Electrical Imaging of DNA Substructures Using Quasi-Static Nanopore Scanning

DNA nanotechnology has advanced beyond sequence design toward precise control of local substructures, such as single-stranded gaps and branched motifs, whose configuration governs mechanical stability and function. However, quantitative interrogation of these dynamic elements at the single-molecule level under native solution conditions remains challenging. Here, we present a quasi-static nanopore scanning strategy that enables deterministic electrical imaging of DNA substructures. Using surface-tethered dual-gap DNA scaffolds, we demonstrate that ionic blockade amplitudes from unstructured single-stranded branches scale with high linearity (R 2 = 0.998) over nearly an order of magnitude in length (10–81 nt), achieving 5-nucleotide resolution. In contrast, base-paired architectures (hairpins and aptamers) exhibit pronounced nonlinear amplification. This work establishes nanopore scanning as a quantitative electrical imaging modality for simultaneous readout of branch length and topology, providing a foundation for quality control, structural validation, and real-time monitoring of complex DNA nanodevices.