Observing Single-Molecule Dynamics at Millimolar Concentrations

Abstract

Single-molecule fluorescence microscopy is a powerful tool for revealing chemical dynamics and molecular association mechanisms, but has been limited to low concentrations of fluorescent species and is only suitable for studying high affinity reactions. Here, we combine nanophotonic zero-mode waveguides (ZMWs) with fluorescence resonance energy transfer (FRET) to resolve single-molecule association dynamics at up to millimolar concentrations of fluorescent species. This approach extends the resolution of molecular dynamics to >100-fold higher concentrations, enabling observations at concentrations relevant to biological and chemical processes, and thus making single-molecule techniques applicable to a tremendous range of previously inaccessible molecular targets. We deploy this approach to show that the binding of cGMP to pacemaking ion channels is weakened by a slower internal conformational change.

Keywords: FRET; kinetics; nucleotides; single-molecule studies; zero-mode waveguide.