De Novo Design of α-helical Peptide Nanopores for Single-Molecule Detection Using Helix Packing Motifs

Nanopore sensing using transmembrane proteins holds great potential for single-molecule detection. The creation of pore-forming proteins is an emerging research direction, and one promising strategy is the bottom-up de novo design of nanopores. However, de novo α-helical nanopores with sufficiently large dimensions to accommodate single-molecule sensing have not been previously reported. Here, we designed and constructed α-helical nanopores based on two helix-packing motifs, GX₆G and GX₃G, which we named FFK and LEK. Electrical recordings revealed that both FFK and LEK form nanopores in lipid membranes with high conductance. FFK exhibited multiple conductance states, whereas LEK formed more monodisperse pores. Although both nanopores assembled via a stepwise monomer-joining mechanism, the assembly and disassembly dynamics of FFK were significantly faster than those of LEK. We further demonstrated the single-molecule sensing capabilities of both pores, showing that they can detect cyclodextrin derivatives, while LEK could additionally sense poly-l-lysine. Overall, these results demonstrate the feasibility of designing functional α-helical nanopores for nanopore sensing technologies.