We investigated the volumetric and kinetic profile of the conformational landscape of a poly dA loop DNA hairpin (Hp) in the presence of salts, osmolytes and crowding media, mimicking the intracellular milieu, using single-molecule FRET methodology. Pressure modulation was applied to explore the volumetric and hydrational characteristics of the free-energy landscape of the DNA Hp, but also because pressure is a stress factor many organisms have to cope with, e.g. in the deep sea where pressures even up to the kbar level are encountered. Urea and pressure synergistically destabilize the closed conformation of the DNA Hp due to a lower molar partial volume in the unfolded state. Conversely, multivalent salts, trimethylamine-N-oxide and Ficoll strongly populate the closed state and counteract deteriorating effects of pressure. Complementary smFRET measurements under immobilized conditions at ambient pressure allowed us to dissect the equilibrium data in terms of folding and unfolding rate constants of the conformational transitions, leading to a deeper understanding of the stabilization mechanisms of the cosolutes. Our results show that the free-energy landscape of the DNA Hp is a rugged one, which is markedly affected by the ionic strength of the solution, by preferential interaction and exclusion of cosolvents as well as by pressure.
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