Methylation-blocked enzymatic recycling amplification for highly sensitive fluorescence sensing of DNA methyltransferase activity

Abstract

Herein, using DNA adenine methylation (Dam) methyltransferase (MTase) as a model analyte, a novel fluorescence sensing strategy was developed for facile, rapid and highly sensitive detection of the activity and inhibition of the target based on methylation-blocked enzymatic recycling amplification. In this sensing system, nicking endonuclease Nt.AlwI with the methylation-sensitive property was selected to achieve signal amplification. In addition, a DNA heteroduplex probe is specially designed to contain the recognition sequences for both Dam MTase and Nt.AlwI. In the absence of Dam MTase, Nt.AlwI cleaves the DNA heteroduplex at only the top strand. At the reaction temperature, the cleaved heteroduplex is unstable and readily separates. The released bottom strand can hybridize with the molecular beacons (MB) and subsequently trigger Nt.AlwI-mediated recycling cleavage of MBs, providing a dramatically amplified fluorescence signal. However, when the heteroduplex is methylated by Dam MTase, the cleaving operation is blocked, resulting in an inconspicuous fluorescence enhancement. Unlike existing signal amplified assays which use at least two enzymes, only one is involved in this amplified strategy. Under optimized conditions, the sensing system reveals a detection limit of 0.05 U mL⁻¹ in a short assay time (65 min), which is much superior to all presently reported methods except for two electrochemical biosensors (0.04 U mL⁻¹). Furthermore, the application of the assay in human serum and screening of Dam MTase inhibition were demonstrated with satisfactory results. Overall, the proposed sensing system shows great potential for further application in biological research, early clinical diagnosis and designed drug therapy.