Graduate School of Bionics, Tokyo University of Technology, 1404-1 Katakura-machi, Hachioji, Tokyo192-0982, Japan.
Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo184-8588, Japan.
Anal Chem. 2023 Feb 21;95(7):3799-3805. doi: 10.1021/acs.analchem.2c05066. Epub 2023 Feb 7.
Global hypomethylation and promoter hypermethylation of tumor-suppressor genes are the hallmarks of cancer. We previously reported a global DNA methylation level sensing system based on dual-color bioluminescence resonance energy transfer (BRET) using methyl-CpG binding domain (MBD)-fused firefly luciferase (Fluc) and unmethyl-CpG binding domain (CXXC)-fused luciferase (Oluc). Moreover, BRET-based hydroxymethylation and hemi-methylation level sensing systems have been developed using hydroxymethyl-CpG and hemi-methyl-CpG binding domain-fused Fluc. These studies suggest that target epigenetic modifications can be simultaneously quantified using target-modification-binding protein-fused luciferases. In this study, we focused on the SnoopTag (SnT)/SnoopCatcher (SnC) protein ligation system to establish a universal design for fusion protein construction for any combination. SnT spontaneously forms an isopeptide bond with SnC; therefore, any kind of fusion protein would be constructed by the SnT/SnC system. To establish the proof of concept, MBD-SnT, CXXC-SnT, and SnC-Oluc were prepared and ligated MBD-SnT or CXXC-SnT to SnC-Oluc. The ligation products of MBD-SnT-SnC-Oluc and CXXC-SnT-SnC-Oluc showed luciferase activity and specific binding activity to methyl-CpG and unmethyl-CpG, respectively. The BRET signal using MBD-SnT-SnC-Oluc and CXXC-SnT-SnC-Oluc increased the amount of methyl-CpG and unmethyl-CpG in genomic DNA, respectively. There was a significant negative correlation between the BRET signals; therefore, the global DNA methylation level was quantified using the BRET signals ( = 0.99, and R.S.D. <3.5%). These results indicate that the SnT/SnC protein ligation system can be utilized to construct target modification-binding protein-fused luciferases in any combination that detects target modifications in genomic DNA based on BRET.
全球抑癌基因的低甲基化和启动子高甲基化是癌症的标志。我们之前报道了一种基于双色生物发光共振能量转移（BRET）的全基因组 DNA 甲基化水平感应系统，该系统使用甲基-CpG 结合域（MBD）融合萤火虫荧光素酶（Fluc）和非甲基-CpG 结合域（CXXC）融合荧光素酶（Oluc）。此外，还开发了基于 BRET 的羟甲基化和半甲基化水平感应系统，使用羟甲基-CpG 和半甲基-CpG 结合域融合的 Fluc。这些研究表明，使用靶标修饰结合蛋白融合荧光素酶可以同时定量检测靶标表观遗传修饰。在本研究中，我们专注于 SnoopTag（SnT）/SnoopCatcher（SnC）蛋白连接系统，以建立用于任何组合融合蛋白构建的通用设计。SnT 与 SnC 自发形成异肽键；因此，任何类型的融合蛋白都可以通过 SnT/SnC 系统构建。为了建立概念验证，制备了 MBD-SnT、CXXC-SnT 和 SnC-Oluc，并将 MBD-SnT 或 CXXC-SnT 连接到 SnC-Oluc 上。MBD-SnT-SnC-Oluc 和 CXXC-SnT-SnC-Oluc 的连接产物均显示出荧光素酶活性，并分别特异性结合甲基化 CpG 和非甲基化 CpG。MBD-SnT-SnC-Oluc 和 CXXC-SnT-SnC-Oluc 的 BRET 信号随基因组 DNA 中甲基化 CpG 和非甲基化 CpG 含量的增加而增加。BRET 信号之间存在显著的负相关；因此，使用 BRET 信号定量检测全基因组 DNA 甲基化水平（=0.99，R.S.D. <3.5%）。这些结果表明，SnT/SnC 蛋白连接系统可用于构建任何组合的靶标修饰结合蛋白融合荧光素酶，该系统基于 BRET 检测基因组 DNA 中的靶标修饰。
