Use of Molecular Beacons in a Simple and Sensitive Fluorescence Assay for CG Methyltransferase Activity

Abstract

DNA methylation plays an important role in the blockage of restriction endonuclease cleavage, regulation of gene expression, cellular differentiation, DNA replication and repair, and chromosomal segregation. Signal amplification assays have been recently developed to assay DNA methyltransferase (MTase) activity with high sensitivity. However, these assays are expensive and have limited utility due to the exogenous reagents required, and due to multiple complex steps involved. In the present study, a simple, sensitive, and cost-effective assay was used to detect CG MTase activity using a molecular beacon (MB) consisting of a fluorophore and a fluorescent quencher attached at each end of its stem. The results showed that AccII cleavage of the MB led to a strong enhancement of fluorescence intensity, which was markedly higher than that of MB in its non-cleaved state. In addition, CG MTase activity, coupled with AccII activity, was monitored with high sensitivity at different concentrations of CG MTase.