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Si us plau utilitzeu sempre aquest identificador per citar o enllaçar aquest document: https://hdl.handle.net/2445/221032
Salt dependent DNA translocation dynamics across nanopores
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[eng] This thesis uses two complementary single-molecule techniques, nanopipette microscopy and optical tweezers, to investigate the impact of various monovalent salts at high concentrations in DNA. The thesis begins by characterizing the conductance and noise properties of nanopipettes in a wide range of concentrations. Once the nanopipettes have been characterized, they are used to conduct DNA translocation experiments to examine how different cations influence their translocation parameters. These experiments also allow us to explore the effects of applied voltage and salt concentration on DNA folding configurations during translocation. In addition, we explore why compact DNA folding configurations have lower dwell times compared to longer folding configurations. Finally, optical tweezers are used to carry out stretching experiments on a DNA fork, providing information on DNA stability under conditions of high ionic strength related to DNA translocation experiments. The thesis is divided into six parts. Part I provides an introduction to the experimental techniques used throughout this work, along with the theoretical frameworks and concepts that will be needed for Parts II, III and IV. This part is divided into three chapters. Chapter 1 is an introduction to the nanofear field. The chapter describes the setup and basic concepts required to perform electrical measurements with nanopipettes and discusses the limits of resolution of the technique. The main sources of noise when experiments with nanopores are carried out are also described. Also, some relevant nanofluidic phenomena are introduced when working at the nanoscale, along with some theoretical foundations on the regulation of surface load. Finally, some previous results of DNA translocation through nanopores are shown. Chapter 2 introduces the optical trap and mini-tweezers configuration that was used to perform the experiments in this thesis. Chapter 3 introduces the basic components and structure of nucleic acids, focusing on DNA, which will be the biomolecule studied throughout the thesis. The chapter concludes by presenting the theoretical foundations of two elastic models used to describe the elastic properties of polynucleotides. Part II contains results of experiments with nanopipettes. It begins with chapter 4, where the conductance of nanopipettes for different salt concentrations is studied, comparing the contributions of conductance in volume and surface area and the effect of pH on surface loading. In addition, two conductivity models are compared to model the conductance of nanopipettes with concentration. In addition, the blinking noise of the nanopipettes is analyzed, exploring how the parameters that describe the noise change with concentration and tension. Chapter 5 presents λ-DNA translocation experiments in different monovalent salts. This chapter investigates the effect of concentration and cation type on translocation parameters such as residence time, electric current blocking, and charge blocking of λ-DNA translocations. The chapter focuses on how the cation-DNA interaction changes with the size of the cation. Chapter 6 studies the different folding configurations that occur during DNA translocation and how they depend on salt tension and concentration. To do this, an analysis of the different levels that occur during λ-DNA translocation and the residence time of the different levels is carried out. The analysis also allows us to extract general data on DNA translocation through nanopipettes. Finally, the causes of the lower dispersion of the dwell time of the more compact folding configurations compared to the longer folding configurations are explored. Part III includes experiments with optical tweezers. In Chapter 7, optical tweezers are used to conduct stretching experiments of long DNA forks at high ionic concentrations of various monovalent salts. The average opening force and the effect of concentration on fork stability are investigated. The chapter concludes with a joint discussion of the results of translocation experiments and optical tweezers at high salt concentrations. Part IV contains the results of a one-month international stay. Chapter 8 includes a brief introduction to the SPRNT (Single-molecule picometer resolution nanopore tweezers) technique, which was used to perform experiments with the Hel308 helicase. In addition, some preliminary experiments of the with the gp41 helicase using SPRNT are presented. Part V contains the final conclusions of the thesis and the future perspectives of the work. Part VI consists of all the Appendices. Appendices E and C complement the results of some of the chapters, while Appendices A, B, F, and D describe in detail the most important experimental protocols and MATLAB codes used in the development of the thesis.
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COLCHERO TRUNIGER, Alejandro. Salt dependent DNA translocation dynamics across nanopores. [consulta: 28 de novembre de 2025]. [Disponible a: https://hdl.handle.net/2445/221032]