Panorama of DNA hairpin folding observed via diffusion-decelerated fluorescence correlation spectroscopy

Beijing National Laboratory for Molecular Sciences and Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
Chemical Communications (Impact Factor: 6.83). 06/2012; 48(59):7413-5. DOI: 10.1039/c2cc31986a
Source: PubMed

ABSTRACT Based on a confocal microscopy platform, we extended the FCS time window by three orders of magnitude to the s timescale by attaching a polystyrene microsphere. We simultaneously monitored the relaxations of multiple intermediates involved in DNA hairpin folding, thus offering a much more detailed view of the kinetics of hairpin folding experimentally.

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    ABSTRACT: The dynamics of two DNA hairpins (5'-TCGCCT-A31-AGGCGA-3' and 5'-TCGCCG-A31-CGGCGA-3') were studied using immobilization-based and diffusion-based single-molecule fluorescence techniques. The techniques enabled separated and detailed investigation of the states and of the transition reactions. Only two states, open and closed, were identified from analysis of the FRET histograms; metastable states with lifetimes longer than the technique resolution (0.3 ms) were not observed. The opening and closing reaction rates were determined directly from the FRET time trajectories and the Gibbs free energies of these states and of the transition state were calculated using the Kramer theory. The rates, which are undoubtedly of transitions between the fully closed and the fully open states, ranged from 2 to 90 s-1, were slower (~10-fold) than rates previously determined from fluorescence correlation spectroscopy. The heights of the barriers for closing were almost identical for the two hairpins. The barrier for opening the hairpin with the stronger stem was higher (4.3 kJ/mol) than that for the hairpin with the weaker stem, in a very good agreement with the difference in stability calculated by the nearest-neighbor method. The barrier for closing the hairpin decreased (~ 8 kJ/mol) and the barrier for opening increased (~ 4 kJ/mol) with increasing NaCl concentration (10-100 mM), indicating that higher ionic strength stabilizes the folded state with respect to the transition state and stabilizes the transition state relative to the unfolded state. The very good agreements in the dynamics measured for free hairpins, for hairpins anchored to origami, and for hairpins anchored to the coverslip and the very good agreement between the two single-molecule techniques demonstrate that neither the origami nor the coverslip influence the hairpin dynamics, supporting a previous demonstration that origami can serve as a platform for biophysical investigations.
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