A Programmable DNA Origami Platform for Organizing Intrinsically Disordered Nucleoporins within Nanopore Confinement

Abstract

Nuclear pore complexes (NPCs) form gateways that control molecular exchange between the nucleus and the 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 nuclear pore proteins rich in Phe-Gly (FG)-repeat domains of different types, such as FxFG and GLFG. While physical modeling and in vitro approaches have provided a framework for explaining how the FG network contributes to the barrier and transport properties of the NPC, it remains unknown whether the number and/or the spatial positioning of different FG-domains along a cylindrical, ∼40 nm diameter transport channel contributes to their collective properties and function. To begin to answer these questions, we have used DNA origami to build a cylinder that mimics the dimensions of the central transport channel and can house a specified number of FG-domains at specific positions with easily tunable design parameters, such as grafting density and topology. We find the overall morphology of the FG-domain assemblies to be dependent on their chemical composition, determined by the type and density of FG-repeat, and on their 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 the DNA central channel mimics, suggestive of how the NPC might establish its permeability properties.

Intrinsically disordered nuclear pore proteins show ideal-polymer morphologies and dynamics

“…We now investigate how the identified ideal polymer behaviour translates into a pore assembly, as here probed by MD simulations on NPC-mimetics based on welldefined numbers and types of FG nups that are grafted in a DNA origami pore scaffold, with an inner radius (∼20 nm) comparable to that of the NPC [44]. We observe that the change in polymer compaction is even more abrupt (in the relevant ǫ pp range) for FG nups that are assembled in a pore geometry (Figures 4 and S7, and Video S3).…”

“…We next investigated the resealing dynamics of the barrier, i.e., how fast and by how much polymers fill the centre of the pore following a perturbation: holes (void of polymers) of 10, 20, and 30 nm diameter are created in an NPC-mimetic pore system containing 48 polymers [44] (Figures 6a and S8, and Video S4).…”

“…4. Investigating polymer morphology in an NPCmimicking geometry containing one type of FG nup [44]. a) MD snapshots of polymers in a pore (polymers in blue; inner pore scaffold in grey; grafting handles in red) for various interaction regimes (48 polymers, N = 300 beads).…”

Biomimetic Compartments Scaffolded by Nucleic Acid Nanostructures

“…[21a] Liu et al used charge neutralization to drive the shape reconfiguration of a biomimetic DNA nanochannel . Fisher et al used DNA nanopores to confine and organize disordered nucleoporins that mimicked the central transport channel of nuclear pore complexes . Recently, a synthetic DNA‐based scramblase was reported capable of transporting lipid molecules between the two bilayer leaflets at a very high speed of ≈10 7 lipids per second .…”

DNA Origami Post‐Processing by CRISPR‐Cas12a

“…Considering DNA-origami structures usually display staple extensions (termed handles) for subsequent chemical modifications (e.g., fluorophores, lipids, and proteins), we reasoned such ssDNA handles must be pre-hybridized to evade Cas12a cleavage. To confirm this, we constructed DNAorigami circles [13] with 8 outward-facing handles. As expected, when premixed with Cy5-labeled complementary ssDNA (anti-handles), the circle retained its fluorophores after 30 minutes of Cas12a digestion (Figure 2 b and S27-S30).…”