Polyarginine-functionalized AP site probes for mechanistic analysis of uracil–DNA glycosylase via nanopore-based single-molecule sensing

Abstract

The interaction between abasic (apurinic/apyrimidinic, AP) sites and aberrant uracil DNA glycosylase (UDG) contributes to cancer progression, establishing AP site recognition and UDG detection as critical tools for bioanalytical applications and clinical diagnostics. However, existing methods face challenges in achieving high sensitivity and specificity. In this work, we developed DNA–R5, a novel DNA adduct formed by conjugating AP sites with polyarginine-5 (R5), which generates distinctive current signatures in nanopore sensing. While signal production remained sequence-independent, it exhibited dependence on arginine unit quantity, linker length, and electrolyte concentration. Characteristic signal patterns enabled clear identification of translocation events through the oscillating features. Quantitative analysis of high-frequency current signatures achieved UDG activity detection with sensitivity reaching 0.0005 U mL⁻¹, demonstrating DNA–R5's utility as a recognition biosensor. This molecular design strategy suggests potential applications for detecting diverse DNA lesions and biomolecular interactions.