Single-molecule studies of nuclear transport using biomimetic nuclear pore complexes.

Abstract

The nuclear pore complex (NPC) is the gatekeeper of the nucleus that regulates the flow of all molecules across the nuclear envelope. Remarkably, this ∼40 nm wide pore is capable of efficient and fast transport while remaining highly selective. The dense central channel of the NPC is filled with a dynamic mesh of intrinsically disordered proteins containing FG repeats (FG-Nups). While small molecules can freely pass this barrier, large biomolecules (>40 kDa) require specific transporter proteins. Our progress in understanding nuclear transport is hindered by the lack of experimental techniques that can probe the structure and dynamics of the disordered proteins and transport receptors inside the NPC channel with sufficient spatiotemporal resolution. We present two bottom-up approaches for constructing biomimetic NPCs that enable us to study different aspects of nuclear transport on the single-molecule level. First, we combine solid-state nanopores with optical detection in zero-mode waveguides. Biomimetic NPCs are constructed by coating nanopores with FG-Nups, allowing us to visualize the translocation of single fluorescently labelled molecules with ultimate sensitivity and specificity. Secondly, we implement a single-molecule FRET assay to study the structural dynamics within the central channel based on a circular DNA origami whose inside is lined with FG-Nups. Our multifaceted approach will provide direct mechanistic and structural insights into the molecular principles that govern nuclear transport.