Evidence of a Three-State Mechanism in DNA Hairpin Folding

DNA hairpins are a model system for biomolecule folding as well as key structures in biology and nanotechnology. However, limitations in traditional solution-phase spectroscopy shorten the window of observable kinetics and cannot account for static heterogeneity. Here, we show that the application of two-dimensional fluorescence lifetime correlation spectroscopy (2DFLCS) to a solution-phase molecule trapped in an anti-Brownian electrokinetic (ABEL) trap bypasses those limitations, enabling kinetic analysis of the dynamics of single solution-phase molecules on a broad range of time scales down to microseconds. The analysis unambiguously shows that DNA hairpin folding proceeds via a three-state system, where hairpins fold initially on the scale of tens to hundreds of microseconds from a random coil to a partially closed intermediate and then form a stable fully closed state.