Biophysical properties of singles-stranded dna studied with single-molecule force spectroscopy
- Autores: Xavier Viader Godoy
- Directores de la Tesis: Felix Ritort Farran (dir. tes.), Maria Mañosas Castejón (codir. tes.)
- Lectura: En la Universitat de Barcelona ( España ) en 2021
- Idioma: español
- Tribunal Calificador de la Tesis: Ramón Eritja Casadellà (presid.), Modesto Orozco López (secret.), Nynke H. Dekker (voc.)
- Programa de doctorado: Programa de Doctorado en Nanociencias por la Universidad de Barcelona
- Materias:
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- Resumen
In this thesis, single-molecule experiments using LOT are employed to extract accurate information about the thermodynamics and kinetics of various molecular systems, with special emphasis on the elastic properties of single-stranded DNA (ssDNA). The thesis is divided in three parts.
Part I provides a general description of the research field as well as the main theoretical framework for the basic concepts that will be developed in parts II and III. In Chapter 2 the miniTweezers and the experimental setup used throughout the thesis is described, as well as the physical basis of its working mechanisms, introducing the phenomenon of optical trapping. Chapter 3 contains a brief introduction of the biomolecules of study in this thesis, with an explanation of their historical discoveries, as well as their structure and function. The main focus of this chapter is on ssDNA, which is the main object of study of the thesis. Chapter 4 introduces the polymer models that are widely used in describing the elasticity of nucleic acids and proteins. Specifically, the Freely-Jointed Chain and Worm-Like Chain models are presented.
Part II deals with the elasticity of single-stranded DNA. This is the main part of the thesis, and it includes chapters 5-7. Chapter 5 is about the study of the elasticity of ideal ssDNA chain, i.e. the one that can be modelled as ideal polymers (presented in Chapter 4). The study of the elasticity of different DNA sequences is presented. The blocking-splint oligo technique is described, a experimental technique developed for studying the elasticity of short (tens of bases) DNA molecules. This study shows the need of using extensible models to succesfully describe ssDNA elasticity over a large range of forces, which explains the previous discrepancies on the elastic parameters obtained in different studies.
We also provide an explanation for the required extensibility of the model: a transition experienced at the nucleotide level: a change in DNA sugar pucker conformation. A simple two-states model is introduced and preeliminary results regarding its energetics are presented. The characterization of the ssDNA elasticity is central for the works developed in the following chapters.
Chapter 6 studies the stacking-unstacking transition for ssDNA, previously observed for certain sequences (mainly purine-rich ones). Several molecules, with different degrees of stacking, are studied by obtaining their force extension curves (FECs). A cooperative helix-coil model including heterogeneity is developed and used to fit the obtained FECs, allowing to obtain elastic parameters to describe the stacked chain. The salt dependence of the unstacking transition is also measured by studying two of the sequences by varying the salt concentration over two decades. The free energy of formation of dsDNA duplexes depends on the salt concentration. The obtained salt dependence on the stacking free-energy of ssDNA provides a possible explanation for the salt dependence of duplex formation.
Chapter 7 deals with the non-specific structures that arise at low forces and high salt concentration when pulling ssDNA molecules longer than $\sim 100$ bases. A helix-coil model with cooperativity is proposed and used to extract some mean-field characteristics of these structures. 8 different sequences are studied, characterizing their elasticity and deviation from the ideal elastic behaviour. The results for a $14$kb molecule for 3 decades of varying \ce{NaCl} and \ce{MgCl2} are also shown. All experimental FECs are fitted to the helix-coil model. The model can be used to predict the formation of secondary structures at zero force. A comparison between the predicted structures from the model and those obtained from Mfold is also investigated.
Part III contains two studies which also need of the correct determination of ssDNA elasticity. In Chapter 8, we study the interaction between the RecQ helicase from E. coli and DNA, i.e. how the RecQ unwinds double-stranded DNA molecules, releasing single-stranded DNA. We obtain some of its kinematic properties as well as study the entropy production of the system using the Fluctuation Theorem. In Chapter 9, the effect of DNA mismatches, i.e. non complementary base pairing, on the stability of DNA is studied. To do so, two types of experiments on several DNA sequences are performed: stretching and releasing the molecule by moving the optical trap (pulling experiments) and monitoring the folding/unfolding of the molecule passively (hopping experiments).
