Photoelectrochemical determination of the activity of M.SssI methyltransferase, and a method for inhibitor screening
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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.
KeywordsPhotoelectrochemical assay DNA methylation Ru(bpy)2(dppz)2 + SnO2 nanoparticle DNA restriction endonuclease S-Adenosyl-L-methionine Polyethylenimine Glutaraldehyde 5-Aza-2′-deoxycytidine Enzyme-linkage reactions
This work is supported by the National Natural Science Foundation of China (21475088, 21507087), PCSIRT (IRT1269), Chenguang Program of Shanghai Municipal Education Commission, and International Joint Laboratory on Resource Chemistry (IJLRC).
Compliance with ethical standards
The author(s) declare that they have no competing interests.
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