Site-specific quantification of 5-carboxylcytosine in DNA by chemical conversion coupled with ligation-based PCR  

作　　者：Qian Wang Jiang-Hui Ding Jun Xiong Yang Feng Bi-Feng Yuan Yu-Qi Feng 

机构地区：[1]Sauvage Center for Molecular Sciences,Department of Chemistry,Wuhan University,Wuhan 430072,China [2]School of Health Sciences,Wuhan University,Wuhan 430071,China

出　　处：《Chinese Chemical Letters》2021年第11期3426-3430,共5页中国化学快报（英文版）

基　　金：supported by the National Natural Science Foundation of China(Nos.22074110,21635006,21721005);the Fundamental Research Funds for the Central Universities(2042021 kf0212).

摘　　要：5-Methylcytosine(5mC)is the most important epigenetic modification in mammals.The active DNA demethylation could be achieved through the ten-eleven translocation(TET)protein-mediated oxidization of 5mC with the generation of 5-hydroxymethylcytosine(5hmC),5-formylcytosine(5fC)and 5-carboxylcytosine(5caC).It has been known that 5mC,5hmC and 5fC play critical roles in modulating gene expression.However,unlike the 5mC,5hmC,and 5fC,the functions of 5caC are still underexplored.Investigation of the functions of 5caC relies on the accurate quantification and localization analysis of 5caC in DNA.In the current study,we developed a method by chemical conversion in conjugation with ligation-based real-time quantitative PCR(qPCR)for the site-specific quantification of 5caC in DNA.This method depends on the selective conversion of 5caC to form dihydrouracil(DHU)by pyridine borane treatment.DHU behaves like thymine and pairs with adenine(DHU-A).Thus,the chemical conversion by pyridine borane leads to the transformation of base paring from 5caC-G to DHU-A,which is utilized to achieve the site-specific detection and quantification of 5caC in DNA.As a proof-of-concept,the developed method was successfully applied in the site-specific quantification of 5caC in synthesized DNA spiked in complex biological samples.The method is rapid,straightforward and cost-effective,and shows promising in promoting the investigation of the functional roles of 5caC in future study.

关 键 词：5-Methylcytosine 5-Carboxylcytidine Dihydrouracil Mass spectrometry PCR Chemical conversion 

分 类 号：Q503[生物学—生物化学] O652[理学—分析化学]