title: Detailed study of DNA hairpin dynamics using single-molecule fluorescence assisted by DNA origami.
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 and ranged from 2 to 90 s(-1), were lower (∼10-fold) than the 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 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.
authors.affiliation: Department of Chemistry and the Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev , Beer Sheva 84105, Israel.
authors.forename: Roman
authors.initials: R
authors.lastname: Tsukanov
authors.forename: Toma E
authors.initials: TE
authors.lastname: Tomov
authors.forename: Rula
authors.initials: R
authors.lastname: Masoud
authors.forename: Hagai
authors.initials: H
authors.lastname: Drory
authors.forename: Noa
authors.initials: N
authors.lastname: Plavner
authors.forename: Miran
authors.initials: M
authors.lastname: Liber
authors.forename: Eyal
authors.initials: E
authors.lastname: Nir
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004058:Diffusion; D031541:Fluorescence Resonance Energy Transfer; D061925:Immobilized Nucleic Acids / Q000737:chemistry / Q000378:metabolism; D055029:Inverted Repeat Sequences; D012965:Sodium Chloride / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D061925:Immobilized Nucleic Acids; D012965:Sodium Chloride; D004247:DNA