Coupling hybridization chain reaction with DNAzyme recycling for enzyme-free and dual amplified sensitive fluorescent detection of methyltransferase activity

- A sensitive fluorescent method for detecting Dam MTase activity is developed.
- Signal amplification is achieved by the integration of HCR with DNAzyme recycling.
- The method is convenient and shows high sensitivity.
- The application of this method for anti-cancer drug screening is also demonstrated.

Aberrant DNA methylation originated from changes in DNA methyltransferase activity can lead to many genetic diseases and tumor types, and the monitoring of methyltransferase activity is thus of great importance in disease diagnosis and drug screening. In this work, by combing hybridization chain reaction (HCR) and metal ion-dependent DNAzyme recycling, we have developed a convenient enzyme-free signal amplification strategy for highly sensitive detection of DNA adenine methyltransferase (Dam MTase) activity and its inhibitors. The Dam MTase-induced methylation and subsequent cleavage of the methylated hairpin DNA probes by DpnI endonuclease lead to the release of ssDNA triggers for HCR formation of many Mg2+-dependent DNAzymes, in which the fluorescently quenched substrate sequences are catalytically and cyclically cleaved by Mg2+ to generate remarkably amplified fluorescent signals for highly sensitive detection of Dam MTase at 7.23 × 10−4 U/mL. In addition, the inhibition of different drugs to Dam MTase activity can also be evaluated with the developed method. With the advantages of simplicity and significant signal amplification over other common methods, the demonstrated biosensing approach thus offers great potential for highly sensitive detection of various methyltransferases and provides a convenient platform for drug screening for therapeutic applications.