AbstractA photoelectrochemical (PEC) method is described for the determination of the activity of M.SssI methyltransferase (MTase). The assay relies on enzyme-linkage reactions and a DNA intercalator Ru(bpy)2(dppz)2+ (where bpy is 2,2′-bipyridine, and dppz is dipyrido[3,2-a:2′,3′-c]phenazine) which both serves as a PEC signal. The PEC electrode was obtained by immobilizing 5′-amino modified DNA strands (containing the methylation recognition site 5′-CCGG-3′) on a polyethylenimine (PEI) coated ITO/SnO2 electrode with glutaraldehyde as crosslinking agent. In the presence of MTase and S-adenosyl-L-methionine, the 5′-CCGG-3′ sequence in the DNA on the electrode is methylated. This protects the DNA strands from the shear of the methylation-sensitive restriction endonuclease HpaII. Consequently, more intact DNA strands remain on the surface of the electrode, providing more sites for Ru(bpy)2(dppz)2+ binding which in turn results in a high PEC response. The result demonstrates that the photocurrent increases linearly with the activity of MTase from 5 to 80 U·mL−1, and the limit of detection is 0.45 U·mL−1. The other MTases does not enhance the photocurrent, suggesting good selectivity of the assay. The method was also applied to rapid evaluate and screen the inhibitors of MTase. This strategy can be utilized to determinate the activity of other DNA MTases with specific DNA sequence. Graphical abstractSchematic presentation of a photoelectrochemical assay based on enzyme-linkage reactions and a photo electrochemical probe combined with the oxalic acid involved cyclic amplification system for the determination of methyltransferase activity.

中文翻译：

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M.SssI甲基转移酶活性的光电化学测定和抑制剂筛选方法

摘要描述了一种用于测定 M.SssI 甲基转移酶 (MTase) 活性的光电化学 (PEC) 方法。该测定依赖于酶联反应和 DNA 嵌入剂 Ru(bpy)2(dppz)2+（其中 bpy 是 2,2'-联吡啶，dppz 是双吡啶并 [3,2-a:2',3'- c]吩嗪），两者都用作 PEC 信号。PEC 电极是通过用戊二醛作为交联剂将 5'-氨基修饰的 DNA 链（包含甲基化识别位点 5'-CCGG-3'）固定在聚乙烯亚胺（PEI）涂层的 ITO/SnO2 电极上获得的。在 MTase 和 S-腺苷-L-甲硫氨酸存在下，电极上 DNA 中的 5'-CCGG-3' 序列被甲基化。这可以保护 DNA 链免受甲基化敏感限制性内切核酸酶 HpaII 的剪切。最后，更多完整的 DNA 链保留在电极表面，为 Ru(bpy)2(dppz)2+ 结合提供更多位点，从而导致高 PEC 响应。结果表明，光电流随MTase活性从5到80 U·mL-1呈线性增加，检测限为0.45 U·mL-1。其他 MTases 不增强光电流，表明该测定具有良好的选择性。该方法还用于快速评估和筛选 MTase 的抑制剂。该策略可用于确定具有特定 DNA 序列的其他 DNA MTase 的活性。图形摘要基于酶联反应和光电化学探针与草酸相结合的光电化学测定的示意图，涉及循环扩增系统，用于测定甲基转移酶活性。