A photo-elutable and template-free isothermal amplification strategy for sensitive fluorescence detection of 5-formylcytosine in genomic DNA  

作　　者：Hongling Yang Yanfei Zhang Zhenning Yu Si-Yang Liu Yuzhi Xu Zong Dai Xiaoyong Zou 

机构地区：[1]School of Chemistry,Sun Yat-sen University,Guangzhou 510275,China [2]Guangdong Provincial Key Laboratory of Sensing Technology and Biomedical Instrument,School of Biomedical Engineering,Sun Yat-sen University,Shenzhen 518107,China [3]Scientific Research Center,The Seventh Affiliated Hospital,Sun Yat-sen University,Shenzhen 518107,China

出　　处：《Chinese Chemical Letters》2023年第3期166-170,共5页中国化学快报（英文版）

基　　金：supported by the Scientific Technology Project of Shenzhen City(Nos.JCYJ20200109142410170 and JCYJ20210324124003008);the National Natural Science Foundation of China(No.21974153);the Scientific Technology Project of Guangzhou City(No.202103000003);the Guangdong Natural Science Foundation(No.2019A1515010587);the Guangdong Science and Technology Plan Project(No.2020B1212060077)。

摘　　要：5-Formylcytosine(5fC), as an important epigenetic modification, plays a vital role in diverse biological processes and multiple diseases by regulating gene expression. Owing to the extremely low abundance of 5fC in all mammalian tissues and high structural similarity with other cytosine derivatives, the precise and sensitive detection of 5fC is challenging. Herein, a photo-elutable and template-free isothermal amplification strategy has been proposed for the sensitive detection of 5fC in genomic DNA based on5fC-specific biotinylation, enrichment, photocleavage, and terminal deoxynucleotidyl transferase(Td T)-assisted fluorescence signal amplification, which is termed 5fC-PTIAS. By introducing the highly specific chemolabeling and the one-step photoelution processes, this strategy possesses a minimal nonspecific background as well as a much higher amplification efficiency. With the high signal-to-noise ratio, this strategy can achieve the accurate quantification of 5fC in various biological samples including mouse brain, kidney, and liver, with a limit of detection(LOD) of 0.025‰ in DNA(S/N=3). These results not only confirm the widespread distribution of 5fC but also indicate its significant variation in different tissues and ages. The bisulfite-and mass spectrometry-free strategy is highly sensitive, selective, and easily mastered, holding great promise in detecting other epigenetic modifications with much lower levels.

关 键 词：5-Formylcytosine DNA demethylation Fluorescence detection Isothermal amplification Magnetic separation 

分 类 号：O657.3[理学—分析化学] Q503[理学—化学]