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Conor Lanphere & Elena Georgiou: Design, assembly, and characterization of membrane-spanning DNA nanopores

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.

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