College of Chemistry and Molecular Sciences, Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China.
School of Public Health, Research Center of Public Health, Renmin Hospital of Wuhan University, Wuhan University, Wuhan 430071, China.
Anal Chem. 2023 Jul 18;95(28):10588-10594. doi: 10.1021/acs.analchem.3c00502. Epub 2023 Jul 4.
-Methyladenosine (mA) is one of the most abundant and prevalent natural modifications occurring in diverse RNA species. mA plays a wide range of roles in physiological and pathological processes. Revealing the functions of mA relies on the faithful detection of individual mA sites in RNA. However, developing a simple method for the single-base resolution detection of mA is still a challenging task. Herein, we report an adenosine deamination sequencing (AD-seq) technique for the facile detection of mA in RNA at single-base resolution. The AD-seq approach capitalizes on the selective deamination of adenosine, but not mA, by the evolved tRNA adenosine deaminase (TadA) variant of TadA8e or the dimer protein of TadA-TadA8e. In AD-seq, adenosine is deaminated by TadA8e or TadA-TadA8e to form inosine, which pairs with cytidine and is read as guanosine in sequencing. mA resists deamination due to the interference of the methyl group at the 6 position of adenosine. Thus, the mA base pairs with thymine and is still read as adenosine in sequencing. The differential readouts from A and mA in sequencing can achieve the single-base resolution detection of mA in RNA. Application of the proposed AD-seq successfully identified individual mA sites in 23S rRNA. Taken together, the proposed AD-seq allows simple and cost-effective detection of mA at single-base resolution in RNA, which provides a valuable tool to decipher the functions of mA in RNA.
