Quantitative and Site-specific Analysis of Adenosine-to-inosine RNA Editing by Ligation-assisted qPCR  

作　　者：TAO Wanbing GU Shuyi XIONG Jun YUAN Bifeng 

机构地区：[1]Department of Occupational and Environmental Health,School of Public Health,Research Center of Public Health,Renmin Hospital of Wuhan University,Wuhan University,Wuhan 430071,P.R.China [2]Department of Radiation and Medical Oncology,Zhongnan Hospital of Wuhan University,Wuhan University,Wuhan 430071,P.R.China [3]College of ChemistryandMolecular Sciences,Wuhan University,Wuhan 430072,P.R.China

出　　处：《Chemical Research in Chinese Universities》2025年第2期288-295,共8页高等学校化学研究(英文版)

基　　金：supported by the National Key R&D Program of China(No.2022YFC3400700);the National Natural Science Foundation of China(Nos.22074110,22277093,22207090);the Key Research and Development Project of Hubei Province,China(No.2023BCB094);the Translational Medicine and Interdisciplinary Research Joint Fund of Zhongnan Hospital of Wuhan University,China(Nos.ZNJC202208,ZLJC2022001).

摘　　要：RNA molecules undergo a variety of modifications,including inosine modification,also called adenosine-to-inosine(A-to-I)RNA editing,which is prevalent across all domains of life.To unravel the roles of A-to-I RNA editing,it is essential to accurately quantify inosine in RNA at specific sites.Here,we developed a ligation-assisted qPCR(LA-PCR)method for the quantitative and site-specific analysis of A-to-I RNA editing.In LA-PCR,adenosine on an edit site pairs with thymidine.In contrast,inosine fails to pair with thymidine,disrupting the nick ligation of the two DNA probes located upstream and downstream from the editing site.The reduction in the liaged products can be quantified through subsequent qPCR,thus enabling the quantification of the A-to-I RNA editing level.The LA-PCR approach was successfully employed to detect and quantify the A-to-I RNA editing at position 2814 in Ino80dos RNA from mouse tissues.A notable elevation in A-to-I RNA editing levels was found across various tissues from sleep-deprived mice in comparison to control mice,suggesting a potential association between A-to-I RNA editing and sleep behavior.The proposed method facilitates the quantitative analysis of A-to-I RNA editing at specific sites,aiding in the elucidation of the functions and mechanisms of A-to-I RNA editing.

关 键 词：A-to-I RNA editing INOSINE RNA modification Quantification Site-specific detection 

分 类 号：Q522[生物学—生物化学]