Xue Wei, Zhang Qiang, Chang Yangyang, Brennan John D, Li Yingfu, Liu Meng
School of Environmental Science and Technology, Key Laboratory of Industrial Ecology and Environmental Engineering, Ministry of Education, Dalian University of Technology Dalian 116024 China
School of Bioengineering, Dalian University of Technology Dalian 116024 China.
Chem Sci. 2022 Apr 20;13(22):6496-6501. doi: 10.1039/d1sc04268h. eCollection 2022 Jun 7.
We report on a paper-based sensor capable of performing template-independent DNA synthesis by terminal deoxynucleotidyl transferase (TdT). Importantly, we observed that TdT efficiently incorporates fluorescently labeled dUTP on to 3'-OH ends of DNA strands in a strictly controllable manner on cellulose paper, in comparison to its distributive mode of DNA synthesis in solution. Due to the high roughness and porous nature of cellulose paper, we attribute this controllable DNA polymerization to the pore confinement effect on the catalytic behaviour of TdT. Taking advantage of this finding, we proposed a paper-assisted TdT (PAT) assay for absolute quantification of alkylated DNA lesions (N7-methylguanine), DNA deamination (cytosine-to-uracil) and DNA oxidation (8-oxo-7,8-dihydroguanine) by combining various DNA glycosylases. This PAT assay provides a low-cost, high throughput and easy to use method for quantifying the absolute levels of various types of DNA lesions, thus making it well-suited for drug development, genotoxicity testing, and environmental toxicology.
我们报道了一种基于纸张的传感器，它能够通过末端脱氧核苷酸转移酶（TdT）进行与模板无关的DNA合成。重要的是，我们观察到，与TdT在溶液中的分散式DNA合成模式相比，它能在纤维素纸上以严格可控的方式将荧光标记的dUTP有效地掺入到DNA链的3'-OH末端。由于纤维素纸具有高粗糙度和多孔性，我们将这种可控的DNA聚合归因于孔对TdT催化行为的限制作用。利用这一发现，我们提出了一种基于纸张辅助的TdT（PAT）分析方法，通过结合各种DNA糖基化酶对烷基化DNA损伤（N7-甲基鸟嘌呤）、DNA脱氨基（胞嘧啶到尿嘧啶）和DNA氧化（8-氧代-7,8-二氢鸟嘌呤）进行绝对定量。这种PAT分析方法提供了一种低成本、高通量且易于使用的方法来定量各种类型DNA损伤的绝对水平，因此非常适合药物开发、遗传毒性测试和环境毒理学研究。
