脱氨基
化学
核糖核酸
肌苷
腺苷
生物化学
碱基对
转移RNA
计算生物学
DNA
基因
生物
酶
作者
Wen-Xuan Shao,Youngsil Min,Wei Chen,Jun Xiong,Xia Guo,Neng-Bin Xie,Shan Zhang,Si-Yu Yu,Conghua Xie,Yu‐Qi Feng,Bi-Feng Yuan
标识
DOI:10.1021/acs.analchem.3c00502
摘要
N6-Methyladenosine (m6A) is one of the most abundant and prevalent natural modifications occurring in diverse RNA species. m6A plays a wide range of roles in physiological and pathological processes. Revealing the functions of m6A relies on the faithful detection of individual m6A sites in RNA. However, developing a simple method for the single-base resolution detection of m6A is still a challenging task. Herein, we report an adenosine deamination sequencing (AD-seq) technique for the facile detection of m6A in RNA at single-base resolution. The AD-seq approach capitalizes on the selective deamination of adenosine, but not m6A, 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. m6A resists deamination due to the interference of the methyl group at the N6 position of adenosine. Thus, the m6A base pairs with thymine and is still read as adenosine in sequencing. The differential readouts from A and m6A in sequencing can achieve the single-base resolution detection of m6A in RNA. Application of the proposed AD-seq successfully identified individual m6A sites in Escherichia coli 23S rRNA. Taken together, the proposed AD-seq allows simple and cost-effective detection of m6A at single-base resolution in RNA, which provides a valuable tool to decipher the functions of m6A in RNA.
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