Orthologous mammalian A3A-mediated single-nucleotide resolution sequencing of DNA epigenetic modification 5-hydroxymethylcytosine

作者：Xia Guo, Jianyuan Wu, Tong‐Tong Ji, Min Wang, Shan Zhang, Jun Xiong, Fang‐Yin Gang, Wei Liu, Yao-Hua Gu, Yu Liu, Neng‐Bin Xie, Bi‐Feng Yuan · 发表于：Chemical Science · 年份：2025 · DOI：10.1039/d4sc08660k · 被引用次数：11 · 研究领域：Epigenetics and DNA Methylation、RNA modifications and cancer、Amino Acid Enzymes and Metabolism

Epigenetic modifications in genomes play a crucial role in regulating gene expression in mammals. Among these modifications, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are recognized as the fifth and sixth nucleobases in genomes, respectively, and are the two most significant epigenetic marks in mammals. 5hmC serves as both an intermediate in active DNA demethylation and a stable epigenetic modification involved in various biological processes. Analyzing the location of 5hmC is essential for understanding its functions. In this study, we introduce an orthologous mammalian A3A-mediated sequencing (OMA-seq) method for the quantitative detection of 5hmC in genomic DNA at single-nucleotide resolution. OMA-seq relies on the deamination properties of two naturally occurring mammalian A3A proteins: green monkey A3A (gmA3A) and dog A3A (dogA3A). The combined use of gmA3A and dogA3A effectively deaminates cytosine (C) and 5mC, but not 5hmC. As a result, the original C and 5mC in DNA are deaminated and read as thymine (T) during sequencing, while the original 5hmC remains unchanged and is read as C. Consequently, the remaining C in the sequence indicates the presence of original 5hmC. Using OMA-seq, we successfully quantified 5hmC in genomic DNA from lung cancer tissue and corresponding normal tissue. OMA-seq enables accurate and quantitative mapping of 5hmC at single-nucleotide resolution, utilizing a pioneering single-step deamination protocol that leverages the high s...