Reconstitution of ultrawide DNA origami pores in liposomes for transmembrane transport of macromolecules

Abstract

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 new 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 22 nm can traverse the pores, whereas larger dextran molecules are blocked. By FRAP measurements and modelling 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, synthetic biology, to drug delivery.

Advanced microfluidic tool for rapid buffer exchange and in situ imaging of phase separation in immobilized giant unilamellar vesicles

“…For example, the lipid composition can be modulated to study, for example, the interplay between LLPS and membrane interactions (Last et al, 2020). Additionally, adjustments in pore size and shape, such as using origami pores (Cochereau et al, 2023;Fragasso et al, 2021;Groeer, Garni, Samanta, & Walther, 2022;Krishnan et al, 2016;Shen et al, 2023) can be implemented to further customize the microenvironment for speci c experiments.…”

Experimental tests of functional molecular regeneration via a standard framework for coordinating synthetic cell building

“…For example, on a quantitative volumetric basis, we estimate that PURE is only capable of remaking ~1% of the peptide bonds needed to instantiate PURE itself; expression capacity will need to be increased ~87-fold to enable sustained self-reproduction (Materials and Methods). However, such quantitative challenges can likely be addressed via exogenous supply of resources directly from the environment, via booting up of a regenerative and auto-sustaining metabolism, or by addition of enzymes and mechanisms that increase the efficiency of protein expression by reducing the number of truncated peptides produced and decreasing the rate of ribosomal stalling [17,[21][22][23][24][25]. Whether PURE can satisfy the qualitative criteria for self-reproduction is less clear.…”