Development of Oxidation Damage Base-Based Fluorescent Probe for Direct Detection of DNA Methylation

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.