DNA-polyarginine probe-enabled nanopore Sensing for ultrasensitive detection of Dam methyltransferase activity and inhibition

Aberrant DNA methyltransferase (DNA MTase) activity correlates with abnormal DNA methylation patterns observed in various cancers, establishing DNA MTase as a therapeutic target for early clinical diagnosis and anticancer/antimicrobial treatment. Current DNA MTase detection methods remain laborious and technically challenging. This study presents a label-free, single-molecule nanopore sensing strategy for sensitive Dam MTase detection utilizing a DNA-polyarginine (DNA-R5) probe. The unique oscillating pattern of DNA-R5 contributes specificity, while other DNA structures cannot output such signatures. Herein, transformation to oscillating signal occurred by Hairpin DNA-R5 releasing the single-strand DNA-R5 with subsequent Dam methylation and DpnI excision. Optimization of mutant protein positioning, hairpin loop length, pH-dependent charge maintenance, and voltage for electroosmosis balance was experimentally validated, with pH 8.0 at 140 mV identified as optimal for signal generation. The integrated use of magnetic beads can lower the Dam detection limit to 0.00005 U/mL. Validation in diluted human serum confirmed the assay implementation while penicillin inhibition studies demonstrated the platform's drug discovery potential. Our proposed approach shows promise for clinical diagnostics, with DNA-R5 serving as a versatile probe for future therapeutic target analysis, the next plan for larger-scale clinical trials was the testing of bacterial lysates in the patient's serum and track the Dam progress before and after drug treatment.