Immuno-DNA binding directed template-free DNA extension and enzyme catalysis for sensitive electrochemical DNA methyltransferase activity assay and inhibitor screening
Sensitive and accurate determination of DNA methyltransferase (DNA Mtase) activity is highly pursued for understanding of fundamental biological processes related with DNA methylation, clinical disease diagnosis and drug discovery. Herein, we proposed a new electrochemical immuno-DNA sensing platform for DNA Mtase activity assay and inhibitor screening. After homogenous DNA methylation by CpG methyltransferase (M.SssI Mtase), the methylated DNA can be specifically recruited onto electrode via its immunological binding with the immobilized anti-5-methylcytosine antibody. The recruited methylated DNA was simultaneously used as substrate to facilitate successive template-free DNA extension and enzyme catalysis for dual-step signal amplification of DNA Mtase activity. The developed immuno-DNA sensing strategy effectively integrates solution-phase DNA methylation, surface affinity binding recognition, and successive template-free DNA extension and enzyme catalysis-based signal amplification, rendering a high specific, sensitive and accurate assay of DNA Mtase activity. A low detection limit of 0.039 U/mL could be achieved with a high selectivity. It was also applied for efficient evaluation of various inhibitors. Current affinity recognition of the immobilized antibody with methylated DNA switches the sensing platform into a DNA operation interface, facilitating the opportunity for combining of various DNA-based signal amplification strategies to improve detection performance. It would be used as a general strategy for the analysis of DNA Mtase activity, inhibitor and more analytes, and is anticipated to show the potential for applications in disease diagnosis and drug discovery.