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Design, assembly, and characterization of membrane-spanning DNA nanopores

Abstract

DNA nanopores are bio-inspired nanostructures that control molecular transport across lipid bilayer membranes. Researchers can readily engineer the structure and function of DNA nanopores to synergistically combine the strengths of DNA nanotechnology and nanopores. The pores can be harnessed in a wide range of areas, including biosensing, single-molecule chemistry, and single-molecule biophysics, as well as in cell biology and synthetic biology. Here, we provide a protocol for the rational design of nanobarrel-like DNA pores and larger DNA origami nanopores for targeted applications. We discuss strategies for the pores’ chemical modification with lipid anchors to enable them to be inserted into membranes such as small unilamellar vesicles (SUVs) and planar lipid bilayers. The procedure covers the self-assembly of DNA nanopores via thermal annealing, their characterization using gel electrophoresis, purification, and direct visualization with transmission electron microscopy and atomic force microscopy. We also describe a gel assay to determine pore–membrane binding and discuss how to use single-channel current recordings and dye flux assays to confirm transport through the pores. We expect this protocol to take approximately 1 week to complete for DNA nanobarrel pores and 2–3 weeks for DNA origami pores.

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Fig. 1: Schematic overview of the design, assembly and characterization of DNA nanopores.
Fig. 2: Overview of several existing DNA nanopores.
Fig. 3: DNA nanopores designed via the nanobarrel or DNA origami method.
Fig. 4: Nanobarrel 2D strand maps showing the connectivity of various designs for nanobarrel-type DNA pores.
Fig. 5: Screenshots taken from CaDNAno showing the two types of lattice network.
Fig. 6: The 72HB DNA origami nanopore designed using the CaDNAno interface with Maya 2015.
Fig. 7: Using CaDNAno to generate the DNA origami structure and staple sequences.
Fig. 8: Gel electrophoretic characterization of DNA nanopores.
Fig. 9: Representative TEM images of nanobarrel and DNA origami nanopores and their interaction with lipid membranes.
Fig. 10: Gel-shift assay for electrophoretic quantification of DNA nanopore binding to SUVs.
Fig. 11: Single-channel current analysis of DNA nanopores using planar lipid bilayer recordings.
Fig. 12: Extracting data from single-channel current traces of DNA nanopores.
Fig. 13: Example traces of the dye flux assay.

Data availability

All data are available from the corresponding author upon reasonable request.

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Acknowledgements

The authors thank J. Ciccone, R. Dickman, and H. Philpott for helping with figures and providing valuable feedback. Furthermore, the authors thank C. Weichbrodt from Nanion Technologies for providing feedback on the section of single-channel current recordings. The Howorka Group receives funding from the EPSRC (EP/ N009282/1), the BBSRC (BB/M025373/1, BB/N017331/1), and the Leverhulme Trust (RPG-2017-015). C.L. and E.G. are supported by the Biotechnology and Biological Sciences Research Council (BB/MO09513/1). C.L. is also supported by the National Physical Laboratory.

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Contributions

C.L., D.O.-S., G.P., and J.R.B. provided the protocols for design and assembly. D.O.-S. and G.P. supplied the protocol for CanDo. C.L., D.O.-S, Y.X., and J.R.B. prepared the protocol for cholesterol lipid anchors. J.R.B. contributed the protocol for the alkyl modification of DNA. C.L. and D.O.-S. generated the protocol for gel electrophoretic characterization. E.G. prepared the protocols for purification of DNA origami structures. Y.X. provided the protocols for TEM analysis, and J.R.B. provided the protocols for AFM. C.L. provided the protocol for gel-binding assays. A.D. wrote the protocol for single-channel current recordings. C.L. provided the protocol for dye flux assays. D.O.-S. compiled the list of and set up materials and equipment. C.L., A.D., and J.R.B. supplied the troubleshooting advice. C.L., D.O.-S., J.R.B., and S.H. wrote the manuscript with input from all authors. C.L., D.O.-S, J.R.B., and S.H. edited the manuscript. C.L., A.D., G.P., and J.R.B. generated the figures, and J.R.B. edited them. All authors were part of the data analysis and discussions.

Corresponding authors

Correspondence to Jonathan R. Burns or Stefan Howorka.

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Competing interests

G.P., J.R.B., and S.H. hold patents on DNA nanopores that have been licensed to Oxford Nanopore Technologies.

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Key references using this protocol

Diederichs, T. et al. Nat. Comm. 10, 5018 (2019): https://doi.org/10.1038/s41467-019-12639-y

Burns, J. R., Seifert, A., Fertig, N. & Howorka, S. Nat. Nanotechnol. 11, 152–156 (2016): https://doi.org/10.1038/nnano.2015.279

Langecker, M. Science 338, 932–936 (2012): https://doi.org/10.1126/science.1225624

Burns, J. R., Stulz, E. & Howorka, S. Nano Lett. 13, 2351–2356 (2013): https://doi.org/10.1021/nl304147f

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Lanphere, C., Offenbartl-Stiegert, D., Dorey, A. et al. Design, assembly, and characterization of membrane-spanning DNA nanopores. Nat Protoc 16, 86–130 (2021). https://doi.org/10.1038/s41596-020-0331-7

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