Probing messenger RNA conformational heterogeneity using single-molecule fluorescence anisotropy

In this letter we describe a method to probe biomolecular conformations and their dynamics at the single molecule level. We show, using fluorescence anisotropy based methods, that the hydrodynamic volume of biomolecules captures the intrinsic heterogeneity within a population. Population distributions of conformations and their dynamics are studied by making anisotropy measurements on one molecule at a time within a confocal volume. The mean anisotropy of mRNA is lowered on addition of salt while the spread remains the same. The intrinsic heterogeneity is revealed when conformational transitions are frozen, resulting in a drastic increase in the spread of the anisotropy. These studies reveal that mRNA samples a broad range of conformations.