Single-Molecule analysis of small molecule involved High-Affinity Biomolecular Interaction Using Self-Crowded Nanopipette.

Exploring biomolecular interactions such as protein-protein, protein-nucleic acid, and protein-ligand dynamics is fundamental for understanding the mechanisms that govern biological processes. Nanopore sensing has emerged as a powerful label-free technique for single-molecule detection and analysis at or near physiological conditions. However, the resistive pulse based nanopore sensing approaches using diluted samples often lack the sensitivity to detect small molecules involved biomolecular interactions. Recently, we developed a quartz nanopipette based nanopore technique for detecting interaction involving small molecule by inducing self-crowding of molecules at the nanopipette tip.(Khatri et al., 2023) Due to the high crowding level, dense conductive pulses were observed with high signal-to-noise ratio. We had successfully probed the weak interactions between two proteins or nucleoside triphosphates. Now, we further expand this technique to study high-affinity biomolecular interactions. Here, I will present our results on streptavidin-biotin binding, one of the well-studied strongest noncovalent interactions. We observed multipeak conductive ionic current spikes upon binding. These distinct current spikes have potential to reveal the binding structure, the stoichiometry, and mechanical stability of the molecular complexes. Therefore, this self-crowding based nanopore-sensing approach opens exciting new opportunities for studying various biomolecular interactions