Resolving Mixtures in Solution by Single-Molecule Rotational Diffusivity

Sensing the size of individual molecules in an ensemble has proven to be a powerful tool to investigate biomolecular interactions and association–dissociation processes. In biologically relevant solution environments, molecular size is often sensed by translational or rotational diffusivity. The rotational diffusivity is more sensitive to the size and conformation of the molecules as it is inversely proportional to the cube of the hydrodynamic radius, as opposed to the inverse linear dependence of the translational diffusion coefficient. Single-molecule rotational diffusivity has been measured with time-resolved fluorescence anisotropy decay, but the ability to sense different sizes has been restricted by the limited number of photons available or has required surface attachment to observe each molecule longer, and the attachment may be perturbative. To address these limitations, we show how to measure and monitor single-molecule rotational diffusivity by combining the solution-phase Anti-Brownian ELectrokinetic (ABEL) trap and maximum likelihood analysis of time-resolved fluorescence anisotropy based on the information inherent in each detected photon. We demonstrate this approach by resolving a mixture of single- and double-stranded fluorescently labeled DNA molecules at equilibrium, freely rotating in a native solution environment. The rotational diffusivity, fluorescence brightness and lifetime, and initial and steady-state anisotropy are simultaneously determined for each trapped single DNA molecule. The time resolution and precision of this method are analyzed using statistical signal analysis and simulations. We present key parameters that define the usefulness of a particular fluorescent label for extracting molecular size information from single-molecule rotational diffusivity measurements.