Single-Base Resolution Detection of N 6 -Methyladenosine in RNA by Adenosine Deamination Sequencing.

N 6 -Methyladenosine (m 6 A) is one of the most abundant and prevalent natural modifications occurring in diverse RNA species. m 6 A plays a wide range of roles in physiological and pathological processes. Revealing the functions of m 6 A relies on the faithful detection of individual m 6 A sites in RNA. However, developing a simple method for the single-base resolution detection of m 6 A is still a challenging task. Herein, we report an adenosine deamination sequencing (AD-seq) technique for the facile detection of m 6 A in RNA at single-base resolution. The AD-seq approach capitalizes on the selective deamination of adenosine, but not m 6 A, 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. m 6 A resists deamination due to the interference of the methyl group at the N 6 position of adenosine. Thus, the m 6 A base pairs with thymine and is still read as adenosine in sequencing. The differential readouts from A and m 6 A in sequencing can achieve the single-base resolution detection of m 6 A in RNA. Application of the proposed AD-seq successfully identified individual m 6 A sites in Escherichia coli 23S rRNA. Taken together, the proposed AD-seq allows simple and cost-effective detection of m 6 A at single-base resolution in RNA, which provides a valuable tool to decipher the functions of m 6 A in RNA.