Dna-Polyarginine Probe-Enabled Nanopore Sensing for Ultrasensitive Detection of Dam Methyltransferase Activity and Inhibition

Abstract

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 and verification were conducted through mutant protein, hairpin sequence, pH and voltage conditions. 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.

Note:
Funding declaration: We acknowledge the funding support from the National Natural Science Foundation of China (No. 22204067, No. 41903020). The Science Foundation of BGI·Research (HX2021103). Talent Research and Development Program of Mianyang Normal University (No. 22204067-1).

Conflict of Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Keywords: nanopore, DNA-R5, Dam MTase, Magnetic Beads