Tao Jeffrey, Zhang Hongquan, Weinfeld Michael, Le X Chris
Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2G3, Canada.
Division of Experimental Oncology, Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Cross Cancer Institute, Edmonton, Alberta T6G 1Z2, Canada.
ACS Meas Sci Au. 2024 May 8;4(4):459-466. doi: 10.1021/acsmeasuresciau.4c00011. eCollection 2024 Aug 21.
DNA glycosylase dysregulation is implicated in carcinogenesis and therapeutic resistance of cancers. Thus, various DNA-based detection platforms have been developed by leveraging the base excision activity of DNA glycosylases. However, the efficacy of DNA-based methods is hampered due to nonspecific degradation by nucleases commonly present in cancer cells and during preparations of cell lysates. In this report, we describe a fluorescence-based assay using a specific and nuclease-resistant three-dimensional DNAzyme walker to investigate the activity of DNA glycosylases from cancer cell lysates. We focus on DNA glycosylases that excise uracil from deoxyuridine (dU) lesions, namely, uracil DNA glycosylase (UDG) and single-stranded monofunctional uracil DNA glycosylase (SMUG1). The limits of detection for detecting UDG and SMUG1 in the buffer were 3.2 and 3.0 pM, respectively. The DNAzyme walker detected uracil excision activity in diluted cancer cell lysate from as few as 48 A549 cells. The results of the UDG inhibitor experiments demonstrate that UDG is the predominant uracil-excising glycosylase in A549 cells. Approximately 500 nM of UDG is present in each A549 cell on average. No fluorescence was generated in the samples lacking DNAzyme activation, indicating that there was no nonspecific nuclease interference. The ability of the DNAzyme walker to respond to glycosylase activity illustrates the potential use of DNAzyme walker technology to monitor and study biochemical processes involving glycosylases.
DNA糖基化酶失调与癌症的发生发展及治疗耐药性有关。因此，人们利用DNA糖基化酶的碱基切除活性开发了各种基于DNA的检测平台。然而，由于癌细胞中普遍存在的核酸酶以及细胞裂解物制备过程中的非特异性降解，基于DNA的方法的效能受到了阻碍。在本报告中，我们描述了一种基于荧光的检测方法，该方法使用一种特异性且耐核酸酶的三维DNAzyme步行器来研究癌细胞裂解物中DNA糖基化酶的活性。我们重点关注从脱氧尿苷（dU）损伤中切除尿嘧啶的DNA糖基化酶，即尿嘧啶DNA糖基化酶（UDG）和单链单功能尿嘧啶DNA糖基化酶（SMUG1）。在缓冲液中检测UDG和SMUG1的检测限分别为3.2和3.0 pM。DNAzyme步行器能够在低至48个A549细胞的稀释癌细胞裂解物中检测到尿嘧啶切除活性。UDG抑制剂实验结果表明，UDG是A549细胞中主要的尿嘧啶切除糖基化酶。平均每个A549细胞中约存在500 nM的UDG。在缺乏DNAzyme激活的样品中未产生荧光，这表明不存在非特异性核酸酶干扰。DNAzyme步行器对糖基化酶活性的响应能力说明了DNAzyme步行器技术在监测和研究涉及糖基化酶的生化过程方面的潜在用途。
