The University of Birmingham, School of Chemistry, Edgbaston, Birmingham, B15 2TT, United Kingdom.
Medical Research Center, Southern University of Science and Technology Hospital, Shenzhen, Guangdong Province, 518055, China.
Bioconjug Chem. 2021 Jan 20;32(1):192-198. doi: 10.1021/acs.bioconjchem.0c00612. Epub 2020 Dec 11.
DNA methyltransferase activity is associated with a host of diseases, including cancers, where global hypomethylation of the genome, as well as marked changes in local DNA methylation patterns, can be both diagnostic and prognostic for the disease. Despite this, we currently lack a method for directly measuring the activity of the DNA methyltransferases, which would support the development of DNA methyltransferase-targeted therapies. Here, we demonstrate an assay for the direct measurement of methyltransferase activity, in real time. We employ a fluorescent methyltransferase cofactor analogue, which when bound by the enzyme to a labeled target DNA sequence results in fluorescence resonance energy transfer (FRET) between the donor dye (DNA) and the acceptor dye (cofactor). We demonstrate that the method can be used to monitor the activity of DNA MTases in real time and can be applied to screen inhibitors of the DNA methyltransferases. We show this in both bulk phase and single molecule imaging experiments, highlighting the potential application of the assay in screening and biophysical studies of methyltransferase function.
DNA 甲基转移酶活性与许多疾病有关，包括癌症，基因组的整体低甲基化以及局部 DNA 甲基化模式的显著变化，都可以作为疾病的诊断和预后指标。尽管如此，我们目前缺乏直接测量 DNA 甲基转移酶活性的方法，这将支持开发针对 DNA 甲基转移酶的治疗方法。在这里，我们展示了一种实时直接测量甲基转移酶活性的方法。我们采用了一种荧光甲基转移酶辅因子类似物，当该酶与标记的靶 DNA 序列结合时，在供体染料（DNA）和受体染料（辅因子）之间产生荧光共振能量转移（FRET）。我们证明该方法可用于实时监测 DNA MTases 的活性，并可用于筛选 DNA 甲基转移酶的抑制剂。我们在批量相和单分子成像实验中都证明了这一点，突出了该测定法在筛选和甲基转移酶功能的生物物理研究中的潜在应用。
