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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\u2010molecule 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\u2013helix packing are advantageous for constructing large\u2010diameter nanopores. The resulting DNA\u2013ALM nanopores exhibited long\u2010lived 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\u2010molecule 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\u2010generation biosensing applications.<\/jats:p>","DOI":"10.1002\/anie.3311099","type":"journal-article","created":{"date-parts":[[2026,5,22]],"date-time":"2026-05-22T13:34:18Z","timestamp":1779456858000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Large\u2010Diameter DNA\u2010Scaffolded Nanopores Enabled by Loosely Packed Peptides for Single\u2010Molecule Sensing"],"prefix":"10.1002","volume":"65","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3446-6058","authenticated-orcid":false,"given":"Zugui","family":"Peng","sequence":"first","affiliation":[{"name":"Department of Biotechnology and Life Science Tokyo University of Agriculture and Technology  Koganei\u2010shi Tokyo Japan"},{"name":"School of Engineering Institute of 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