State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, 730000, China.
Key Laboratory of Special Function Materials and Structure Design (MOE), Lanzhou University, Lanzhou, 730000, China.
Anal Bioanal Chem. 2022 Sep;414(23):6989-7000. doi: 10.1007/s00216-022-04267-1. Epub 2022 Aug 19.
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 U/mL for UDG and 3.3 × 10 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.
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 有助于研究早期临床诊断。然而，报道的用于多种 DNA 糖基化酶检测的方法存在应用昂贵的单分子仪器、繁琐的磁分离和复杂设计等问题。本研究基于 3'-OH 末端触发的多色荧光编码的产生，开发了一种仅用 3 种必需 DNA 链的简单荧光法，用于选择性和灵敏地检测多种 DNA 糖基化酶活性。该方法对 UDG 和 hAAG 的检测限分别低至 5.5×10 U/mL 和 3.3×10 U/mL，低于报道的荧光方法。此外，该方法还可进一步用于检测人宫颈癌细胞系（HeLa 细胞）、正常人肾上皮细胞（293T 细胞）中的多种 DNA 糖基化酶，并测量 UDG 和 hAAG 的酶动力学参数。
