DNA-origami scaffold for NPC mimics

Abstract

In eukaryotic cells, the segregation of nuclear information from the cytosol is carried out by the Nuclear Envelope, a double lipid membrane which protects the genes from cytoplasmic mutagens. To establish a connection between the cytoplasm and nucleoplasm, tiny pores named Nuclear Pore Complexes (NPCs) span through such membrane. The biological function of the NPC consists in regulating the bidirectional transportation of small molecules, like ions and transcriptional proteins, between the two compartments by imparting a selective barrier. FG-Nups are key proteins in this process, as they line the inner channel of the NPC and are responsible for the selective behaviour. Despite many models have attempted to explain the mechanism behind selectivity and substantially agree on the importance of FG-Nups in the context of nuclear transport, the exact details about how FG-Nups arrange within the NPC lumen and interact with translocating cargoes are still debated. In the current project, we illustrate an in-vitro approach to study the behaviour of purified FG- Nups, that takes advantage of DNA-origami nanotechnology. We show that it is possible to recreate a minimalistic version of the NPC by using an octagonally shaped DNA-origami as a scaffold while keeping low concentrations of magnesium in solution (< 1mM). The NPC mimic was built by coupling FG-Nups to specified locations along the inner wall of the origami. We characterized the DNA-origami structure, under different buffer conditions, before and after functionalization, by means of negative staining transmission electron microscopy and liquid atomic force microscopy. A volumetric comparison between the bare and FG Nup-coated DNA-origami provide quantitative confirmation of the success of the protein administration. We show that the incubation of a 10 times excess of proteins per binding site on the DNA-origami successfully couples the complexes together. Finally, we present our preliminary attempts at inserting a cholesterol-modified version of the DNA-origami molecule into both large and small vesicles, as well as planar lipid bilayers.