DNA-Origami NanoTrap for Studying the Selective Barriers Formed by Phenylalanine-Glycine-Rich Nucleoporins

Abstract

DNA nanotechnology provides a versatile and powerful tool to dissect the structure-function relationship of biomolecular machines like the nuclear pore complex (NPC), an enormous protein assembly that controls molecular traffic between the nucleus and cytoplasm. To understand how the intrinsically disordered, Phe-Gly-rich nucleoporins (FG-nups) within the NPC establish a selective barrier to macromolecules, we built a DNA-origami NanoTrap. The NanoTrap comprises precisely arranged FG-nups in an NPC-like channel, which sits on a baseplate that captures macromolecules that pass through the FG network. Using this biomimetic construct, we determined that the FG-motif type, grafting density, and spatial arrangement are critical determinants of an effective diffusion barrier. Further, we observed that diffusion barriers formed with cohesive FG interactions dominate in mixed-FG-nup scenarios. Finally, we demonstrated that the nuclear transport receptor, Ntf2, can selectively transport model cargo through NanoTraps composed of FxFG but not GLFG Nups. Our NanoTrap thus recapitulates the NPC's fundamental biological activities, providing a valuable tool for studying nuclear transport.

Figure 1.

A DNA-origami NanoTrap built from two pre-assembled parts, a channel and a baseplate. (A) Cartoon models and negative-stain EM images of the channel. (B) Cartoon models and negative-stain EM images of the baseplate. (C) Two teeth (blue) with sticky ends (blue arrows) mediate the baseplate attachment onto the channel, which contains two cavities with complementary sticky ends (red arrows), resulting in the formation of a NanoTrap (cartoon model and negative-stain EM images shown on the right). (D) Up to four layers of DNA handles (12 handles per layer) protrude from the channel wall (green curls), serving as anchor points for anti-handle-conjugated proteins. The baseplate displays three “bait” oligonucleotides (red curls/dots) to capture nanoscale objects carrying “prey” oligonucleotides. (E) Cartoon models and EM images showing the immobilization of prey-oligo-modified AuNPs (dark spots) inside the NanoTrap. Circles indicate standalone baseplates. Scale bars = 50 nm.

Figure 2.

Assembly and characterization of nucleoporin-gated NanoTraps. (A) Two central channel FG-nup domains of yeast origin, Nsp1 (FxFG-rich) and Nup100 (GLFG-rich), were cloned and expressed in E.coli as MBP-SUMO-nup-SNAP fusions. Such SNAP-tag bearing nucleoporins were conjugated with benzylguanine modified DNA oligonucleotides (BG-DNA) and verified by SDS-PAGE. (B) Cartoon models (top) and negative-stain EM images (bottom) of an ungated (empty) and various protein-gated NanoTraps. Scale bar = 50 nm.

Figure 3.

The influence of FG-nup type, density and geometric distribution on the barrier permeability. (A) Schematic diagrams showing the permeability assay using an FG-nup-gated NanoTrap and fluorescently tagged macromolecules of different sizes (106 kD MBP-GFP-SNAP-prey, 53 kD GFP-SNAP-prey, and 7 kD Alexa488-prey). (B) The barrier strengths of different gating proteins (48 copies per NanoTrap) against the 53 kD GFP-SNAP-prey. (C) Size-selective diffusion barriers formed by 48 copies of nucleoporins (Nup100 or Nsp1) within a NanoTrap. The penetration levels of the GFP-SNAP-prey (53 kD GFP) and Alexa488-prey (7 kD oligo) into empty NanoTraps were both set as 100%, serving as references (REF) for quantifying penetration levels of the GFP (53 and 106 kD) and Alexa488 labeled (7 kD) molecules into nup-gated NanoTraps, respectively. (D) Permeability of NanoTraps (4 nM, 48×handles per trap) formed at different nucleoporin (Nup100 or Nsp1) concentrations (0–800 nM), tested against the 53 kD GFP-SNAP-prey. Fitted curves are guides to the eye. (E) Permeability of NanoTraps containing 12–48 copies of Nup100 or Nsp1 tested against the 53 kD GFP-SNAP-prey. The exact nup arrangement is shown by the schematic drawing at the top of each lane (blue: Nup100, green: Nsp1, 12 nups/layer). Fitted curves are guides to the eye. Statistical data are plotted to show mean ± standard error of the mean (SEM) from three trials.

Figure 4.

Different nucleoporin arrangements affect barrier permeability. (A) Permeability of NanoTraps with the FG-nups located near the entrance (top) or the baseplate (bottom) of the NanoTrap, tested against the 53kDa GFP-SNAP-prey. The exact nup arrangement is shown by the schematic drawing at the top of each lane (blue: Nup100, green: Nsp1, 12 nups/layer). Statistical data are plotted to show mean ± SEM. Statistical significance was determined by a two-tailed Student’s t-test; n=3; NS: not significant (P≥0.05); **: P<0.01. (B) Permeability of NanoTraps containing single or mixed types of FG-nups, tested against the 53kDa GFP-SNAP-prey. The exact nup arrangements are shown by the schematic drawings at the top of each group of bars (blue: Nup100, green: Nsp1, 12 nups/layer). Data are plotted to show mean ± SEM. Difference between mixed-nup and Nup100-NanoTraps was analyzed by two-way ANOVA and Tukey’s multiple comparison; n=3; NS: not significant (P≥0.05); *: P<0.05; ***:P<0.001.

Figure 5.

Ntf2-mediated cargo transport through FG-nup gated NanoTraps. (A) Schematics of an NTR-bound cargo (Ntf2-GFP-SNAP-prey, only a monomeric chain of the homodimer is shown) and an NTR-free control molecule (MBP-GFP-SNAP-prey). (B) Permeability of Nsp1₄₈ and Nup100₄₈-NanoTraps to the Ntf2-fused molecule (140 kD), compared with the 106 kD NTR-free control. Data are plotted to show mean ± SEM from three trials.