Evaluation of DNA Methyltransferase Activity and Inhibition via Isothermal Enzyme-Free Concatenated Hybridization Chain Reaction

Methyltransferase (MTase)-catalyzed DNA methylation plays a vital role in the biological epigenetic processes of key diseases and has attracted increasing attention, making the amplified detection of MTase activity of great significance in clinical disease diagnosis and treatment. Herein, we developed an isothermal, enzyme-free, and autonomous strategy for analyzing MTase activity based on concatenated hybridization chain reaction (C-HCR)-mediated Forster resonance energy transfer (FRET). In a typical C-HCR procedure without MTase (Dam), Y-shaped initiator DNA activates upstream HCR-1 to assemble a double-stranded DNA (dsDNA) copolymeric nanowire consisting of multiple tandem DNA trigger units that motivate downstream HCR-2 to successively bring a fluorophore donor/acceptor (FAM/TAMRA) pair into close proximity, leading to the generation of an amplified FRET readout signal. The target Dam MTase and auxiliary DpnI endonuclease can sequentially and specifically recognize/methylate and cleave the Y-shaped in...