Novel thermostable phosphorylation and adenylation one-step catalytic enzyme, and preparation method and application thereof

Abstract

The invention relates to a novel thermostable phosphorylation and adenylation one-step catalytic enzyme, and a preparation method and application thereof. The novel thermostable phosphorylation and adenylation one-step catalytic enzyme is characterized in that two genes for coding T4 PNK and Mth RNA Ligase are subjected to genetic engineering modification, and a Linker is introduced into two coding genes to prepare a fusion protein of T4 PNK and Mth RNA Ligase. Activity test and identification show that the fusion protein has the catalytic activity of the T4 PNK and the Mth RNA Ligase at the same time, a hydroxyl terminal substrate can be innovatively and directly catalyzed into an adenylation product, and a phosphorylation substrate can still be catalyzed into the adenylation product.

Description

technical field [0001] The invention relates to the technical field of adenylylation modification of nucleic acids, in particular to a novel thermostable phosphorylation and adenylylation one-step catalyzing enzyme and its preparation method and application. Background technique [0002] Phosphorylation at the end of a nucleic acid sequence refers to the process in which a phosphate group is covalently attached to the end of a nucleic acid. Adenylation of nucleic acid refers to the process in which adenosine monophosphate (AMP) molecules are covalently attached to the ends of nucleic acids. Phosphorylated nucleic acid is a nucleic acid containing a 5'-phosphate group, including phosphorylated DNA and phosphorylated RNA. It is a substrate for various nucleic acid ligases to catalyze nucleic acid cyclization or ligation reactions, and is widely used in molecular biology. [0003] Adenylylated nucleic acid is a nucleic acid containing a 5'-adenosine pyrophosphate cap with a hi...

AI Technical Summary

Problems solved by technology

[0021] (1) The scheme of directly synthesizing adenylylated DNA or RNA by chemical synthesis requires multi-step reactions, the steps are cumbersome, and technicians with high professional technical knowledge and operational ability are required to perform the synthesis, which is not suitable for ordinary laboratories Independent preparation; (2) The price of synthetic raw materials is high; (3) The reaction efficiency is not high when synthesizing DNA or RNA larger than 11 bases, and the product needs to be separated from the substrate and the product with a difference of 1 base by PAGE electrophoresis However, the purification process is time-consuming and laborious, further increasing the cost of synthesis

(4) At present, there is no domestic company that provides direct chemical synthesis of adenylylated DNA or RNA, and ordering from foreign synthesis companies is expensive and has a long lead time

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