RNA polymerase mutants that can utilize chemically modified nucleotides

Abstract

The present invention provides a T7 RNA polymerase mutant by introducing a novel mutation, which is selected from: the arginine at position 632 in the amino acid sequence constituting the wild-type T7 RNA polymerase shown in SEQ ID NO: 1 is replaced by a cysteine Amino acid substituted mutant (R632C), the T7 RNA polymerase mutant has DNA-dependent RNA polymerase activity, and compared with the wild-type T7 RNA polymerase, the T7 RNA polymerase mutant can be modified by various 2' three Nucleoside phosphates are used as synthetic substrates. As well as methods and kits for synthesizing the mutants, nucleic acids containing one or more modified nucleotides.

Description

[0001] field of invention [0002] The invention relates to the field of enzyme engineering, in particular the invention relates to an improved mutant of T7 RNA polymerase, and the mutation leads to an improvement in the performance of the enzyme in synthesizing RNA by using modified nucleoside triphosphates. Background technique [0003] T7 RNA polymerase (E.C. 2.7.7.6.) is a monomeric, phage-encoded, DNA-directed RNA polymerase that catalyzes RNA formation in the 5'→3' direction. During transcription initiation, T7 recognizes a specific promoter sequence, the T7 promoter. T7 consists of 883 amino acids and has a molecular weight of 99 kDa. At the amino acid sequence level, T7 has a high degree of homology with T3 RNA polymerase and a lesser degree of homology with SP6 RNA polymerase. The three-dimensional structure of T7 is very similar to other polymerases with different template and substrate specificities, despite low sequence similarity. T7 consists of different domai...

AI Technical Summary

Problems solved by technology

Unfortunately, wild-type T7 RNA polymerase (T7RNAP), the enzyme commonly used in this RNA in vitro transcription synthesis reaction, incorporates chemically modified nucleotides very inefficiently

Therefore, protein modification of T7 RNA polymerase through genetic engineering to find T7 RNA polymerase mutants that can efficiently incorporate 2'-O-methyl nucleotides is a solution to the current inability to generate 2' in large quantities through enzymatic synthesis. The key to the '-O-methyl-modified RNA problem'

[0011] The earliest T7 RNA polymerase mutants designed by protein engineering that can incorporate 2'-O-methyl nucleotides, such as E593G / V685A, although they can incorporate 2'-OMe-ATP, 2'-OMe CTP, 2 Three kinds of 2'-O-methyl nucleotides such as '-OMe UTP, but cannot be incorporated into 2'-OMe GTP, so it is impossible to synthesize completely 2'-O-methyl modified RNA molecules, and due to the continuous Reduced synthesis ability leads to very low efficiency of enzymatic synthesis of 2'-O-methyl-modified RNA, and only short-chain products can be synthesized

Although the R425C mutant discovered by Kennedy et al. solved the problem of 2'-OMe GTP incorporation, its synthesis efficiency is still very low, which is far from meeting the needs of in vitro transcription and synthesis of a large amount of complete 2'-O-methyl modified RNA.

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