| Abstract |
Abnormal DNA methylations such as hypermethylation on tumor suppressor genes and global hypomethylation have been recognized as hallmarks of cancer. Previously, we reported a bioluminescence resonance energy transfer (BRET)-based global DNA methylation level assay using a methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc) and unmethylated CpG-binding domain-fused firefly luciferase (CXXC-Fluc). The BRET signal between MBD-Fluc and BOBO-3 DNA intercalating dye depends on the methylated CpG contents, whereas the BRET signal between CXXC-Fluc and BOBO-3 depends on the unmethylated CpG contents. Therefore, the global DNA methylation level can be quantified using the BRET assay. However, these assays must be performed separately, because the same luciferase fuses to both MBD and CXXC. In this study, we developed a one-step quantification assay of global DNA methylation based on a multicolor BRET assay using MBD-Fluc and CXXC-fused Oplophorus luciferase (CXXC-Oluc). We demonstrated that MBD-Fluc and CXXC-Oluc simultaneously excite BOBO-3 and BOBO-1 DNA intercalating dyes on genomic DNA, respectively. Moreover, the BRET signals produced from MBD-Fluc and CXXC-Oluc depended on the methylation status of the CpG contents. These results demonstrate that global DNA methylation can be quantified by this multicolor BRET assay in a single tube. Graphical abstract ᅟ.
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