Large-Diameter DNA-Scaffolded Nanopores Enabled by Loosely Packed Peptides for Single-Molecule Sensing

Creating alternative nanopores is crucial for advancing single-molecule methodologies. Peptide nanopores scaffolded by DNA nanostructures have emerged as a promising class of chemically synthesizable nanopores with organized geometries. However, no such nanopores have yet achieved single-molecule detection due to size limitations. Here, we overcome this limitation by employing the alamethicin (ALM) peptide, based on the hypothesis that peptides with looser helix-helix packing are advantageous for constructing large-diameter nanopores. The resulting DNA-ALM nanopores exhibited long-lived open states and conductance values comparable to those of natural protein nanopores. To further enhance pore functionality, we introduced asymmetric electrostatic forces by harnessing the intrinsic helix dipole of ALM, which facilitated pore expansion and improved ion transport efficiency. This modification led to enhanced conductance levels and enabled single-molecule sensing of biomolecules, including DNA and peptides. Overall, this work demonstrates a generalizable strategy for constructing functional, chemically synthesizable nanopores, providing a foundation for programmable nanopore design and next-generation biosensing applications.