College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan, Shandong 250014, China.
School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi'an, Shaanxi 710021, P. R. China.
Anal Chem. 2020 Aug 4;92(15):10223-10227. doi: 10.1021/acs.analchem.0c01880. Epub 2020 Jul 21.
DNA methylation has become a promising epigenetic biomarker for cancer diagnosis, prognosis, and therapy monitoring. Herein, we demonstrate for the first time the development of a new oxidation damage base (8-oxo-7,8-dihydroguanine (8-oxoG))-modified fluorescent probe for direct detection of DNA methylation. This fluorescent probe is labeled with carboxy-X-rhodamine (ROX) and black hole quencher 2 (BHQ2) at the 5' and 3' termini, respectively, with one 8-oxoG base modification in the middle position, and it can discriminate the methylated cytosine from the unmethylated cytosine. The presence of target methylated DNA may induce the recycle cleavage of fluorescent probes with the assistance of human 8-oxoG DNA glycosylase 1 (hOGG1) enzyme, resulting in an enhanced fluorescence signal. In comparison with the reported bisulfite treatment-based indirect approaches, this fluorescent probe can be used for direct detection of DNA methylation under isothermal conditions without the requirement of a stringent primer/template design, any thermal cycling, and ligation procedures, greatly simplifying the experimental processes. Moreover, this fluorescent probe exhibits good specificity and high sensitivity, and it can distinguish a 0.01% methylation level even in the presence of excess unmethylated DNA. Furthermore, this fluorescent probe can be used to detect DNA methylation in genomic DNA extracted from human colon cancer cells, holding great potential in epigenetic study and early clinical diagnosis.
DNA 甲基化已成为癌症诊断、预后和治疗监测有前途的表观遗传生物标志物。在此，我们首次展示了一种新的氧化损伤碱基（8-氧代-7,8-二氢鸟嘌呤（8-氧代鸟嘌呤））修饰的荧光探针的开发，用于直接检测 DNA 甲基化。该荧光探针在 5'和 3'末端分别用羧基-X-罗丹明（ROX）和黑洞猝灭剂 2（BHQ2）标记，在中间位置有一个 8-氧代鸟嘌呤碱基修饰，可以区分甲基化的胞嘧啶和非甲基化的胞嘧啶。靶标甲基化 DNA 的存在可能会在人 8-氧代鸟嘌呤 DNA 糖基化酶 1（hOGG1）酶的协助下诱导荧光探针的循环切割，从而增强荧光信号。与报道的基于亚硫酸氢盐处理的间接方法相比，该荧光探针可以在等温条件下直接检测 DNA 甲基化，无需严格的引物/模板设计、任何热循环和连接步骤，大大简化了实验过程。此外，该荧光探针具有良好的特异性和高灵敏度，即使在存在过量未甲基化 DNA 的情况下，也可以区分 0.01%的甲基化水平。此外，该荧光探针可用于检测人结肠癌细胞中提取的基因组 DNA 中的 DNA 甲基化，在表观遗传学研究和早期临床诊断中具有很大的应用潜力。
