Construction of DNA template sequences to generate fluorescent gold nanoclusters for the sensitive detection of DNA methyltransferase activity bioassay†

Abstract

Abnormal DNA methylation mediated by DNA methyltransferases is one of the most common epigenetic modifications, and abnormal DNA methyltransferase activity is often responsible for serious diseases such as cancer. For rapid, sensitive and efficient detection of DNA methyltransferase activity, a bioassay system using gold nanoclusters (DNA-AuNCs) as output has been developed. In this study, dumbbell DNA substrate is recognized and methylated by methyltransferase followed by cleavage by endonuclease (GlaI). In the presence of terminal deoxynucleotidyl transferase (TdT), the poly-A DNA product eventually becomes the template for the reduction of gold nanoclusters and then generated with a strong fluorescence signal. The assay is highly sensitive and requires no amplification or complex material synthesis steps. The detection limit is 0.077 U mL⁻¹. The bioassay showed good detection efficiency in both human serum, cell lysates and human-derived cells. Moreover, it can be used for screening and evaluation of M.SssI MTase inhibitors and hence has great potential use in disease diagnosis and drug discovery. The method was universal and allowed for other biological target detection by simply replacing the sequence of the substrate DNA recognition site; thus the proposed assay has a broad scope of application in both bioassay and drug screening.