Dynamic force spectroscopy of DNA hairpins. I. Force kinetics and free energy landscapes

We investigate the thermodynamics and kinetics of DNA hairpins that fold/unfold under the action of applied mechanical force. We introduce the concept of the molecular free energy landscape and derive simplified expressions for the force dependent Kramers-Bell rates. To test the theory we have designed a specific DNA hairpin sequence that shows two-state cooperative folding under mechanical tension and carried out pulling experiments using optical tweezers. We show how we can determine the parameters that characterize the molecular free energy landscape of such sequence from rupture force kinetic studies. Finally we combine such kinetic studies with experimental investigations of the Crooks fluctuation relation to derive the free energy of formation of the hairpin at zero force.Comment: 28 pages, 12 figure

Single-molecule derivation of salt dependent base-pair free energies in DNA

Accurate knowledge of the thermodynamic properties of nucleic acids is crucial to predicting their structure and stability. To date most measurements of base-pair free energies in DNA are obtained in thermal denaturation experiments, which depend on several assumptions. Here we report measurements of the DNA base-pair free energies based on a simplified system, the mechanical unzipping of single DNA molecules. By combining experimental data with a physical model and an optimization algorithm for analysis, we measure the 10 unique nearest-neighbor base-pair free energies with 0.1 kcal mol-1 precision over two orders of magnitude of monovalent salt concentration. We find an improved set of standard energy values compared with Unified Oligonucleotide energies and a unique set of 10 base-pair-specific salt-correction values. The latter are found to be strongest for AA/TT and weakest for CC/GG. Our new energy values and salt corrections improve predictions of DNA unzipping forces and are fully compatible with melting temperatures for oligos. The method should make it possible to obtain free energies, enthalpies and entropies in conditions not accessible by bulk methodologies.Comment: Main text: 27 pages, 4 figures, 2 tables. Supporting Information: 51 pages, 19 figures, 4 table