We developed a bio-sensing system for global DNA methylation levels that can be applied to cancer diagnostics. The paper has been published in Analytica Chimica Acta (Vol. 990, p. 168-173, 2017)1.
DNA methylation of cytosine in CpG dinucleotides has important biological roles in regulation of gene expression. In cancer cells, hypomethylation has been reported in transposable elements, which constitute approximately 45% of the human genome; therefore, global DNA methylation levels are potential biomarkers for cancer diagnostics. We previously developed a bio-sensing system for detecting methylated CpG content in genomic DNA based on bioluminescence resonance energy transfer (BRET) using methyl-CpG-binding domain (MBD)-fused luciferase (MBD-luciferase)2. In the system, BRET between the artificial protein and DNA intercalating dye is detected on the methylated CpG site on genomic DNA; thus, methylated CpG content can be detected by the BRET signal (Fig. 1 left). However, a calibration curve was required to quantify global DNA methylation levels since the system does not detect unmethylated CpG content.
Fig. 1 A bio-sensing system for global DNA methylation levels using CXXC- and MBD-fused luciferase.
We then constructed a new unmethyl-CpG-binding domain (MLL CXXC)-fused luciferase (CXXC-luciferase) and developed a bio-sensing system for detecting unmethylated CpG content on genomic DNA on the same platform (Fig. 1 right). We demonstrated that global DNA methylation levels can be quantified by measuring BRET signals from CXXC-luciferase and MBD-luciferase without a calibration curve. In the future, the bio-sensing system could be applied for home cancer tests.
1. Yoshida W., Baba Y., Banzawa K., Karube I. A quantitative homogeneous assay for global DNA methylation levels using CpG-binding domain- and methyl-CpG-binding domain-fused luciferase. Anal. Chim. Acta (2017) 990, 168-173
2. Yoshida W., Baba Y., Karube I. Global DNA Methylation Detection System Using MBD-Fused Luciferase Based on Bioluminescence Resonance Energy Transfer Assay. Anal. Chem. (2016) 88, 9264-9268