Fluorescence biosensor for DNA methyltransferase activity and related inhibitor detection based on methylation-sensitive cleavage primer triggered hyperbranched rolling circle amplification

Abstract

Highly sensitive and selective detection of DNA adenine methylation methyltransferase (Dam MTase) activity is essential for clinical diagnosis and treatment as Dam MTase can catalyze DNA methylation and has a profound effect on gene regulation. In this study, a fluorescence biosensor has been developed for label-free detection of Dam MTase activity via methylation-sensitive cleavage primers triggered hyperbranched rolling circle amplification (HRCA). A hairpin DNA probe (HP) with a Dam MTase specific recognition sequence on the stem acting as a substrate has been designed. This substrate probe can be methylated by the target in the system and subsequently cleaved by DpnI, which results in the release of the primer release probe (RP) and hence in turn triggers the subsequent HRCA reaction. As the HRCA products contain many double-strand DNA (dsDNA) with different lengths, and the SYBR Green I can be embedded in the dsDNA to produce a strong fluorescence signal. However, in the absence of the target, the presence of the probe HP in the form of a hairpin cannot induce the HRCA reaction, and only weak fluorescence intensity can be detected. Under the optimized conditions, the fluorescence of the system has a linear relationship with the logarithm of the concentration of Dam MTase in the range of 2.5–70 U/mL with a detection limit of 1.8 U/mL. The Dam MTase can be well distinguished from other MTase analogs. The developed sensor was applied to detect target in serum and E. coli cell lysate, and the standard recovery rates were in the range of 96%–105%. The results showed that this method has great potential for assessing Dam MTase activity in complex biological samples. In addition, the method has been applied to detect the related inhibitors with high efficiency.