河南师范大学李凌君团队通过与非自然碱基特异性配对实现致突变甲基化DNA加合物的扩增、富集和测序。相关研究成果于2022年10月26日发表在《美国化学会杂志》。

3-甲基胸腺嘧啶（m3T）是一种已知的化学稳定且具有强诱变作用的DNA碱基加合物。然而，迄今为止缺乏确定m3T的测序方法。两个主要障碍包括m3T阻止DNA延伸的能力及其低丰度。

为了应对这些挑战，研究人员报告了一种非自然碱基配对策略。新的m3T-TPT3碱基对的Vmax/Km值比m3T-A碱基对高82倍。TPT3核碱基可以与m3T碱基相对地特异性结合，并且在商业DNA聚合酶的存在下，所产生的m3T–TPT3碱基对可以有效地延伸以产生全长产物。

重要的是，m3T–TPT3对的特征使替代PCR扩增能够将精确位置的m3T损伤转移到非自然碱基对中，实现Sanger测序以及基于生物素-链霉亲和素的m3T损伤富集。总之，该工作首次为m3T的扩增、富集和测序提供了一种简单、方便和实用的方法。

附：英文原文

Title: Amplification, Enrichment, and Sequencing of Mutagenic Methylated DNA Adduct through Specifically Pairing with Unnatural Nucleobases

Author: Wuyuan Zhu, Honglei Wang, Xiaohuan Li, Wenchao Tie, Bianbian Huo, Anlian Zhu, Lingjun Li

Issue&Volume: October 26, 2022

Abstract: 3-Methylthymine (m3T) is a long-known chemically stable but strongly mutagenic DNA base adduct; however, the sequencing method to determine m3T is lacking so far. Two of the main obstacles include the capacity of m3T to stall DNA elongation and its low abundance. To address the challenges, we report an unnatural base pairing strategy in this paper. A new m3T–TPT3 base pair was developed with a Vmax/Km value 82-fold higher than that of the m3T-A pair. The TPT3 nucleobase can be specifically incorporated opposite the m3T base, and the resulting m3T–TPT3 base pair can be effectively extended to give full-length products in the presence of commercial DNA polymerases. Importantly, the feature of the m3T–TPT3 pair enables a replacement PCR amplification to transfer m3T lesions at the exact positions into unnatural base pairs, which permits Sanger sequencings as well as biotin–streptavidin-based enrichments of m3T lesions. Taken together, this work offers a simple, convenient, and practical method for amplification, enrichment, and sequencing of m3T for the first time.

DOI: 10.1021/jacs.2c06110

Source: https://pubs.acs.org/doi/10.1021/jacs.2c06110