Graduate School of Bionics, Tokyo University of Technology, 1404-1 Katakura-machi, Hachioji, Tokyo 192-0982, Japan.
School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakura-machi, Hachioji, Tokyo 192-0982, Japan.
Anal Chem. 2022 Jun 21;94(24):8618-8624. doi: 10.1021/acs.analchem.1c05619. Epub 2022 Jun 3.
5-Methylcytosine (5mC) plays an important role in the regulation of gene expression. Ten-eleven translocation (TET) continuously oxidizes 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). High levels of 5hmC are found in the brain and embryonic stem cells, while global hydroxymethylation levels are reduced in several cancer cells. Moreover, alterations in hydroxymethylation levels occur in neurological diseases, such as Alzheimer's disease and Parkinson's disease. In this study, a convenient sensing method for the determination of global hydroxymethylation levels was developed. A bioluminescence resonance energy transfer (BRET) assay for global methylation level determination has been previously reported. In the assay, BOBO-3 DNA intercalating dye is excited by the bioluminescence of methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc); that is, the BRET signal depends on the content of methylated CpG on genomic DNA. To develop a hydroxymethylation level sensing method, SET- and RING-associated (SRA) domain of ubiquitin-like with PHD and RING finger domains 2 (UHRF2)-fused Fluc (UHRF2 SRA-Fluc) was prepared. UHRF2 SRA is known to bind to both hydroxymethylated and methylated CpG sites; thus, MBD was utilized to mask the methylated CpG on genomic DNA. We demonstrated that the BRET signal between UHRF2 SRA-Fluc and BOBO-3 depends on the global hydroxymethylation level in the presence of MBD ( = 0.99, and relative standard deviation < 2.3%). The limit of detection for hydroxymethylated genomic DNA was 0.75 ng μL. In this assay, the global hydroxymethylation level was quantified within 40 min in a single tube, indicating that the assay would be utilized not only for clinical diagnostics but also for the elucidation of 5hmC functions.
5- 甲基胞嘧啶（5mC）在基因表达调控中发挥重要作用。Ten-eleven 易位（TET）将 5mC 连续氧化为 5-羟甲基胞嘧啶（5hmC）、5- 甲酰胞嘧啶（5fC）和 5- 羧基胞嘧啶（5caC）。高浓度的 5hmC 存在于大脑和胚胎干细胞中，而在几种癌细胞中整体羟甲基化水平降低。此外，在神经退行性疾病如阿尔茨海默病和帕金森病中，羟甲基化水平发生改变。在这项研究中，开发了一种用于测定整体羟甲基化水平的便捷传感方法。先前已经报道了用于测定整体甲基化水平的生物发光共振能量转移（BRET）测定法。在该测定法中，BOBO-3 DNA 嵌入染料由甲基-CpG 结合域融合的萤火虫荧光素酶（MBD-Fluc）的生物发光激发；也就是说，BRET 信号取决于基因组 DNA 上甲基化的 CpG 的含量。为了开发羟甲基化水平传感方法，制备了包含 SET 和 RING 相关（SRA）结构域的泛素样物与含有 PH 和 RING 指结构域 2（UHRF2）的融合荧光素酶（UHRF2 SRA-Fluc）。已知 UHRF2 SRA 结合羟甲基化和甲基化的 CpG 位点；因此，MBD 用于掩蔽基因组 DNA 上的甲基化 CpG。我们证明，在存在 MBD 的情况下，UHRF2 SRA-Fluc 与 BOBO-3 之间的 BRET 信号取决于整体羟甲基化水平（=0.99，相对标准偏差 <2.3%）。羟甲基化基因组 DNA 的检测限为 0.75ng μL。在该测定法中，在单个管中在 40 分钟内定量了整体羟甲基化水平，表明该测定法不仅可用于临床诊断，而且还可用于阐明 5hmC 的功能。
