Department of Biomedical Engineering, City University of Hong Kong, 83 Tat Chee Avenue, Hong Kong, Special Administrative Region, 999077, Hong Kong, China.
City University of Hong Kong Shenzhen Research Institute, Shenzhen, 518000, China.
Mikrochim Acta. 2023 Nov 16;190(12):468. doi: 10.1007/s00604-023-06050-0.
Human 8-oxoguanine DNA glycosylase (hOGG1) is an essential enzyme that recognizes and removes 8-oxoguanine (8-oxoG), a common DNA oxidative damage caused by reactive oxygen species, to maintain genomic integrity of living organisms. Abnormal expression of hOGG1 has been proved to be associated with different diseases such as cancer and neurogenerative disorders, making it a potential biomarker and therapeutic target. In this study, we report the development of a novel strategy for detecting hOGG1 activity based on CRISPR/Cas12a trans-cleavage triggered by cleavage ligation of a dumbbell DNA probe (DBP) designed with a 3' overhang and an 8-oxoG modification. When hOGG1 is present, it cleaves the DBP at the 8-oxoG site, forming a 5' phosphate termini and exposing a single-strand region allowing complementary to the 3' overhang. After hybridization, the 3' and 5' termini in the juxtaposition are ligated by T4 DNA ligase, leading to a closed DBP for CRISPR/Cas12a-crRNA to recognize and initiate the trans-cleavage of the surrounding ssDNAs with fluorophore and quencher. The method achieves a limit of detection (LOD) with 370 μU/mL and high selectivity. Furthermore, it demonstrates a good compatibility for detecting hOGG1 activity in cell lysates, suggesting a good performance for further application in disease diagnosis and scientific research.
人 8-氧鸟嘌呤 DNA 糖基化酶（hOGG1）是一种必需的酶，可识别并清除活性氧引起的常见 DNA 氧化损伤 8-氧鸟嘌呤（8-oxoG），以维持生物的基因组完整性。异常表达的 hOGG1 已被证明与癌症和神经退行性疾病等不同疾病有关，使其成为潜在的生物标志物和治疗靶点。在这项研究中，我们报告了一种基于 CRISPR/Cas12a 转切割的新型 hOGG1 活性检测策略的发展，该策略由设计带有 3'突出和 8-氧代鸟嘌呤修饰的哑铃 DNA 探针（DBP）的切割连接触发。当存在 hOGG1 时，它会在 8-oxoG 位点切割 DBP，形成 5'磷酸末端并暴露出允许互补的单链区域3'突出。杂交后，毗邻的 3'和 5'末端由 T4 DNA 连接酶连接，导致封闭的 DBP 被 CRISPR/Cas12a-crRNA 识别并启动周围 ssDNA 的转切割，带有荧光团和猝灭剂。该方法的检测限（LOD）为 370 μU/mL，具有高选择性。此外，它在细胞裂解物中检测 hOGG1 活性的兼容性良好，表明在疾病诊断和科学研究中的进一步应用具有良好的性能。
