Single‐Molecule Detection and Characterization of Non‐Canonical DNA Structures With α‐Hemolysin‐Based Protein Nanopore Technology

ABSTRACT Nanopore‐based approaches have emerged as versatile, cost‐effective platforms for nanoscale biosensing and single‐molecule analysis, delivering high sensitivity and throughput. These capabilities are underpinned by two intrinsic attributes: internal geometries commensurate with the physical dimensions of targeted molecules, and the ability to engineer the sensing interface with atomic precision—achieved through mutagenesis in biological nanopores or controlled functionalization in synthetic systems. Ultra‐sensitive molecular detection is achieved through analysis of ionic current fluctuations across the nanopore, where the frequency, relative amplitude, and duration of current blockades report on the size and charge of the analyte, and the underlying Markovian dynamics stemming from intrinsic conformational changes or interactions with the nanopore. In this paper, we highlight recent developments of the α‐hemolysin (α‐HL) protein nanopore, to observe and perform real‐time monitoring of the folding dynamics and conformational distribution of various DNA motifs, at the single‐molecule level, in the presence of various environmental factors. The α‐HL's potential to study the interactions of small molecule ligands and various nucleic acid structures, underscores impactful biologically relevance for future screening of drugs acting on such motifs, and underlines a transformative potential in precision medicine, bridging biological detection with potential applications in disease treatment and bioengineering.