The general objective of this thesis is the use of single-molecule fluorescence (SMF) techniques to study the dynamics and stability of different chemical and biochemical systems.
Most of its content is focused on the optimization of Fluorescence Correlation Spectroscopy (FCS) technique and its application to investigate host-guest systems. Several studies were carried out on the interaction between fluorescent dyes or dye-labelled molecules of different nature and macromolecules of interest such as cyclodextrins, micelles or DNA oligonucleotides. In all cases, the results discussed here led to significative contributions in order to move forward in the understanding of the involved mechanisms in this type of supramolecular associations. At the same time, it is intended to lay the groundwork for the use of FCS as a tool in the study of a wide range of host-guest supramolecular systems.
Single-molecule FRET in combination with Total Internal Reflection Fluorescence Microscopy (smFRET/TIRF) is also used as another SMF technique, complementary to FCS. The immobilization of the molecules on a glass surface enables the observation for longer periods of time in comparison with the free-diffusion setup used in FCS. This approach allows the study of processes such as the folding of ARN aptamers, which are too slow to be addressed by FCS.
Chapter I is dedicated to a brief introduction into single-molecule fluorescence detection with detailed description of the FCS technique (setup, main equations in the data analysis, etc). It also includes a detailed discussion on the influence of impurities in FCS. Some practical aspects concerning FCS measurements, such as calibration of the confocal volume, sample preparation, influence of cover-slide thickness, etc. were gathered through the different experiments and appear in the last section of this chapter.
Chapter II includes studies on several host-guest systems using FCS together with the more conventional absorption and fluorescence spectroscopies. The factors that control structure and stability of supramolecular host-guest assemblies were discussed through three different publications.
Chapter III is also based on a publication in which the role of a high brightness ratio of the dye during the association process is discussed in order to enhance the sensitivity of FCS in the determination of the rate constants of inclusion complexes.
In Chapter IV, FCS was used to investigate the affinity and dynamics in the binding of synthetic agents to the minor groove of specific DNA sequences. Part of the most relevant results has been already published as a communication.
Chapter V collects the results obtained during a research internship in the Laboratory for Biophysics and Biomolecular Dynamics of Dr. Penedo, at the University of St. Andrews (Scotland). In this chapter smFRET/TIRF is applied to identify conformational transitions and characterize conformational changes in the chemical denaturation pathway of a riboswitch (mRNA) as well as to provide information about the kinetics of the process.
This thesis is presented in the style of a series of published papers. Five original publications are reproduced in full in chapters II, III and as a part of chapter IV
