Engineering Research Center of Molecular and Neuro Imaging, Ministry of Education, School of Life Science and Technology, Xidian University, Xi'an 710071, China.
Institute of Medical Engineering, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an 710061, China.
Lab Chip. 2024 Mar 26;24(7):1987-1995. doi: 10.1039/d4lc00096j.
Uracil-DNA glycosylase (UDG) is a base excision repair (BER) enzyme, which catalyzes the hydrolysis of uracil bases in DNA chains that contain uracil and -glycosidic bonds of the sugar phosphate backbone. The expression of UDG enzyme is associated with a variety of genetic diseases including cancers. Hence, the identification of UDG activity in cellular processes holds immense importance for clinical investigation and diagnosis. In this study, we employed Cas12a protein and enzyme-assisted cycle amplification technology with a test strip to establish a precise platform for the detection of UDG enzyme. The designed platform enabled amplifying and releasing the target probe by reacting with the UDG enzyme. The amplified target probe can subsequently fuse with crRNA and Cas12a protein, stimulating the activation of the Cas12a protein to cleave the signal probe, ultimately generating a fluorescent signal. This technique showed the ability for evaluating UDG enzyme activity in different cell lysates. In addition, we have designed a detection probe to convert the fluorescence signal into test strip bands that can then be observed with the naked eye. Hence, our tool presented potential in both biomedical research and clinical diagnosis related to DNA repair enzymes.
尿嘧啶-DNA 糖基化酶（UDG）是一种碱基切除修复（BER）酶，它能催化 DNA 链中含有尿嘧啶和糖磷酸主链 -糖苷键的尿嘧啶碱基的水解。UDG 酶的表达与多种遗传疾病有关，包括癌症。因此，鉴定细胞过程中的 UDG 活性对临床研究和诊断具有重要意义。在这项研究中，我们使用 Cas12a 蛋白和酶辅助循环扩增技术与测试条，建立了一种用于检测 UDG 酶的精确平台。该设计的平台通过与 UDG 酶反应，实现了目标探针的扩增和释放。扩增的目标探针随后可以与 crRNA 和 Cas12a 蛋白融合，刺激 Cas12a 蛋白的激活，切割信号探针，最终产生荧光信号。该技术显示了在不同细胞裂解物中评估 UDG 酶活性的能力。此外，我们还设计了一个检测探针，将荧光信号转换为测试条带，然后可以用肉眼观察。因此，我们的工具在与 DNA 修复酶相关的生物医学研究和临床诊断中具有潜在应用价值。
