Reconstitution of Ultrawide DNA Origami Pores in Liposomes for Transmembrane Transport of Macromolecules

Molecular traffic across lipid membranes is a vital process in cell biology that involves specialized biological pores with a great variety of pore diameters, from fractions of a nanometer to >30 nm. Creating artificial membrane pores covering similar size and complexity will aid the understanding of transmembrane molecular transport in cells, while artificial pores are also a necessary ingredient for synthetic cells. Here, we report the construction of DNA origami nanopores that have an inner diameter as large as 30 nm. We developed methods to successfully insert these ultrawide pores into the lipid membrane of giant unilamellar vesicles (GUVs) by administering the pores concomitantly with vesicle formation in an inverted-emulsion cDICE technique. The reconstituted pores permit the transmembrane diffusion of large macromolecules, such as folded proteins, which demonstrates the formation of large membrane-spanning open pores. The pores are size selective, as dextran molecules with a diameter up to 28 nm can traverse the pores, whereas larger dextran molecules are blocked. By FRAP measurements and modeling of the GFP influx rate, we find that up to hundreds of pores can be functionally reconstituted into a single GUV. Our technique bears great potential for applications across different fields from biomimetics, to synthetic biology, to drug delivery.