Henan Key Laboratory of Organic Functional Molecule and Drug Innovation, NMPA Key Laboratory for Research and Evaluation of Innovative Drug, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007, China.
State Key Laboratory of Cell Differentiation Regulation and Target Drug, Henan Normal University, Xinxiang 453007, China.
J Am Chem Soc. 2022 Nov 9;144(44):20165-20170. doi: 10.1021/jacs.2c06110. Epub 2022 Oct 26.
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.
3-甲基胸腺嘧啶（m3T）是一种已知的化学稳定性强但诱变作用强的 DNA 碱基加合物；然而，迄今为止还没有确定 m3T 的测序方法。存在的两个主要障碍包括 m3T 停止 DNA 延伸的能力及其含量低。为了解决这些挑战，我们在本文中报告了一种非天然碱基配对策略。开发了一种新的 m3T-TPT3 碱基对，其 Vmax/Km 值比 m3T-A 对高 82 倍。TPT3 核苷碱基可以特异性地与 m3T 碱基配对，并且在存在商业 DNA 聚合酶的情况下，所得的 m3T-TPT3 碱基对可以有效地延伸以给出全长产物。重要的是，m3T-TPT3 对的特征使得取代 PCR 扩增能够将 m3T 损伤精确地转移到非天然碱基对中，这允许进行 Sanger 测序以及 m3T 损伤的生物素-链霉亲和素富集。总之，这项工作首次为 m3T 的扩增、富集和测序提供了一种简单、方便和实用的方法。
