Universal Design of Luciferase Fusion Proteins for Epigenetic Modifications Detection Based on Bioluminescence Resonance Energy Transfer
Universal Design of Luciferase Fusion Proteins for Epigenetic Modifications Detection Based on Bioluminescence Resonance Energy Transfer
复制标题
基于生物发光共振能量转移的表观遗传修饰检测荧光素酶融合蛋白的通用设计
DOI：
10.1021/acs.analchem.2c05066
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发表时间：
2023
期刊：
影响因子：
7.4
通讯作者：
Yoshida W.
中科院分区：
文献类型：
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作者：
Miyata T.;Shimamura H.;Asano R.;Yoshida W.
肿瘤抑制基因的整体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）的全球 DNA 甲基化水平传感系统，使用甲基 CpG 结合域（MBD）融合的萤火虫荧光素酶（Fluc）和非甲基 CpG 结合域（CXXC）融合的 Oplophorus 荧光素酶（Oluc）。此外，使用羟甲基-CpG 和半甲基-CpG 结合域融合的 Fluc 开发了基于 BRET 的羟甲基化和半甲基化水平传感系统。这些研究表明，可以使用靶标修饰结合蛋白融合荧光素酶同时定量靶标表观遗传修饰。在本研究中，我们重点关注 SnoopTag (SnT)/SnoopCatcher (SnC) 蛋白质连接系统，以建立任何组合的融合蛋白构建的通用设计。 SnT 自发地与 SnC 形成异肽键；因此，任何种类的融合蛋白都可以通过SnT/SnC系统构建。为了进行概念验证，制备了 MBD-SnT、CXXC-SnT 和 SnC-Oluc，并将 MBD-SnT 或 CXXC-SnT 连接到 SnC-Oluc。 MBD-SnT-SnC-Oluc和CXXC-SnT-SnC-Oluc的连接产物分别表现出荧光素酶活性以及对甲基-CpG和非甲基-CpG的特异性结合活性。使用MBD-SnT-SnC-Oluc和CXXC-SnT-SnC-Oluc的BRET信号分别增加了基因组DNA中甲基-CpG和非甲基-CpG的量。 BRET信号之间存在显着的负相关性；因此，使用 BRET 信号对全局 DNA 甲基化水平进行定量（R2= 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意组合的靶标修饰结合蛋白融合荧光素酶，基于 BRET 检测基因组 DNA 中的靶标修饰。
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)-fusedOplophorusluciferase (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 (R2= 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.