Derivation of nearest-neighbor DNA parameters in magnesium from single molecule experiments.

DNA hybridization is an essential molecular reaction in biology with many applications. The nearest-neighbor (NN) model for nucleic acids predicts DNA thermodynamics using energy values for the different base pair motifs. These values have been derived from melting experiments in monovalent and divalent salt and applied to predict melting temperatures of oligos within a few degrees. However, an improved determination of the NN energy values and their salt dependencies in magnesium is still needed for current biotechnological applications seeking high selectivity in the hybridization of synthetic DNAs. We developed a methodology based on single molecule unzipping experiments to derive accurate NN energy values and initiation factors for DNA. A new set of values in magnesium is derived, which reproduces unzipping data and improves melting temperature predictions for all available oligo lengths, in a range of temperature and salt conditions where correlation effects between the magnesium bound ions are weak. The NN salt correction parameters are shown to correlate to the GC content of the NN motifs. Our study shows the power of single-molecule force spectroscopy assays to unravel novel features of nucleic acids such as sequence-dependent salt corrections.

Elastic Properties of Nucleic Acids by Single-Molecule Force Spectroscopy.

We review the current knowledge on the use of single-molecule force spectroscopy techniques to extrapolate the elastic properties of nucleic acids. We emphasize the lesser-known elastic properties of single-stranded DNA. We discuss the importance of accurately determining the elastic response in pulling experiments, and we review the simplest models used to rationalize the experimental data as well as the experimental approaches used to pull single-stranded DNA. Applications used to investigate DNA conformational transitions and secondary structure formation are also highlighted. Finally, we provide an overview of the effects of salt and temperature and briefly discuss the effects of contour length and sequence dependence.

A Temperature-Jump Optical Trap for Single-Molecule Manipulation.

To our knowledge, we have developed a novel temperature-jump optical tweezers setup that changes the temperature locally and rapidly. It uses a heating laser with a wavelength that is highly absorbed by water so it can cover a broad range of temperatures. This instrument can record several force-distance curves for one individual molecule at various temperatures with good thermal and mechanical stability. Our design has features to reduce convection and baseline shifts, which have troubled previous heating-laser instruments. As proof of accuracy, we used the instrument to carry out DNA unzipping experiments in which we derived the average basepair free energy, entropy, and enthalpy of formation of the DNA duplex in a range of temperatures between 5°C and 50°C. We also used the instrument to characterize the temperature-dependent elasticity of single-stranded DNA (ssDNA), where we find a significant condensation plateau at low force and low temperature. Oddly, the persistence length of ssDNA measured at high force seems to increase with temperature, contrary to simple entropic models.

Universal axial fluctuations in optical tweezers.

Optical tweezers (OTs) allow the measurement of fluctuations at the nanoscale, in particular fluctuations in the end-to-end distance in single molecules. Fluctuation spectra can yield valuable information, but they can easily be contaminated by instrumental effects. We identify axial fluctuations, i.e., fluctuations of the trapped beads in the direction of light propagation, as one of these instrumental effects. Remarkably, axial fluctuations occur on a characteristic timescale similar to that of conformational (folding) transitions, which may lead to misinterpretation of the experimental results. We show that a precise measurement of the effect of force on both axial and conformational fluctuations is crucial to disentangle them. Our results on axial fluctuations are captured by a simple and general formula valid for all OT setups and provide experimentalists with a general strategy to distinguish axial fluctuations from conformational transitions.

Free-energy inference from partial work measurements in small systems.

Fluctuation relations (FRs) are among the few existing general results in nonequilibrium systems. Their verification requires the measurement of the total work performed on a system. Nevertheless in many cases only a partial measurement of the work is possible. Here we consider FRs in dual-trap optical tweezers where two different forces (one per trap) are measured. With this setup we perform pulling experiments on single molecules by moving one trap relative to the other. We demonstrate that work should be measured using the force exerted by the trap that is moved. The force that is measured in the trap at rest fails to provide the full dissipation in the system, leading to a (incorrect) work definition that does not satisfy the FR. The implications to single-molecule experiments and free-energy measurements are discussed. In the case of symmetric setups a second work definition, based on differential force measurements, is introduced. This definition is best suited to measure free energies as it shows faster convergence of estimators. We discuss measurements using the (incorrect) work definition as an example of partial work measurement. We show how to infer the full work distribution from the partial one via the FR. The inference process does also yield quantitative information, e.g., the hydrodynamic drag on the dumbbell. Results are also obtained for asymmetric dual-trap setups. We suggest that this kind of inference could represent a previously unidentified and general application of FRs to extract information about irreversible processes in small systems.