A novel phosphorylated adenylase and its preparation method and application

Abstract

A new type of phosphorylated adenylase and its preparation method and application, a prokaryotic inducible expression plasmid of Adenylase (PF0353) and T4 PNK (T4 Polynucleotide Kinase) fusion enzyme was constructed, and the target fusion enzyme was obtained through expression and purification, The enzyme has strong water solubility, simple expression and purification, and the obtained fusion enzyme has both the activities of T4 polynucleotide kinase and adenylase, and can directly catalyze the reaction of 5'-hydroxy DNA in the presence of ATP to obtain 5'-hydroxyl DNA in one step. 'Adenylylated DNA. The method for preparing adenylated DNA by using the fusion enzyme has low cost, high reaction efficiency, simplicity and rapidity, and can be used flexibly in molecular biology techniques.

Description

technical field [0001] The present invention specifically relates to the technical field of adenylation modification of nucleic acid, in particular to a novel phosphorylated adenylase and a preparation method and application thereof. Background technique [0002] Nucleic acid adenylation (Adenylation) refers to the process of covalent attachment of adenosine monophosphate (AMP) molecules to the ends of nucleic acids. Adenylated nucleic acid is a nucleic acid containing a 5'5'-adenosine pyrophosphate cap. It is an intermediate product of nucleic acid ligation catalyzed by nucleic acid ligase. It has a high-energy pyrophosphate bond and is an activated form of nucleic acid molecule. It has many applications in molecular biology. Adenylated nucleic acids include adenylated DNA and adenylated RNA, and currently, adenylated DNA (AppDNA) is widely used. [0003] AppDNA is widely used in molecular biology applications such as adapter preparation for RNA sequencing libraries (espe...

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Problems solved by technology

[0019] (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 carry out 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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