Thermodynamics and Kinetics of Nucleic Acids Folding
[eng] This doctoral work investigates nucleic acids' thermodynamic and kinetic properties. The main objective is the characterization of the energetics and the folding mechanisms driving the hybridization of DNA and RNA molecules. A rigorous study of these processes is key to understanding the...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2023 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/197921 |
| Acceso en línea: | https://hdl.handle.net/2445/197921 http://hdl.handle.net/10803/688234 |
| Access Level: | acceso abierto |
| Palabra clave: | Biofísica Espectroscòpia molecular Àcids nucleics Termodinàmica Mecànica estadística Biophysics Molecular spectroscopy Nucleic acids Thermodynamics Statistical mechanics |
| Sumario: | [eng] This doctoral work investigates nucleic acids' thermodynamic and kinetic properties. The main objective is the characterization of the energetics and the folding mechanisms driving the hybridization of DNA and RNA molecules. A rigorous study of these processes is key to understanding the diversity of behaviors observed for nucleic acids and predicting their main features. The thesis is organized into four main parts. In Part I, we overview single-molecule force spectroscopy and some of the most common and relevant experimental techniques (Chapter 2). Among these, we describe optical trapping with laser optical tweezers, the experimental method used to carry out this work. The experimental setup is also accurately described (Chapter 3). Then, we discuss the biological concepts and the statistical tools used in the thesis. This includes fluctuation relations (Chapter 4), nucleic acids structure, thermodynamic modeling of the unzipping experiments (Chapter 5), and the transition state theory for two-state systems in thermodynamic equilibrium (Chapter 6). In Part II, we report the results of calorimetry force spectroscopy experiments on long DNA hairpins. We studied the temperature dependence of free energy, entropy, and enthalpy by carrying out unzipping experiments in the temperature range of 7 – 42°C. Even though the effects of temperature are known to be non-negligible, an accurate characterization of the thermodynamics parameters at the single base pair level still needs to be improved. Therefore, we developed a powerful method to accurately assess the temperature dependence of the entropy and enthalpy parameters, ultimately permitting us to measure the specific heat change per base pair. In Part III, we report the study of the energetics and kinetics of RNA folding, focusing on the complex mechanisms underlying RNA hybridization. By mechanically unzipping a long RNA hairpin, we derived the ten nearest-neighbor base pair RNA free energies in sodium and magnesium (Chapter 8). To characterize the irreversibility of the unzipping–rezipping process and the folding dynamics, we hypothesize that stem-loops structures forming along the unpaired RNA strands drive the folding (Chapter 9). This phenomenon is modeled by introducing a barrier energy landscape of the stem-loop structures forming along the complementary strands, which compete against the formation of the native hairpin. Finally, in Part IV, the results of pulling experiments of short RNA hairpins at low temperatures are reported. After reaching 5 – 7°C, short RNA sequences designed to fold as simple duplexes exhibit misfolded states competing with the native fold. Despite different sequences forming different misfolded structures, all of them share common features: they are very compact and brittle, and their stability does not depend on the presence of monovalent or divalent ions. RNA cold misfolding appears to be a general phenomenon questioning our understanding of RNA folding. |
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