title: A three-state mechanism for DNA hairpin folding characterized by multiparameter fluorescence fluctuation spectroscopy.
abstract: The folding of a dye-quencher labeled DNA hairpin molecule was investigated using fluorescence autocorrelation and cross-correlation spectroscopy (FCS) and photon counting histogram analysis (PCH). The autocorrelation and cross-correlation measurements revealed the flow and diffusion times of the DNA molecules through two spatially offset detection volumes, the relaxation time of the folding reaction, and the total concentration of DNA molecules participating in the reaction. The PCH measurements revealed the equilibrium distribution of DNA molecules in folded and unfolded conformations and the specific brightnesses of the fluorophore in each conformational state. These measurements were carried out over a range of NaCl concentrations, from those that favored the open form of the DNA hairpin to those that favored the closed form. DNA melting curves obtained from each sample were also analyzed for comparison. It was found that the reactant concentrations were depleted as the reaction progressed and that the equilibrium distributions measured by FCS and PCH deviated from those obtained from the melting curve analyses. These observations suggest a three-state mechanism for the DNA hairpin folding reaction that involves a stable intermediate form of the DNA hairpin. The reaction being probed by FCS and PCH is suggested to be a rapid equilibrium between open and intermediate conformations. Formation of the fully closed DNA hairpin is suggested to occur on a much longer time scale than the FCS and PCH measurement time. The closed form of the hairpin thus serves as a sink into which the reactants are depleted as the reaction progresses.
authors.affiliation: Department of Chemistry, Colorado State University, Fort Collins, 80523, USA.
authors.forename: Jaemyeong
authors.initials: J
authors.lastname: Jung
authors.forename: Alan
authors.initials: A
authors.lastname: Van Orden
mesh_terms: D004247:DNA / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D004247:DNA