A cascade strand displacement amplification strategy for highly sensitive and label-free detection of DNA methyltransferase activity

DNA methyltransferase (MTase) is regarded as a promising biomarker and therapeutic target, which plays an important regulatory role in many biological processes. Here, a cascade strand displacement amplification strategy was explored for highly sensitive and label-free fluorescent detection of DNA MTase activity. Firstly, the hairpin recognition probe (RP) with a long stem was designed. DNA MTase catalyzed the RP methylation, and then DpnI endonuclease cleaved the methylated RP, releasing a trigger strand. Subsequently, it as a primer hybridized with hairpin probe 1 and triggered strand displacement amplification reaction (SDAR), producing numerous replaced strands. And the replaced strands hybridized with hairpin probe 2 and triggered next SDAR, producing a larger number of G-rich sequences. After that, N-methylmesoporphyrin IX was selectively intercalated into the G-quadruplex structures which were folded from the G-rich sequences, obtaining a significantly enhanced fluorescent signal. However, RP was stable, and could not produce primer for the subsequent cascade SDAR in the absence of DNA MTase. The effective amplification efficiency of cascade SDAR improved the detection sensitivity (detection limit: 0.0073 U/mL). Moreover, this strategy well evaluated the inhibitions of 5-fluorouracil, benzylpenicillin and gentamycin on DNA MTase activity. The results indicated this method held great potential in new method exploration and early disease diagnosis.