Direct sequencing of DNA 5-methylcytosine by engineered dioxygenase NTET-assisted eNAPS
Shan Zhang, Neng‐Bin Xie, Li Zeng, Fang‐Yin Gang, Yao-Hua Gu, Min Wang, Xia Guo, Tong‐Tong Ji, Jun Xiong, Bi‐Feng Yuan
Abstract
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.