Single-molecule fluorescence studies of the dynamics in supramolecular systems of biological interest

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

Evidence of cyclodextrin aggregation obtained with Fluorescence Correlation Spectroscopy

The 14th International Electronic Conference on Synthetic Organic Chemistry session Polymer ChemistryWe have studied the complexation between an adamantane derivative labelled with the fluorescent probe Alexa 488 and the three natural cyclodextrins (-, - and -CD) by Fluorescence Correlation Spectroscopy (FCS), demonstrating the ability of this technique to detect association and to determine the corresponding equilibrium constants with no need for changes in the fluorescence properties of the guest. At low CD concentrations the observed increase of the diffusion time of the probe is mainly due to the complexation of the adamantyl moiety, but further changes are observed when increasing CD concentration that are attributed to the formation of CD aggregates. These aggregates appear at quite low CD concentrations and seem to be formed by a small number of CD molecules. These results show the potential of FCS for the study of CD self-assembly, a recently-recognized phenomenon that could be used to improve certain applications of CDsThe authors thank the Ministerio de Ciencia e Innovación and the Xunta de Galicia for financial support (CTQ2007-68057-C02-02/BQU, INCITE09E2R209064ES, INCITE09262304PR, 2009/029). J. Bordello and S. Freire thank Ministerio de Educación and Xunta de Galicia for scholarship

Single‐Molecule Approach to DNA Minor‐Groove Association Dynamics

This is the peer reviewed version of the following article: Bordello, J. , Sánchez, M. I., Vázquez, M. E., Mascareñas, J. L., Al‐Soufi, W. and Novo, M. (2012), Single‐Molecule Approach to DNA Minor‐Groove Association Dynamics. Angew. Chem. Int. Ed., 51: 7541-7544, which has been published in final form at https://doi.org/10.1002/anie.201201099. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsGetting in the groove: Fluorescence correlation spectroscopy reveals that the dynamics of the association process of the bisbenzamidine minor‐groove binder BBA‐OG (blue with green star, see scheme) to dsDNA is not controlled by diffusion, but by the insertion of the binder into the groove at the specific site (red), as shown by the rate constants for each step of the binding eventMinisterio de Ciencia e Innovación. Grant Numbers: CTQ2010‐21369, SAF2007‐61015, SAF2010‐20822‐C02, CTQ2009‐14431/BQU, CSD2007‐00006 Xunta de Galicia. Grant Numbers: INCITE09262304PR, INCITE09E2R209064ES, IN845B‐2010/094, INCITE09 209 084PR, GRC2010/12, PGIDIT08CSA‐047209PRS

Fluorescence-Labeled Bis-benzamidines as Fluorogenic DNA Minor-Groove Binders: Photophysics and Binding Dynamics

NOTICE: This is the peer reviewed version of the following article: Bordello, J., Sánchez, M. I., Vázquez, M. E., Mascareñas, J. L., Al-Soufi, W. and Novo, M. (2015), Fluorescence-Labeled Bis-benzamidines as Fluorogenic DNA Minor-Groove Binders: Photophysics and Binding Dynamics. Chem. Eur. J., 21: 1609–1619 [doi: 10.1002/chem.201404926]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for self-archiving.In recent decades there has been great interest in the design of highly sensitive sequence-specific DNA binders. The eligibility of the binder depends on the magnitude of the fluorescence increase upon binding, related to its photophysics, and on its affinity and specificity, which is, in turn, determined by the dynamics of the binding process. Therefore, progress in the design of DNA binders requires both thorough photophysical studies and precise determination of the association and dissociation rate constants involved. We have studied two bis-benzamidine (BBA) derivatives labeled by linkers of various lengths with the dye Oregon Green (OG). These fluorogenic binders show a dramatic fluorescence enhancement upon binding to the minor groove of double-stranded (ds) DNA, as well as significant improvement in their sequence specificity versus the parent BBA, although with decreased affinity constants. Detailed photophysical analysis shows that static and dynamic quenching of the OG fluorescence by BBA through photoinduced electron transfer is suppressed upon insertion of BBA into the minor groove of DNA. Fluorescence correlation spectroscopy yields precise dynamic rate constants that prove that the association process of these fluorogenic binders to dsDNA is very similar to that of BBA alone and that their lower affinity is mainly a consequence of their weaker attachment to the minor groove and the resultant faster dissociation process. The conclusions of this study will allow us to go one step further in the design of new DNA binders with tunable fluorescence and binding propertiesJ.B. and M.I.S thank the Spanish MCINN for their research scholarships. The authors are thankful for financial support from Spanish grants (CTQ2010-21369, SAF2013-41943-R and CTQ2012-31341), the Xunta de Galicia (GPC2013/052, GRC2013-041, CN 2012/314), the ERDF, and the European Research Council (Advanced Grant Nº 340055). Support of COST Actions CM1105 and CM1306 and the orfeo-cinqa network is also kindly acknowledgedS