Department of Biology, Payame Noor University, Tehran, Iran.
Department of Chemistry, Payame Noor University, Tehran, Iran.
Spectrochim Acta A Mol Biomol Spectrosc. 2020 Mar 5;228:117731. doi: 10.1016/j.saa.2019.117731. Epub 2019 Nov 2.
DNA methylation mediated by DNA methyltransferase (MTase) enzyme is internal cell mechanism which regulate the expression or suppression of crucial genes involve in cancer early diagnosis. Herein, highly sensitive fluorescence biosensing platform was developed for monitoring of DNA Dam MTase enzyme activity and inhibition based on fluorescence signal on mechanism. The specific Au NP functionalized oligonucleotide probe with overhang end as a template for the synthesis of fluorescent silver nanoclusters (Ag NCs) was designed to provide the FRET occurrence. Following, methylation and cleavage processes by Dam MTAse and DpnI enzymes respectively at specific probe recognition site could resulted to release of AgNCs synthesizer DNA fragment and returned the platform to fluorescence signal-on state through interrupting in FRET. Subsequently, amplified fluorescence emission signals of Ag NCs showed increasing linear relationship with amount of Dam MTase enzyme at the range of 0.1-20 U/mL and the detection limit was estimated at 0.05 U/mL. Superior selectivity of experiment was illustrated among other tested MTase and restriction enzymes due to the specific recognition of MTase toward its substrate. Furthermore, the inhibition effect of applied Dam MTase drug inhibitors screened and evaluated with satisfactory results which would be helpful for discovery of antimicrobial drugs. The real sample assay also showed the applicability of proposed method in human serum condition. This novel strategy presented an efficient and cost effective platform for sensitive monitoring of DNA MTase activity and inhibition which illustrated its great potential for further application in medical diagnosis and drug discovery.
DNA 甲基化由 DNA 甲基转移酶（MTase）酶介导，是一种内部细胞机制，可调节参与癌症早期诊断的关键基因的表达或抑制。在此，基于机制的荧光信号，开发了一种用于监测 DNA Dam MTase 酶活性和抑制的高灵敏荧光生物传感平台。设计了具有悬垂末端的特定 Au NP 功能化寡核苷酸探针作为荧光银纳米团簇（Ag NCs）合成的模板，以提供 FRET 发生。随后，Dam MTAse 和 DpnI 酶分别在特定探针识别位点的甲基化和切割过程导致 AgNCs 合成器 DNA 片段的释放，并通过中断 FRET 将平台恢复到荧光信号开启状态。随后，Ag NCs 的放大荧光发射信号显示出与 0.1-20 U/mL 范围内 Dam MTase 酶量的增加线性关系，检测限估计为 0.05 U/mL。由于 MTase 对其底物的特异性识别，实验表现出优于其他测试的 MTase 和限制酶的优越选择性。此外，应用 Dam MTase 药物抑制剂的抑制效果进行了筛选和评估，结果令人满意，这将有助于发现抗菌药物。实际样品测定也表明了所提出方法在人血清条件下的适用性。该新策略为敏感监测 DNA MTase 活性和抑制提供了一种高效且具有成本效益的平台，表明其在医学诊断和药物发现中的进一步应用具有巨大潜力。
