]VIOLLET S，FUCHS R T，MUNAFO D B，et al.T4 RNA ligase 2 truncated active site mutants：improved tools for RNA analysis[J].BMC Biotechnol，2011，11(72.
[2]LOHMAN G J，TABOR S，NICHOLS N M.DNA ligases[J]. Curr Protoc Mol Biol，2011，Chapter 3(Unit314.
[3]JOHNSON A，O′DONNELL M.DNA ligase：Getting a grip to seal the deal[J].Current Biology，2005，15(3)：R90-R2.
[4]CHEREPANOV A V，DE VRIES S.Dynamic mechanism of nick recognition by DNA ligase[J].European Journal of Biochemistry，2002，269(24)：5993-9.
[5]TUMBALE P P，JURKIW T J，SCHELLENBERG M J，et al.Two-tiered enforcement of high-fidelity DNA ligation[J].Nature Communications，2019，10(1)：5431.
[6]FOY C A，PARKES H C.Emerging Homogeneous DNA-based Technologies in the Clinical Laboratory[J].Clinical Chemistry，2001，47(6)：990-1000.
[7]BLONDAL T，THORISDOTTIR A，UNNSTEINSDOTTIR U，et al.Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties[J].Nucleic Acids Res，2005，33(1)：135-42.
[8]ZHELKOVSKY A M，MCREYNOLDS L A.Structure-function analysis of Methanobacterium thermoautotrophicum RNA ligase-engineering a thermostable ATP independent enzyme[J].BMC Molecular Biology，2012，13(1)：10.

Claims (14)

  1. 核酸连接酶，其包含与SEQ ID NO：1的氨基酸序列相比，在选自79位、281位、370位和372位的一个或多个位置具有突变的氨基酸序列。
  2. 权利要求1的核酸连接酶，其中所述79位的突变是Arg替换为Ala，所述281位的突变是Arg替换为Ala，所述370位的突变是Lys替换为除Lys外的任何天然氨基酸，并且所述372位的突变是Lys替换为除Lys外的任何天然氨基酸。
  3. 权利要求1的核酸连接酶，其包含选自SEQ ID NOs：2-7的氨基酸序列。
  4. 权利要求3的核酸连接酶，其包含SEQ ID NO：2或3的氨基酸序列，其中Xaa是非极性氨基酸，优选是Ala；或者，其包含SEQ ID NO：4或5的氨基酸序列。
  5. 权利要求3的核酸连接酶，其包含SEQ ID NO：6的氨基酸序列。
  6. 权利要求5的核酸连接酶，其中第370和372位的Xaa是Ala。
  7. 权利要求3的核酸连接酶，其包含SEQ ID NO：7的氨基酸序列，其中第79和281位的Xaa是非极性氨基酸，优选是Ala。
  8. 权利要求7的核酸连接酶，其中第370和372位的Xaa是非极性氨基酸，优选是Ala。
  9. 核酸分子，其编码权利要求1-8的任一项的核酸连接酶。
  10. 载体，其包含权利要求9的核酸分子。
  11. 重组细胞，其中引入了权利要求9的核酸或权利要求10的载体。
  12. 用于连接单链DNA和/或RNA的组合物，其包含权利要求1-8的任一项的核酸连接酶。
  13. 用于连接单链DNA和/或RNA的试剂盒，其包含权利要求1-8的任一项的核酸连接酶。
  14. 权利要求1-8的任一项核酸连接酶在制备用于连接单链DNA和/或RNA的产品中的用途。
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