Fluorescence turn-off strategy for sensitive detection of DNA methyltransferase activity based on DNA-templated gold...

DNA methylation results in a variety of human diseases and the DNA methylation process is mediated by DNA methyltransferases, which have therefore become potential targets for disease treatment. In this study, a turn-off nanogold biological probe system was successfully created for determining the activity of DNA methyltransferases (M.SssI MTase). A dumbbell-shaped DNA probe with a site-recognizable region of M. SssI MTase and a fluorescent signal probe based on a DNA-templated gold nanocluster (DNA-AuNC) probe combined for the quantitative detection of M. SssI MTase. This dumbbell-shaped DNA probe was methylated by M. SssI MTase, and the dumbbell-shaped DNA probe with a methyl group was recognized by an endonuclease (GlaI) and cleaved into hairpin DNA. The dGTP was added to the 3'-OH terminus of hairpin DNA fragments in the presence of terminal deoxynucleotidyl transferase (TdT), and the hairpin DNA was extended with a G-rich sequence that can be used as an inactivation probe. When the inactivation probe was combined with the signal probe, the fluorescent signal disappeared due to the photoinduced electron transfer effect. Methyltransferase activity was then detected based on the turn-off principle of the fluorescence signal from the DNA-AuNCs. The bioprobe enabled sensitive detection of M. SssI MTase with a detection limit of 0.178 U mL^-1 and good specificity. The bioprobe demonstrated good detection efficiency in both human serum and cell lysates, and its unique fluorescence turn-off mechanism provided good resistance to interference, thus increasing its potential application in complex biological samples. Moreover, it is suitable for screening and assessing the inhibitory activity of M. SssI MTase inhibitors, and therefore has significant potential for disease diagnosis and drug discovery.