Photoelectrochemical determination of the activity of M.SssI methyltransferase, and a method for inhibitor screening

Abstract

A 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 abstract Schematic 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.

Keywords: 5-Aza-2′-deoxycytidine; DNA methylation; DNA restriction endonuclease; Enzyme-linkage reactions; Glutaraldehyde; Photoelectrochemical assay; Polyethylenimine; Ru(bpy)2(dppz)2 +; S-Adenosyl-L-methionine; SnO2 nanoparticle.