Single Molecule FRET Studies of DNA Hairpin Folding

Abstract

Hairpins are the simplest structures for investigating fundamental aspects of nucleic acid folding mechanisms. For hairpins exhibiting two-state behavior, all of the mechanistic information is contained in the transition path, the rare event in single molecule trajectories when the free energy barrier between folded and unfolded states is actually crossed. The first step toward observing transition paths is to determine the transition path time. Average transition path times have recently been directly determined in proteins using the Gopich/Szabo maximum likelihood method (Gopich, Szabo J. Phys. Chem. B 2009) to analyze folding/unfolding photon trajectories in single molecule FRET experiments (Chung et al. Science 2012; Chung and Eaton Nature 2013), while the only experimental study of transition paths in nucleic acids used optical tweezer measurements to determine an upper bound of 50 μs in trajectories for a variety of structures (Neupane et al. Phys. Rev. Lett. 2012). Neupane et al. also reconstructed the free energy surface for an indirect determination of average transition path times from Szabo's analytical theory for diffusive barrier crossing. We use single molecule FRET to study a fast-folding DNA hairpin with 2 A-T and 2 G-C base pairs in the stem and 21 T's in the loop immobilized on a polyethylene glycol-coated glass surface via a biotin-streptavidin-biotin linkage. The folding time for this hairpin in 500mM NaCl is 530 μs. Maximum likelihood analysis of 780 transitions in photon trajectories with an average detection rate of 750 photons/ms yields an upper bound of 4 μs for the average transition path time, compared to the value of ∼3.6±0.8 μs predicted by Neupane et al. from hairpins with 9-30 base pair stems and 4 T's in the loop. Current experiments use viscogens to slow the transition path for directly determining the actual value.