A real-time assay for CpG-specific cytosine-C5 methyltransferase activity

Abstract

A real-time assay for CpG-specific cytosine-C5 methyltransferase activity has been developed. The assay applies a break light oligonucleotide in which the methylation of an unmethylated 5′-CG-3′ site is enzymatically coupled to the development of a fluorescent signal. This sensitive assay can measure rates of DNA methylation down to 0.34 ± 0.06 fmol/s. The assay is reproducible, with a coefficient of variation over six independent measurements of 4.5%. Product concentration was accurately measured from fluorescence signals using a linear calibration curve, which achieved a goodness of fit (R2) above 0.98. The oligonucleotide substrate contains three C5-methylated cytosine residues and one unmethylated 5′-CG-3′ site. Methylation yields an oligonucleotide containing the optimal substrate for the restriction enzyme GlaI. Cleavage of the fully methylated oligonucleotide leads to separation of fluorophore from quencher, giving a proportional increase in fluorescence. This method has been used to assay activity of DNMT1, the principle maintenance methyltransferase in human cells, and for the kinetic characterization of the bacterial cytosine-C5 methyltransferase M.SssI. The assay has been shown to be suitable for the real-time monitoring of DNMT1 activity in a high-throughput format, with low background signal and the ability to obtain linear rates of methylation over long periods, making this a promising method of high-throughput screening for inhibitors.

The Replication Focus Targeting Sequence (RFTS) Domain Is a DNA-competitive Inhibitor of Dnmt1

“…To test this hypothesis, we synthesized an internally quenched, fluorescent hairpin DNA substrate (16). In this restriction enzyme-coupled assay, methylation of a hemimethylated CpG site forms a GlaI site and generates fluorescence in real time (Fig.…”

“…Dnmt1 Activity Assay-Dnmt1 activity was measured using an assay related to that developed by Wood et al (16) in which a hemimethylated, internally quenched, fluorescent hairpin DNA substrate is incubated with Dnmt1 and AdoMet in the presence of GlaI, which cleaves the fully methylated product. Assays (0.1 ml) were performed in triplicate in 96-well half-area black plates at 37°C.…”

“…Kinetic analysis established that Dnmt1 without the RFTS domain functioned with a K m of 1 nM for an internally quenched oligonucleotide substrate. This represents a Ͼ100-fold binding advantage with respect to recent assays with hemimethylated oligonucleotide substrates (9,16 2 To whom correspondence may be addressed. E-mail: sirano.dhepaganon@ utoronto.ca.…”

Biosensors based on molecular beacons

“…Many schemes for enzyme determination involving MBs utilise the DNA cleavage processes induced by nucleases (e.g. S1 nuclease, DNase I and mung bean nuclease) (Li et al, 2000b;Ma et al, 2008), DNA methylation by methylating enzymes and their inhibitors (Li et al, 2007;Ye & Stivers, 2010;Wood et al, 2010), DNA ligation catalysed by ligases Ma et al, 2008;He et al, 2011), DNA glycosylase removal of uracil bases inducing strand release , DNA phosphorylation , etc. A label-free fluorescence turn-on assay for the detection of endonuclease (Zhou et al, 2012a) has been designed based on G-quadruplex DNAzyme signal amplification.…”

Electrogenerated chemiluminescence biosensing method for methyltransferase activity using tris(1, 10-phenanthroline) ruthenium-assembled graphene oxide

“…The above methods usually challenged with some shortcomings, such as time-consuming, DNA-consuming, laborious treatment and lower sensitivity. Recently, the design of a hairpin DNA probe as a substrate of MTase has demonstrated a potential application in MTase activity detection [14,[22][23][24], which may overcome these limitations. It is still highly required to develop sensitive, simple biosensing methods for MTase quantification and activity assay by taking the advantage of hairpin DNA probe.…”