The assembly of peptides into membrane-spanning nanopores might be promoted by scaffolds to pre-organize the structures. Such scaffolds could enable the construction of uniform pores of various sizes and pores with controlled permutations around a central axis. Here, we show that DNA nanostructures can serve as scaffolds to arrange peptides derived from the octameric polysaccharide transporter Wza to form uniform nanopores in planar lipid bilayers. Our ring-shaped DNA scaffold is assembled from short synthetic oligonucleotides that are connected to Wza peptides through flexible linkers. When scaffolded, the Wza peptides form conducting nanopores of which only octamers are stable and of uniform conductance. Removal of the DNA scaffold by cleavage of the linkers leads to a rapid loss of the nanopores from the lipid bilayer, which shows that the scaffold is essential for their stability. The DNA scaffold also adds functionality to the nanopores by enabling reversible and permanent binding of complementary tagged oligonucleotides near the nanopore entrance.
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E.S. acknowledges support from the European Union's Horizon 2020 Programme under grant 655660 (Hybripore). The work was supported by the BBSRC and a European Research Council Advanced Grant. The authors thank O. Wilner and K. R. Mahendran for their helpful discussions, E. Johnson for help with the TEM measurements and I. Liko and C. V. Robinson for their assistance in mass spectrometry.
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Supplementary Methods, Supplementary Data 1–4 and Supplementary References
Supplementary Video 1
OxDNA2 coarse-grained MD simulation of the DNA scaffold without external force applied to the arms. Duration, 0.152 ms; frame rate, 16.4 µs–1.
Supplementary Video 2
OxDNA2 coarse-grained MD simulation of the DNA scaffold with an external force applied to all terminal 3ʹ and 5ʹ nucleotides in the direction perpendicular to the plane of the ring leading to a down-pointing orientation of all arms within 5 µs. Duration, 30 µs; frame rate, 11.6 µs–1.
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Spruijt, E., Tusk, S.E. & Bayley, H. DNA scaffolds support stable and uniform peptide nanopores. Nature Nanotech 13, 739–745 (2018). https://doi.org/10.1038/s41565-018-0139-6
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