Molecular Interactions within Nanoconfinement of Model DNA Nanostructures Controlled by Compensatory Kinetics as Revealed by Single-Molecule Fluorescence Analysis

Molecular interactions under steric confinement are important in chemistry, biology, biotechnology, and medicine. The impact of nanoconfinement on the underpinning kinetics and affinities is, however, unclear. While theoretical frameworks predict any effect only for very fast diffusion-limited association kinetics, experimental studies report that molecules can be trapped inside confined spaces to increase the effective local concentration and impact binding kinetics. Understanding is furthermore complicated by poorly comparable confinement geometries and reactions. Here, we determine the kinetics and affinities for interactions slower than the diffusion limit using highly modular DNA origami nanopores as model nanoconfinement systems. The pores feature either inside or outside their narrow lumen a single receptor, which can bind to three differently sized biomolecular ligands. We conduct kinetic binding analysis at the single-molecule resolution using fluorescence correlation spectroscopy to readily acquire large datasets and help overcome limitations of other single-molecule approaches. Nanoconfinement is found to hinder ligand association and dissociation, even below the diffusion limit. Yet, both suppressed kinetics compensate for each other to yield the same overall equillibrium affinity as nonconfined receptors, while local concentration enhancement by ligand trapping was not observed. We expect our insights and experimental strategy to guide the development of biosensing nanopores and help advance the understanding of biological nanochannels.