FRET-Based Method for Direct, Real-Time Measurement of DNA Methyltransferase Activity.
FRET-Based Method for Direct, Real-Time Measurement of DNA Methyltransferase Activity.
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DOI：
10.1021/acs.bioconjchem.0c00612
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发表时间：
2020-12
影响因子：
4.7
通讯作者：
Yi Long;K. Ubych;E. Jagu;R. Neely
中科院分区：
文献类型：
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作者：
Yi Long;K. Ubych;E. Jagu;R. Neely
DNA甲基转移酶活性与许多疾病有关，包括癌症，其中基因组的整体低甲基化以及局部DNA甲基化模式的显着变化可以作为疾病的诊断和预后。尽管如此，我们目前缺乏一种直接测量DNA甲基转移酶活性的方法，这将支持DNA甲基转移酶靶向治疗的发展。在这里，我们展示了一种直接测量甲基转移酶活性的实时测定方法。我们采用了一种荧光甲基转移酶辅助因子类似物，当该酶与标记的靶DNA序列结合时，会在供体染料（DNA）和受体染料（辅助因子）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 mtase的活性，并可用于筛选DNA甲基转移酶抑制剂。我们在体相和单分子成像实验中都证明了这一点，强调了该检测在甲基转移酶功能筛选和生物物理研究中的潜在应用。
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.