Evidence of a Three-State Mechanism in DNA Hairpins 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 stat-ic heterogeneity. Here, we show that the application of 2-Dimenstional 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 timescales down to micro-seconds. The analysis unambiguously shows that DNA hairpin folding proceeds via a three-state system, where hairpins fold initially on the scale of 10s-100s of microseconds from a random coil to a partially closed intermediate, and then form a sta-ble fully closed state.