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DNA scaffolds support stable and uniform peptide nanopores

Nature Nanotechnology volume 13, pages 739–745 (2018)Cite this article

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Abstract

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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Fig. 1: Design, assembly and characterization of the DNA scaffolds for peptide nanopores.
Fig. 2: Attachment of the Wza peptides to oligonucleotides and annealing of peptide-bearing DNA scaffolds.
Fig. 3: Electrical properties of DNA-scaffolded octameric Wza peptide nanopores.
Fig. 4: The DNA scaffold stabilizes Wza peptide nanopores.
Fig. 5: The DNA scaffold is a versatile docking site for tagged oligonucleotides.

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Acknowledgements

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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Author notes

  1. Evan Spruijt

    Present address: Institute for Molecules and Materials, Radboud University, Nijmegen, The Netherlands

Authors and Affiliations

  1. Chemistry Research Laboratory, University of Oxford, Oxford, UK

    Evan Spruijt & Hagan Bayley

  2. Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, UK

    Samuel E. Tusk

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Contributions

E.S. and H.B. conceived and designed the experiments and wrote the paper. E.S. performed the experiments and simulations and analysed data. S.T. performed photobleaching experiments and analysed data. All the authors discussed and commented on the manuscript.

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Correspondence to Evan Spruijt or Hagan Bayley.

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Supplementary Information

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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