Single-Molecule Analysis of Glycopeptide Antibiotic-Target Interactions Using Nanopore-Based Biomimetic Probes

Glycopeptide antibiotics serve as critical last-resort drugs against resistant pathogens. However, emerging resistance─primarily due to altered interactions between glycopeptides and their bacterial targets─has begun to compromise their clinical effectiveness. Understanding the dynamic, molecular-level interactions between glycopeptide antibiotics and their targets is essential for elucidating resistance mechanisms and guiding the design of next-generation therapeutics. Conventional techniques, however, lack the resolution to capture these transient binding events in real time. In this study, we present a nanopore-based biomimetic probe strategy to study glycopeptide-peptidoglycan interactions at the single-molecule level. A series of biomimetic peptidoglycan precursor peptides (BPGPPs) with the sequence of (DE)_nA^DEK(G₅)^DA^DA have been designed. Using these BPGPPs and the α-hemolysin (α-HL) nanopore, we achieved real-time single-molecule recordings of BPGPP-glycopeptide complex dissociation for a range of commercially available glycopeptides. The binding affinity of each glycopeptide toward BPGPPs is reflected by the mean dwell time of the complex within the α-HL nanopore. Furthermore, by investigating terminal residue mutations in BPGPPs, we gained insight into natural resistance mechanisms and potential trends in future glycopeptide-resistant strains. This research offers a time- and space-resolved single-molecule method for investigating glycopeptide-peptidoglycan interactions and holds promise for guiding the development of next-generation antimicrobial agents.