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

doi:10.1007/s40242-024-4165-7

Chemical Research in Chinese Universities ›› 2025, Vol. 41 ›› Issue (2): 288-295.doi: 10.1007/s40242-024-4165-7

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

TAO Wanbing^1,2,3, GU Shuyi¹, XIONG Jun¹, YUAN Bifeng^1,2,3

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 Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China

Received:2024-07-15 Accepted:2024-08-16 Online:2025-04-01 Published:2025-03-31
Contact: XIONG Jun,jxiong@whu.edu.cn;YUAN Bifeng,bfyuan@whu.edu.cn E-mail:jxiong@whu.edu.cn;bfyuan@whu.edu.cn
Supported by:
This work was 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), and the Translational Medicine and Interdisciplinary Research Joint Fund of Zhongnan Hospital of Wuhan University, China (Nos. ZNJC202208, ZLJC2022001).

Abstract

Abstract: 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.

Key words: A-to-I RNA editing, Inosine, RNA modification, Quantification, Site-specific detection

TAO Wanbing, GU Shuyi, XIONG Jun, YUAN Bifeng. Quantitative and Site-specific Analysis of Adenosine-to-inosine RNA Editing by Ligation-assisted qPCR[J]. Chemical Research in Chinese Universities, 2025, 41(2): 288-295.

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

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