Label-Free and Sensitive Electrochemical Detection of DNA Methylation Damage Based on DNA Glycosylase Recognition and Excision Coupled with Exonuclease III Amplification

Abstract

Because cytotoxic DNA methylated bases are chemically inert and small in size, limited biosensing strategies, especially with high sensitivity, were established to detect DNA methylation damage. In this work, a label-free electrochemical assay was proposed for the sensitive detection of DNA methylation damage in dsDNA film on indium tin oxide electrode. The human alkyladenine DNA glycosylase was employed to recognize and selectively remove DNA methylated bases, generating apurinic site. Subsequently, exonuclease Ⅲ not only further converted the methylation sites into strand breaks, but also progressively removed nucleotides from the 3′ to 5′ end starting from the apurinic sites. These processes could maximally amplify DNA methylation damage, also confirmed by gel electrophoresis and fluorescence measurements. The damaged DNA film bound much less electrochemical indicator, Ru(bpy)2(dppz)2+, and accompanied by an anodic current drop. As a result, DNA methylation damage produced with as low as 10 μM of methyl methanesulfonate can be detected, which was 100-fold lower than the previously reported photoelectrochemical sensing strategy. Other DNA base modification products showed much less decrease of oxidation current, demonstrating its good selectivity. This strategy can be utilized to sensitively and rapidly assess the genotoxicity of environmental pollutants.