A quantitative homogeneous assay for global DNA methylation levels using CpG-binding domain- and methyl-CpG-binding...

Global DNA methylation levels have been considered as biomarkers for cancer diagnostics because transposable elements that constitute approximately 45% of the human genome are hypomethylated in cancer cells. We have previously reported a homogeneous assay for measuring methylated CpG content of genomic DNA based on bioluminescence resonance energy transfer (BRET) using methyl-CpG-binding domain (MBD)-fused luciferase (MBD-luciferase). In this study, a homogeneous assay for measuring unmethylated CpG content of genomic DNA in the same platform was developed using CXXC domain-fused luciferase (CXXC-luciferase) that specifically recognizes unmethylated CpG. In this assay, CXXC-luciferase recognizes unmethylated CpG on genomic DNA, whereby BRET between luciferase and the fluorescent DNA intercalating dye is detected. We demonstrated that the BRET signal depended on the genomic DNA concentration (R² = 0.99) and unmethylated CpG content determined by the bisulfite method (R² = 0.97). There was a significant negative correlation between the BRET signal of the CXXC-luciferase-based assay and that of the MBD-luciferase-based assay (R² = 0.92). Moreover, we demonstrated that the global DNA methylation level determined using the bisulfite method was dependent on the ratio of the BRET signal in the MBD-luciferase-based assay to the total BRET signal in the MBD-luciferase- and CXXC-luciferase-based assays (R² = 0.99, relative standard deviation < 2.2%, and analysis speed < 35 min). These results demonstrated that global DNA methylation levels can be quantified by calculating the BRET signal ratio without any calibration curve.