In Vitro Measurements of Single-Molecule Transport Across an Individual Biomimetic Nuclear Pore Complex

Abstract

Nuclear pore complexes (NPCs) regulate the exchange of RNA and proteins between the nucleus and the cytoplasm in eukaryotic cells. We present a new method to investigate NPC transport at the single molecule level. We construct a biomimetic NPC by covalently bonding natively unfolded Phe-Gly rich (FG) domains of human nucleoporins (Nups) to a solid-state nanopore. Trans-pore ionic current measurements provide a probe to monitor single molecules traversing the pore. Importantly, we find that translocation events are indeed observed for transport receptors (Impβ) whereas the passage of non-specific proteins (BSA) is inhibited. A single type of Nups is thus sufficient to form a transport barrier that exhibits the selectivity found in NPCs. We find a translocation time for transporter molecule Impβ of 3 ms for both Nup153 and Nup98, which is 20-fold slower than the passage time through a bare pore, but comparable to in vivo measurements on NPCs. By reproducing key features of the NPC, our biomimetic approach opens the way to study a wide variety of nucleocytoplasmic transport processes at the single-molecule level in vitro.∗∗Kowalczyk et al, Nature Nanotechnology, under review