DNA Hairpin Hybridization under Extreme Pressures: A Single-Molecule FRET Study

Organisms have evolved to live in a variety of complex environments, which clearly has required cellular biology to accommodate to extreme conditions of hydraulic pressure and elevated temperature. In this work, we exploit single-molecule Forster resonance energy transfer (FRET) spectroscopy to probe structural changes in DNA hairpins as a function of pressure and temperature, which allows us to extract detailed thermodynamic information on changes in free energy (ΔG°), free volume (ΔV°), enthalpy (ΔH°), and entropy (ΔS°) associated with DNA loop formation and sequence-dependent stem hybridization. Specifically, time-correlated single-photon counting experiments on freely diffusing 40A DNA hairpin FRET constructs are performed in a 50 μm × 50 μm square quartz capillary cell pressurized from ambient pressure up to 3 kbar. By pressure-dependent van't Hoff analysis of the equilibrium constants, ΔV° for hybridization of the DNA hairpin can be determined as a function of stem length (n_stem = 7-10) with single base-pair resolution, which further motivates a simple linear deconstruction into additive stem (ΔV°_stem = ΔV°_bp x n_stem) and loop (ΔV°_loop) contributions. We find that increasing pressure destabilizes the DNA hairpin stem region [ΔV°_bp = +1.98(16) cm³/(mol bp)], with additional positive free volume changes [ΔV°_loop = +7.0(14) cm³/mol] we ascribe to bending and base stacking disruption of the 40-dA loop. From a van't Hoff temperature-dependent analysis of the DNA 40A hairpin equilibria, the data support a similar additive loop/stem deconstruction of enthalpic (ΔH° = ΔH°_loop + ΔH°_stem) and entropic (ΔS° = ΔS°_loop + ΔS°_stem) contributions, which permits insightful comparison with predictions from nearest-neighbor thermodynamic models for DNA duplex formation. In particular, the stem thermodynamics is consistent with exothermically favored (ΔH°_stem < 0) and entropically penalized (ΔS°_stem < 0) hydrogen bonding but with additional enthalpic (ΔH°_loop > 0) and entropic (ΔS°_loop > 0) contributions due to loop bending effects consistent with distortion of dA base stacking in the 40-dA linker.