Generation of 3'-OH terminal-triggered encoding of multicolor fluorescence for simultaneous detection of different DNA glycosylases.
Generation of 3'-OH terminal-triggered encoding of multicolor fluorescence for simultaneous detection of different DNA glycosylases.
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DOI：
10.1007/s00216-022-04267-1
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发表时间：
2022
影响因子：
4.3
通讯作者：
Xingguo Chen
中科院分区：
文献类型：
--
作者：
Huige Zhang;Zixi Gao;Fei He;Jingfeng Lan;Hailong Chai;Shiqian Zhang;Xianwei Zuo;Hongli Chen;Xingguo Chen
尿嘧啶DNA糖基化酶（UDG）和人烷基腺嘌呤DNA糖基化酶（hAAG）是启动DNA损伤修复的重要DNA糖基化酶，DNA糖基化酶的异常表达与帕金森病、多种癌症和人类免疫缺陷等多种疾病密切相关。同时检测UDG和hAAG有助于临床早期诊断的研究。然而，已报道的用于多个DNA糖基化酶测定的方法受到昂贵的单分子仪器的应用、繁琐的磁性分离和复杂的设计的影响。在此，我们开发了一种简单的荧光方法，只有三个必要的DNA链的选择性和灵敏度的检测多个DNA糖基化酶活性的基础上产生的3 '-OH末端触发编码的荧光。该方法对UDG和hAAG的检出限分别为5.5 × 10-5 U/mL和3.3 × 10- 3U/mL，均低于文献报道的荧光法。此外，它还可用于检测人宫颈癌细胞系（HeLa细胞）、正常人肾上皮细胞（293 T细胞）和生物体液中的多种DNA糖基化酶，并测定UDG和hAAG的酶动力学参数。
Uracil DNA glycosylase (UDG) and human alkyladenine DNA glycosylase (hAAG) are the important DNA glycosylases for initiating the repair of DNA damage, and the aberrant expression of DNA glycosylases is closely associated with various diseases, such as Parkinson's disease, several cancers, and human immunodeficiency. The simultaneous detection of UDG and hAAG is helpful for the study of early clinical diagnosis. However, the reported methods for multiple DNA glycosylase assay suffer from the application of an expensive single-molecule instrument, labor-tedious magnetic separation, and complicated design. Herein, we develop a simple fluorescence method with only three necessary DNA strands for the selective and sensitive detection of multiple DNA glycosylase activity based on the generation of 3'-OH terminal-triggered encoding of multicolor fluorescence. The method can achieve the detection limits of 5.5 × 10-5 U/mL for UDG and 3.3 × 10-3 U/mL for hAAG, which are lower than those of the reported fluorescence methods. Moreover, it can be further used to detect multiple DNA glycosylases in the human cervical carcinoma cell line (HeLa cells), normal human renal epithelial cells (293 T cells), and biological fluid and measure the enzyme kinetic parameters of UDG and hAAG.