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

A protein and substrate technology, applied in fixed-point targeted RNA editing, locating the substrate sequence information that ADAR protein binds efficiently in cells, and obtaining field, can solve the problem of inability to effectively use ADAR protein to act on substrate information acquisition, and it is difficult to obtain widely The clinical application and the low proportion of chimeras can achieve the effects of shortening the experimental time, increasing the yield, and increasing the number of constructions

Active Publication Date: 2022-03-04
时夕(广州)生物科技有限公司
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A method for obtaining a highly efficient binding substrate sequence of adar protein in cells, as well as the substrate sequence and application
  • A method for obtaining a highly efficient binding substrate sequence of adar protein in cells, as well as the substrate sequence and application
  • A method for obtaining a highly efficient binding substrate sequence of adar protein in cells, as well as the substrate sequence and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of ADAR1 Protein High Affinity Substrate Sequence and RNA Level Targeted Editing in HEK293 Cell Line

[0042] The overall process of the experimental method of the present invention is as follows: figure 1 As shown, the experimental sequencing library data analysis process is as follows figure 2 As shown, compared with other published methods, the results show that the method of the present invention greatly shortens the time-consuming experiments (such as image 3 ); the present embodiment takes the ADAR protein family ADAR1 protein as an example to further illustrate the present invention; specifically, the method includes the following steps:

[0043] 1. Cell preparation and protein and RNA cross-linking reaction

[0044] HEK293 cells were cultured in a 15 cm cell culture dish and overexpressed FLAG-tagged ADAR1. After 48 hours, the cells were washed with PBS and the cross-linking reaction of protein and RNA was performed on a 254 nm ultravi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6809
CPCC12Q1/6809C12N15/11C12N15/113C12Q2531/113C12Q2525/191C12Q2535/122C12Q2537/165C12Q2521/327
Inventor 张锐杨文兵宋玉龙
Owner 时夕(广州)生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap