- Here we provide high resolution study of DNA hairpin dynamics achieved by probability distribution analysis (PDA) of diffusion-based single-molecule Förster resonance energy transfer (sm-FRET) histograms. The opening and closing rates of three hairpins both free and attached to DNA origami were determined. The agreement with rates previously obtained using the total internal reflection (TIRF) technique and between free hairpins and hairpins attached to origami validated the PDA and demonstrated that the origami had no influence on the hairpin dynamics. From comparison of rates of four DNA hairpins, differing only in stem sequence, and from comparison with rates calculated using nearest-neighbor method and standard transition state theory, we conclude that the unfolding reaction resembles that of melting of DNA duplex with a corresponding sequence and that the folding reaction depends on counterion concentration and not on stem sequence. Our validation and demonstration of the PDA method will encourage its implementation in future high-resolution dynamic studies of freely diffusing biomolecules.