• Orbit mini

    Simultaneous recording from four lipid bilayers

2021 - Design, assembly, and characterization of membrane-spanning DNA nanopores

 Icon Orbit  Orbit 16 and Icon Orbit Mini Orbit mini publication in Nature Protocols (2021)

Lanphere C., Offenbartl-Stiegert D., Dorey A., Pugh G., Georgiou E., Xing Y., Burns J.R., Howorka S.


Nature Protocols (2021) doi: 10.1038/s41596-020-0331-7


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.

Download here.

Back to Overview

We use cookies on our website. Some of them are essential for the operation of the site, while others help us to improve this site and the user experience (tracking cookies). You can decide for yourself whether you want to allow cookies or not. Please note that if you reject them, you may not be able to use all the functionalities of the site.