A loop-mediated cascade amplification strategy for highly sensitive detection of DNA methyltransferase activity

Abstract

DNA methyltransferase (MTase) is a predictive cancer biomarker and drug target, and the sensitive detection of DNA MTase activity is crucial to early cancer diagnosis and therapy. In this work, we developed a loop-mediated cascade amplification strategy for highly sensitive fluorescent detection of DNA MTase activity based on strand displacement amplification (SDA) and exponential rolling circle amplification (ERCA). Firstly, we designed a long stem-loop probe (LSLP) which contains a methylation site for DNA MTase recognition, a long stem for ensuring the stability of probe, and a loop for initiating subsequent amplification. The loop and part of stem of LSLP acted as a trigger strand for subsequent signal output process. And the trigger strand was fully enclosed in loop of LSLP by the long stem, avoiding the nonspecific amplification caused by the leakage of probe. The LSLP was methylated by DNA MTase and then was specifically cleaved by DpnI endonuclease, producing a trigger strand. Under the synergetic action of polymerase and nicking enzyme, the trigger strand initiated SDA, producing many primers. The produced primers initiated ERCA, synthesizing numerous G-quadruplex sequences. The G-quadruplex sequences interacted with N-methylmesoporphyrin IX, obtaining an enhanced fluorescent signal. The method could detect as low as 8.1×10−5U/mL DNA MTase. Furthermore, this assay was successfully used to assess the inhibition effect of inhibitors for DNA MTase activity. These results show that our system has a great potential in early cancer diagnosis and therapy.