Gang Fang-Yin, Xie Neng-Bin, Wang Min, Zhang Shan, Ji Tong-Tong, Liu Wei, Guo Xia, Gu Shu-Yi, Yuan Bi-Feng
Department of Occupational and Environmental Health, School of Public Health, Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China.
Research Center of Public Health, Renmin Hospital of Wuhan University, Wuhan University, Wuhan 430060, China.
Anal Chem. 2024 Dec 31;96(52):20559-20567. doi: 10.1021/acs.analchem.4c05030. Epub 2024 Dec 16.
5-Methylcytosine (5mC) is the most significant DNA modification present in mammalian genomes. Understanding the roles of 5mC in diverse biological processes requires quantitative detection at single-base resolution. In this study, we engineered the repressor of the silencing 1 (ROS1) protein derived from to enhance its 5mC glycosylase/lyase activity, resulting in the creation of the engineered ROS1 (eROS1) protein. Leveraging the unique properties of eROS1, we introduced a method termed engineered ROS1 sequencing (eROS1-seq) for bisulfite-free and quantitative detection of 5mC in DNA at single-base resolution. In eROS1-seq, the eROS1 protein selectively cleaves 5mC while leaving unmodified cytosine (C) intact, followed by the incorporation of dTTP, which subsequently results in sequencing as thymine (T). This method effectively differentiates between C and 5mC. Unlike conventional bisulfite sequencing (BS-seq), which predominantly converts cytosines, eROS1-seq specifically transforms 5mC into T, thereby avoiding potential imbalances in the nucleobase composition of the sequencing library. Using eROS1-seq, we successfully achieved quantitative and site-specific detection of 5mC in the genomic DNA of lung cancer tissue. Overall, the eROS1-seq approach is bisulfite-free and straightforward, making it a valuable tool for the quantitative detection of 5mC at single-base resolution.
5-甲基胞嘧啶（5mC）是哺乳动物基因组中存在的最重要的DNA修饰。了解5mC在多种生物学过程中的作用需要在单碱基分辨率下进行定量检测。在本研究中，我们对源自的沉默1（ROS1）蛋白进行工程改造，以增强其5mC糖基化酶/裂解酶活性，从而产生了工程化的ROS1（eROS1）蛋白。利用eROS1的独特特性，我们引入了一种称为工程化ROS1测序（eROS1-seq）的方法，用于在单碱基分辨率下对DNA中的5mC进行无亚硫酸氢盐的定量检测。在eROS1-seq中，eROS1蛋白选择性切割5mC，同时使未修饰的胞嘧啶（C）保持完整，随后掺入dTTP，其随后导致测序为胸腺嘧啶（T）。该方法有效地区分了C和5mC。与主要转化胞嘧啶的传统亚硫酸氢盐测序（BS-seq）不同，eROS1-seq特异性地将5mC转化为T，从而避免了测序文库中核碱基组成的潜在不平衡。使用eROS1-seq，我们成功地在肺癌组织的基因组DNA中实现了5mC的定量和位点特异性检测。总体而言，eROS1-seq方法无亚硫酸氢盐且操作简单，使其成为在单碱基分辨率下定量检测5mC的有价值工具。
