Method for obtaining substrate sequence efficiently bound in ADAR protein cells, substrate sequence and application

Abstract

The invention discloses a method for obtaining a substrate sequence efficiently bound in ADAR protein cells, the substrate sequence and application. The method comprises the following steps: S1, cellpreparation and cross-linking reaction of protein and RNA; S2, co-immunoprecipitation and double-stranded RNA digestion, linking and reverse transcription; S3, cDNA cyclization, library building and high-throughput sequencing; S4, ADAR substrate assembly; and S5, evaluation of an efficient binding substrate. The invention develops a method irCLASH for obtaining the substrate sequence efficiently bound in ADAR protein cells for the first time, the blank of the existing ADAR1 binding substrate research is filled up from the perspectives of experiments and bioinformatics, compared with other existing methods for researching the interaction between protein and RNA, the method has the advantages that the yield of chimeric reads can be remarkably increased, the construction number of ADAR protein acting substrates can be increased to the maximum extent, meanwhile, the experiment consumed time is shortened, and the experiment operation is simplified.

Description

technical field [0001] The present invention relates to the technical field of molecular biology, specifically, to the technical field of protein-RNA interaction, and more specifically, to a method for acquiring and locating substrate sequence information for efficient binding of ADAR proteins in cells and a substrate sequence, for site-specific targeted RNA editing. Background technique [0002] Gene mutation is the key cause of many diseases and cancers. At present, there are more than 3,000 gene-specific mutations associated with the occurrence of diseases. With the development of next-generation sequencing technology, whether it is genetically acquired gene mutations or acquired growth Gene mutations in certain somatic cells during development can be obtained through next-generation sequencing technology, and mutation sites directly related to the occurrence of diseases can be found through association analysis. However, at present, various tumors have different mutatio...

AI Technical Summary

Problems solved by technology

However, due to the lack of double-stranded RNA substrate research on ADAR, it is difficult to use ADAR protein combined with adRNA to repair disease-related G-to-A mutations.

At the same time, the current methods used to study the interaction between protein and RNA, such as hiCLIP and CLASH, etc., have a small proportion of chimeras obtained and take a long time to experiment, and cannot be effectively used to obtain information on the substrates of ADAR proteins

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