Viral cDNA-based extension for highly sensitive fluorescence detection of DNA methyltransferase activity

Abstract A simple, innovative yet cycle amplification-free strategy was developed for highly sensitive fluorescence detection of activity of DNA methyltransferases and inhibitors, in which cDNA-based extension of potato virus X was used. Briefly, a partial hybrid double strand DNA (dsDNA) probe with the sequence of 5′-GATC-3′ was methylated and cleaved into four parts by dam MTase and DpnI. The newborn 5′-termini of the dsDNA complemented to 3′-end of viral cDNA and initiated dsDNA synthesis by DNA polymerase. The dsDNA product of about 6430-bp-long combined with SG and strong green fluorescence could be detected. However, in the absence of dam MTase, methylation/cleavage could not be initiated and viral cDNA was digested by Exonuclease I, and no fluorescence signal was observed. A correlation between the fluorescence intensity and dam MTase activity was obtained in a range from 0.025 to 5 U/mL of dam MTase, and the detection limit was 0.0087 U/mL. The inhibition study indicated that gentamicin and 5-flurouracil could inhibit the dam MTase activity with IC 50 values of 2.24 μM and 2.75 μM, respectively. The proposed assay showed the potential as an accessible platform for simple, rapid and sensitive detection of DNA MTase activity and screening its inhibitors.