碱基切除修复嵌合 d/l-DNA 探针能够实时监测活细胞中胸腺嘧啶 DNA 糖基化酶活性,Journal of the American Chemical Society

当前位置： X-MOL 学术 › J. Am. Chem. Soc. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

碱基切除修复嵌合 d/l-DNA 探针能够实时监测活细胞中胸腺嘧啶 DNA 糖基化酶活性
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2023-07-26 , DOI: 10.1021/jacs.3c03010
Wenrui Zhong ₁ , Jonathan T Sczepanski ₁

Affiliation

碱基切除修复 (BER) 途径是基因组完整性的前线捍卫者，通过参与 5-甲基胞嘧啶的擦除，在表观遗传调控中发挥核心作用。这种生物学和临床意义导致了对能够监测 BER 活动（尤其是活细胞中的 BER 活动）的分析方法的需求。不幸的是，主要源自核酸的主流方法由于对核酸酶降解和其他脱靶相互作用的敏感性而大多与细胞内使用不相容。这些限制阻碍了 BER 酶的重要生物学研究和许多临床应用。在此，我们报告了一种使用独特的嵌合d / l -DNA 架构构建生物稳定 BER 探针的简单方法，该架构利用了镜像l -DNA 的生物正交特性。我们证明嵌合 BER 探针在活细胞内具有出色的稳定性，它们成功地用于监测相对 BER 活性、评估小分子 BER 抑制剂的效率以及研究酶突变体。值得注意的是，我们报告了第一个用于实时监测活细胞中胸腺嘧啶 DNA 糖基化酶 (TDG) 介导的 5-甲酰基胞嘧啶和 5-羧基胞嘧啶 BER 的荧光探针的例子，为研究 DNA 提供了急需的工具 (de)甲基化生物学。嵌合探针为实时监测活细胞中的 BER 活性提供了一种强大且高度通用的方法，这应该能够实现广泛的基础研究和临床应用。

"点击查看英文标题和摘要"

Chimeric d/l-DNA Probes of Base Excision Repair Enable Real-Time Monitoring of Thymine DNA Glycosylase Activity in Live Cells

The base excision repair (BER) pathway is a frontline defender of genomic integrity and plays a central role in epigenetic regulation through its involvement in the erasure of 5-methylcytosine. This biological and clinical significance has led to a demand for analytical methods capable of monitoring BER activities, especially in living cells. Unfortunately, prevailing methods, which are primarily derived from nucleic acids, are mostly incompatible with intracellular use due to their susceptibility to nuclease degradation and other off-target interactions. These limitations preclude important biological studies of BER enzymes and many clinical applications. Herein, we report a straightforward approach for constructing biostable BER probes using a unique chimeric d/l-DNA architecture that exploits the bioorthogonal properties of mirror-image l-DNA. We show that chimeric BER probes have excellent stability within living cells, where they were successfully employed to monitor relative BER activity, evaluate the efficiency of small molecule BER inhibitors, and study enzyme mutants. Notably, we report the first example of a fluorescent probe for real-time monitoring of thymine DNA glycosylase (TDG)-mediated BER of 5-formylcytosine and 5-carboxylcytosine in living cells, providing a much-needed tool for studying DNA (de)methylation biology. Chimeric probes offer a robust and highly generalizable approach for real-time monitoring of BER activity in living cells, which should enable a broad spectrum of basic research and clinical applications.

更新日期：2023-07-26

点击分享查看原文

点击收藏

公开下载

阅读更多本刊新发论文本刊介绍/投稿指南