T4 RNA ligase 2 mutants and preparation method and use thereof
Provided are T4 RNA ligase 2 mutants and preparation method and use thereof, relating to the field of biology and enzyme engineering. The T4 ligase 2 mutants have mutation at least one site of positions 62, 103, 166, 168, 193, 217, 260, 297, 298, 303, 306, 311, 313 and 318 of sequence of the wild-type T4 RNA ligase 2. Compared with the wild type, the mutants have remarkably improved thermal stability, and can maintain relatively high ligase activity at higher temperatures such as 45° C., and 50° C. In addition to ligating natural nucleic acid sequences, the mutants further can ligate non-natural nucleic acids with modification at high temperatures. The mutants can synthesize ribonucleic acids at elevated temperatures, so as to effectively reduce the production of byproducts caused by base mismatching, thus improving the efficiency of RNA synthesis, and facilitating large-scale production of high-quality RNAs.
1 . A T4 RNA ligase 2 mutant, comprising compared with wild-type T4 RNA ligase 2 mutation at any one or more sites of positions 62, 103, 166, 168, 193, 217, 260, 297, 298, 303, 306, 311, 313 and 318 of an amino acid sequence of the wild-type T4 RNA ligase 2, wherein the T4 RNA ligase is at least 85% sequence identity to SEQ ID NO: 1 or 2:
the amino acid sequence of the wild-type T4 RNA ligase 2 is as set forth in SEQ ID NO: 1 or 2; and
wherein the T4 RNA ligase 2 is an RNA ligase isolated from T4 bacteriophage.
2 . The T4 RNA ligase 2 mutant according to claim 1 , wherein the mutation at position 62 comprises A62W;
the mutation at position 103 comprises P103G;
the mutation at position 166 comprises N166P;
the mutation at position 168 comprises L168F;
the mutation at position 193 comprises N193K;
the mutation at position 217 comprises any one of R217A, R217Q, R217N and R217P;
the mutation at position 260 comprises C260D;
the mutation at position 297 comprises T297A;
the mutation at position 298 comprises S298E;
the mutation at position 303 comprises T303D;
the mutation at position 306 comprises any one of Q306A, Q306D and Q306E;
the mutation at position 311 comprises any one of S311E, S311D, S311A and S311V; and
the mutation at position 313 comprises I313V.
3 . The T4 RNA ligase 2 mutant according to claim 2 , wherein the T4 RNA ligase 2 mutant has any one or a combination of following mutation combinations relative to the wild-type T4 RNA ligase 2: N193K-S311D, N193K-R217P, N193K-T303D, N193K-T297A, T297A-Q306D, N193K-I313V, R217P-S298E, R217P-T297A, R217P-I313V, and S311E-I313V.
4 . The T4 RNA ligase 2 mutant according to claim 2 , wherein the T4 RNA ligase 2 mutant has any one or a combination of following mutation combinations relative to the wild-type T4 RNA ligase 2: R217P-T297A-S311E, R217P-S298E-S311D, R217P-T297A-I313V, N193K-R217P-C260D, R217P-S311E-I313V, R217P-T303D-I313V, N193K-R217P-T303D-S311E, N193K-R217P-T303D-I313V, N193K-R217P-T303D-V318T, N193K-R217P-S311E-I313V, N193K-R217P-T303D-S311E-I313V, A62W-N193K-R217P-T303D-S311E-I313V, P103G-N193K-R217P-T303D-S311E-I313V, N166P-N193K-R217P-T303D-S311E-I313V, L168F-N193K-R217P-T303D-S311E-I313V and N193K-R217P-C260D-T303D-S311E-I313V-V318T, L168F-N193K-R217P-C260D-S311E-T303D-I313V-V318T, A62W-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, P103G-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, A62W-P103G-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, A62W-P103G-N166P-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, A62W-N166P-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, P103G-N166P-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T and N166P-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T.
5 . The T4 RNA ligase 2 mutant according to claim 2 , wherein the T4 RNA ligase 2 mutant has any one or a combination of following mutations relative to the wild-type T4 RNA ligase 2: A62W, P103G, N166P, L168F, R217P, R217A, R217N, R217Q and C260D.
6 . The T4 RNA ligase 2 mutant according to claim 4 , wherein the T4 RNA ligase 2 mutant has any one of following mutation or combinations relative to the wild-type T4 RNA ligase 2: N193K-R217P-C260D, N193K-R217P-S311E-I313V, N193K-R217P-T303D-S311E-I313V, L168F-N193K-R217P-T303D-S311E-I313V and N193K-R217P-C260D-T303D-S311E-I313V-V318T, L168F-N193K-R217P-C260D-S311E-T303D-I313V-V318T, A62W-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, P103G-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, A62W-P103G-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, A62W-P103G-N166P-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, A62W-N166P-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T, P103G-N166P-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T and N166P-L168F-N193K-R217P-C260D-T303D-S311E-I313V-V318T.