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.

中文翻译：

---

用于整体DNA甲基化定量的多色生物发光共振能量转移测定

异常的DNA甲基化，例如肿瘤抑制基因上的高甲基化和整体性低甲基化已被认为是癌症的标志。以前，我们报道了使用甲基CpG结合域融合萤火虫荧光素酶（MBD-Fluc）和未甲基化CpG结合域融合萤火虫荧光素酶（CXXC-Fluc）的基于生物发光共振能量转移（BRET）的全球DNA甲基化水平测定）。MBD-Fluc和BOBO-3 DNA嵌入染料之间的BRET信号取决于甲基化的CpG含量，而CXXC-Fluc和BOBO-3之间的BRET信号取决于未甲基化的CpG含量。因此，可以使用BRET分析来量化总体DNA甲基化水平。但是，由于相同的萤光素酶与MBD和CXXC融合，因此必须分别进行这些测定。在这项研究中，Oplophorus萤光素酶（CXXC-Oluc）。我们证明了MBD-Fluc和CXXC-Oluc同时激发了基因组DNA上的BOBO-3和BOBO-1 DNA嵌入染料。而且，由MBD-Fluc和CXXC-Oluc产生的BRET信号取决于CpG含量的甲基化状态。这些结果表明，可以通过在单个试管中进行此多色BRET分析来定量总体DNA甲基化。