Zhang Shan, Xie Neng-Bin, Zeng Li, Gang Fang-Yin, Gu Yao-Hua, Wang Min, Guo Xia, Ji Tong-Tong, Xiong Jun, Yuan Bi-Feng
Department of Occupational and Environmental Health, School of Public Health, Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, State Key Laboratory of Metabolism and Regulation in Complex Organisms, Wuhan University Wuhan 430071 China
Hubei Provincial Center for Disease Control and Prevention & NHC Specialty Laboratory of Food Safety Risk Assessment and Standard Development Wuhan 430079 China.
Chem Sci. 2025 Jul 11. doi: 10.1039/d5sc03634h.
DNA methylation (5-methylcytosine, 5mC) represents the most prevalent modification in mammals, which is closely linked to disease pathogenesis and cancer development. Single-base resolution sequencing and quantitative analysis of 5mC are essential for elucidating its biological functions. However, current methods are still limited by resolution, sequencing bias, and false positives. In this study, we engineered a TET-like protein (NTET), yielding a recombinant engineered NTET (eNTET), to improve both its oxidation activity and sequence compatibility for 5mC. Combined with pyridine borane reduction, we developed engineered NTET-assisted pyridine borane sequencing (eNAPS) to quantitatively detect 5mC in DNA at single-base resolution. In eNAPS, 5mC is oxidized by eNTET to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), which are further reduced to dihydrouracil (DHU) by pyridine borane and read as thymine (T) in the subsequent sequencing. The direct conversion of 5mC-to-T allows for precise mapping of 5mC in DNA at single-base resolution. Compared with conventional bisulfite sequencing (BS-seq), eNAPS exhibits advantages such as non-destruction, enhanced sensitivity, improved accuracy, and greater efficiency. Using the eNAPS method, we achieved quantitative analysis of 5mC at single-base resolution in genomic DNA of lung tumor and tumor-adjacent normal tissues. Overall, eNAPS is a mild and bisulfite-free method with high accuracy, making it a valuable tool for investigating the dynamic interplay of 5mC in epigenetic regulation and disease pathogenesis.
DNA甲基化（5-甲基胞嘧啶，5mC）是哺乳动物中最普遍的修饰形式，它与疾病发病机制和癌症发展密切相关。对5mC进行单碱基分辨率测序和定量分析对于阐明其生物学功能至关重要。然而，目前的方法仍然受到分辨率、测序偏差和假阳性的限制。在本研究中，我们设计了一种类TET蛋白（NTET），得到重组工程化NTET（eNTET），以提高其对5mC的氧化活性和序列兼容性。结合吡啶硼烷还原，我们开发了工程化NTET辅助吡啶硼烷测序（eNAPS），以单碱基分辨率定量检测DNA中的5mC。在eNAPS中，5mC被eNTET氧化为5-甲酰基胞嘧啶（5fC）和5-羧基胞嘧啶（5caC），它们被吡啶硼烷进一步还原为二氢尿嘧啶（DHU），并在随后的测序中被读取为胸腺嘧啶（T）。5mC直接转化为T使得能够在单碱基分辨率下精确绘制DNA中的5mC图谱。与传统的亚硫酸氢盐测序（BS-seq）相比，eNAPS具有无损、灵敏度提高、准确性改善和效率更高等优点。使用eNAPS方法，我们在肺肿瘤和肿瘤邻近正常组织的基因组DNA中实现了5mC的单碱基分辨率定量分析。总体而言，eNAPS是一种温和且无亚硫酸氢盐的方法，具有很高的准确性，使其成为研究5mC在表观遗传调控和疾病发病机制中动态相互作用的有价值工具。
