Programmable Assembly of Intrinsically Disordered FG-nucleoporins in DNA Origami Channel

Nuclear pore complexes (NPCs) form gateways that control molecular exchange between the nucleus and cytoplasm. They impose a diffusion barrier to macromolecules and enable the selective transport of nuclear transport receptors with bound cargo. The underlying mechanisms that establish these permeability properties remain to be fully elucidated, but require unstructured nucleoporins/nups rich in Phe-Gly (FG)-repeats. The FG-nup collective consists of thousands of FG domains of different types like FxFG and GLFG. While physical modeling and in vitro approaches have provided a framework for explaining the barrier and transport properties of the NPC, there remains a need for experimental platforms that can interrogate precise numbers of unique FG-nups in an NPC-mimicking geometry. In particular, it remains unknown how the confinement of FG-domains within a cylindrical nanochannel impacts their collective properties and function. Here, I used DNA origami to build nucleoporins on DNA (NuPODs) that house a specified number of FG-domains at specific positions within a ~45-nm diameter cylinder with easily tunable design parameters, such as grafting density and topology. I found the overall morphology of the FG-nup assemblies to be dependent on the chemical composition, determined by the type and density of FG-domain, and on the architectural confinement provided by the DNA cylinder, largely consistent with here presented molecular dynamics simulations based on a coarse-grained polymer model. In addition, high speed atomic force microscopy reveals local and reversible FG-domain condensation that transiently occludes the lumen of NuPODs, thus providing a new perspective on how the NPC establishes its unique permeability properties.