Hybridization-based CpG methylation level detection using methyl-CpG-binding domain–fused luciferase

Abstract

Hypermethylation of tumor-suppressor genes and global hypomethylation, which is related to methylation level at the retroelement, have been recognized as features of the cancer genome. In this study, we developed a hybridization-based CpG methylation level detection method using methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc). In this method, methylated probe oligonucleotides were used to capture target oligonucleotides. Fully methylated and hemimethylated double-stranded DNA (dsDNA) was formed by hybridization of the methylated captured oligonucleotides with methylated or unmethylated target oligonucleotides, respectively. MBD-Fluc specifically binds to fully methylated dsDNA but not to hemimethylated dsDNA; therefore, methylated target oligonucleotides can be detected by measuring the luciferase activity of the bound MBD-Fluc. Using the corresponding methylated probe oligonucleotides, the CpG methylation levels of SEPT9, BRCA1, and long interspersed nuclear element-1 (LINE-1) oligonucleotides were quantified. Moreover, we demonstrated that the emission detection signal was not affected by the methylation state of the overhang region of the target oligonucleotide, which was not hybridized to the probe oligonucleotide, indicating that methylated CpG of the target region could be accurately detected. Unmethylated-CpG-binding domain-fused luciferases and 5-hydroxymethyl-CpG-binding domain-fused luciferases have been constructed, suggesting that other modified bases can be detected by the same platform.