Novel phosphorylated adenosine acylase as well as preparation method and application thereof

Abstract

The invention relates to a novel phosphorylated adenosine acylase and a preparation method and application thereof, prokaryotic inducible expression plasmid of Adenylase (PF0353) and T4 PNK (T4 Polynucleotide Kinase) fusion enzyme is constructed, the target fusion enzyme is obtained through expression and purification, the enzyme is high in water solubility and simple in expression and purification, the obtained fusion enzyme has the activity of T4 polynucleotide kinase and adenosine acylase at the same time, the 5'-hydroxy DNA can be subjected to the direct catalytic reaction in the presence of ATP to obtain the 5'-adenylate acylation DNA by using the one-step method, and the method for preparing the adenylate acylation DNA by using the fusion enzyme has advantages of low cost, high reaction efficiency, simpleness, convenience and rapidness, and can be flexibly used by the molecular biological technology.

Description

Technical field [0001] The present invention specifically relates to the field of adenosine acylation modification of nucleic acids, and more particularly to a novel phosphorylated adenproductase and preparation method thereof. Background technique [0002] Adenylation of the nucleic acid refers to a process of covalently attached to the end of the nucleic acid by a phosphate adenosine (AMP) molecule. Adenylated nucleic acids are nucleic acids containing a 5'5'-adenosine pyrophosphate-based cap, an intermediate product of nucleic acid ligase catalyzed, has a high energy pyrophosphate bond, a nucleic acid molecule in an activation form, in Molecular biology has more applications. Adenylated nucleic acids include both adenylated DNA and adenylated RNA, and is currently more applications with adenylation DNA (APPDNA). [0003] AppDNA is widely used in RNA sequencing libraries (especially miRNA sequencing library) joints, single-stranded DNA connections, specific ligase catalytic lig...

AI Technical Summary

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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