A method for obtaining a highly efficient binding substrate sequence of adar protein in cells, as well as the substrate sequence and application

Abstract

The invention discloses a method for obtaining a highly efficient intracellular binding substrate sequence of ADAR protein, as well as the substrate sequence and application. The method comprises the following steps: S1. cell preparation and protein and RNA cross-linking reaction; S2. co-immunoprecipitation and double-stranded RNA digestion, ligation, and reverse transcription; S3. cDNA circularization, library construction and high-throughput sequencing; S4. ADAR substrate assembly; S5. Evaluation of efficient binding substrates. The present invention firstly develops a method irCLASH to obtain the highly efficient binding substrate sequence of ADAR protein in the cell. Compared with the RNA interaction method, it can significantly increase the yield of chimera reads, maximize the number of ADAR protein substrates, shorten the time-consuming experiment, and simplify the experimental operation.

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 the experiment takes a long time, and they cannot be effectively used to obtain information on the substrates of ADAR proteins

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