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.

Characterizing Single‐Molecule FRET Dynamics with Probability Distribution Analysis

“…[4,5] Although our hairpin data were consistent with a two-state model via the chi-squared metric, we also observed a systematic deviation between our data and the model. Recent studies of DNA hairpin dynamics have identified putative intermediates in the folding pathway, [36,37] which could explain this discrepancy. However, we caution that the detection of such intermediates by PDA should involve the use of novel experimental controls, and not simply an increase in the number of free parameters.…”

“…This could be due to more complex kinetic schemes, as identified in recent biophysical studies of DNA hairpins. [36,37] While some of these behaviors are expected to occur on timescales too short to be detected by PDA (e.g. multiple loop orientations in the open conformation), longerlived states (e.g.…”

RNA Folding: Conformational Statistics, Folding Kinetics, and Ion Electrostatics

“…The system is simple enough for exhaustive detailed studies yet sufficiently sophisticated to serve as a paradigm for more complex RNA folding kinetics. There have been extensive studies on DNA hairpin folding kinetics using laser temperature-jump (4,55) and fluorescence correlation spectroscopy (8,43,44,52) techniques. In contrast, there are only very limited experiments on RNA hairpin (and pseudoknot) folding kinetics (54,64,69,111,113,114).…”

The Role of Loop Stacking in the Dynamics of DNA Hairpin Formation

“…19 This hypothesis has been challenged by several recent studies that suggest a more complex pathway, involving intermediate states containing misfolded or partially folded configurations. [20][21][22] Additionally, there have been diverse, and sometimes contradictory, experimental observations and interpretations of hairpin formation kinetics. Fluorescence energy transfer and fluorescence correlation spectroscopy measurements 23 have reported an Arrhenius temperature dependency with a positive activation enthalpy for the hairpin closing rates.…”