Sensitive detection of methylated DNA and methyltransferase activity based on the lighting up of FAM-labeled DNA quenched fluorescence by gold nanoparticles

Abstract

DNA methylation of cytosine bases, which is catalyzed by methyltransferase enzymes, involve biochemical processes that contribute to gene expression and gene regulation in cells. Detection of abnormal patterns of both methylated DNA and methyltransferase enzyme activity at early stages could be considered as promising targets for early cancer diagnosis. In the present study, a novel and facile method is introduced for the sensitive detection of the M.SssI methyltransferase (M.SssI MTase) enzyme and methylated DNA based on the fluorescence recovery of FAM-labeled DNA coupled with gold nanoparticles (AuNPs). Thiol-modified probes were functionalized with AuNPs, which brought the FAM fluorophore into the close proximity of the AuNPs. This led to the overlap between the FAM fluorescence emission and AuNPs absorption spectra, introducing a FRET occurrence and causing fluorescence quenching. The hybridization of the probe and its complementary target provided specific CpG sites for M.SssI MTase enzyme activity. The methylation process gradually converted the quenched FAM fluorophore into an emissive fluorophore upon the addition of the MTase enzyme, and the observed fluorescence recovery proved the efficiency of the assay for the detection of MTase enzyme. The fluorescence intensity showed an increasing trend with M.SssI MTase enzyme activity in the range of 1–8 U mL⁻¹ with a detection limit of 0.14 U mL⁻¹. The addition of methylated ssDNA targets to a ssDNA FAM-labeled probe resulted in a DNA duplex formation, leading to a strong fluorescence signal emission due to the recovery of the fluorophore signal. Conversely, the unmethylated ssDNA target caused no changes in the fluorescence signal. In the presence of methylated DNA targets, the biosensor could specifically recognize it and accordingly trigger the methylated targets through a fluorescence enhancement in the range of 5–100 pM by monitoring the increase in the fluorescence intensity with a detection limit of 2.2 pM. The obtained results showed that the assay could realize the detection of M.SssI MTase and methylated DNA effectively in diluted human serum samples. Human serum conditions showed no significant interference with the assay performance, indicating that the present method has great potential for further application in real samples.