Department of Chemistry, Stanford University, Stanford, CA, 94305, USA.
Department of Chemistry, University of California, Davis, Davis, CA, 95616, USA.
Angew Chem Int Ed Engl. 2020 May 4;59(19):7450-7455. doi: 10.1002/anie.202001516. Epub 2020 Mar 17.
Direct measurement of DNA repair enzyme activities is important both for the basic study of cellular repair pathways as well as for potential new translational applications in their associated diseases. NTH1, a major glycosylase targeting oxidized pyrimidines, prevents mutations arising from this damage, and the regulation of NTH1 activity is important in resisting oxidative stress and in suppressing tumor formation. Herein, we describe a novel molecular strategy for the direct detection of damaged DNA base excision activity by a ratiometric fluorescence change. This strategy utilizes glycosylase-induced excimer formation of pyrenes, and modified DNA probes, incorporating two pyrene deoxynucleotides and a damaged base, enable the direct, real-time detection of NTH1 activity in vitro and in cellular lysates. The probe design was also applied in screening for potential NTH1 inhibitors, leading to the identification of a new small-molecule inhibitor with sub-micromolar potency.
直接测量 DNA 修复酶活性对于细胞修复途径的基础研究以及在相关疾病中的潜在新的转化应用都很重要。NTH1 是一种主要的针对氧化嘧啶的糖苷酶，可防止由此类损伤引起的突变，并且 NTH1 活性的调节对于抵抗氧化应激和抑制肿瘤形成很重要。本文描述了一种新的分子策略，可通过比率荧光变化直接检测受损 DNA 碱基切除活性。该策略利用糖苷酶诱导的并五苯的二聚体形成，并且包含两个并五苯脱氧核苷酸和一个受损碱基的修饰 DNA 探针，可实现体外和细胞裂解物中 NTH1 活性的直接实时检测。该探针设计还应用于筛选潜在的 NTH1 抑制剂，从而鉴定出一种具有亚微摩尔效力的新型小分子抑制剂。
