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Si us plau utilitzeu sempre aquest identificador per citar o enllaçar aquest document: https://hdl.handle.net/2445/191404
Statistical and thermodynamical properties of DNA unzipping experiments with optical tweezers
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[eng] Molecular biophysics is a scientific discipline that studies biomolecules. This discipline has experienced a revolution thanks to the development of single- molecule techniques. These techniques allows us to obtain new and valuable information that complements the traditional bulk assays. Optical tweezers is an experimental technique that uses the radiation pressure of light to exert forces on a tiny dielectric microsphere. Biomolecules can be bonded to such microspheres in order to perform pulling experiments at the single- molecule level. Minitweezers is a dual counter-propagating laser tweezers instrument that measures the force exerted on the microsphere by conservation of light momentum. The instrument has high stability and resolution (0.1 pN in force and 0.5 nm in distance) in the measurements. The properties of the DNA molecule can be studied with the Minitweezers at the single-molecule level. The DNA is a biomolecule that forms a double helix that stores the genetic information of the cells. DNA unzipping experiments consist in pulling apart the two strands of DNA by exerting mechanical forces on the extremities of the molecule. In such process, basepairs (bp) are disrupted sequentially, showing a succession of cooperative unzipping regions (CUR) of different sizes (between 1–100 bp). In a DNA unzipping experiment, we measure the force vs. distance curve (FDC) of the molecule, which has a characteristic sawtooth-like shape that is sequence-dependent. The FDC is analyzed with a Bayesian approach to infer the size distribution of the CURs. The experimental accuracy does not allow to observe CURs of sizes below 10 bp. Furthermore, the unzipping of one bp at a time can only be achieved by having an optical trap stiffness value higher than 0.1 N/m, which corresponds to the stiffness of a single nucleotide of DNA. This has been deduced from a toy model specifically introduced to study the CUR size distributions. In addition, the FDCs are theoretically predicted by the nearest-neighbor (NN) model adapted to unzipping experiments. The NN model describes the hybridization reaction of two strands of DNA. By fitting the experimental FDCs to the model, the unique 10 NN bp free energies are obtained with 0.1 kcal mol−1 precision between 10 mM–1 M of monovalent salt concentration. The results show that the unzipping FDCs and the melting temperatures of oligos are correctly described with a specific salt correction for each of the 10 NN bp free energies. Differently from the previous experiments, the unzipping of DNA can also be performed at con- trolled force. These last type of experiments exhibit large hysteresis and irreversibility. The free energy landscape is a tool that helps to understand the unzipping at controlled force. Finally, the work presented in this thesis can be extended to find practical applications of DNA unzipping, such as sequencing of DNA by force, and measurement of thermodynamic properties of biomolecules in conditions not accessible by bulk methodologies.
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HUGUET CASADES, Josep maria. Statistical and thermodynamical properties of DNA unzipping experiments with optical tweezers. [consulta: 9 de desembre de 2025]. [Disponible a: https://hdl.handle.net/2445/191404]