Spectroscopic Characterization of Mitochondrial G-Quadruplexes.

Guanine quadruplexes (G4s) are highly polymorphic four-stranded structures formed within guanine-rich DNA and RNA sequences that play a crucial role in biological processes. The recent discovery of the first G4 structures within mitochondrial DNA has led to a small revolution in the field. In particular, the G-rich conserved sequence block II (CSB II) can form different types of G4s that are thought to play a crucial role in replication. In this study, we decipher the most relevant G4 structures that can be formed within CSB II: RNA G4 at the RNA transcript, DNA G4 within the non-transcribed strand and DNA:RNA hybrid between the RNA transcript and the non-transcribed strand. We show that the more abundant, but unexplored, G6AG7 (37%) and G6AG8 (35%) sequences in CSB II yield more stable G4s than the less profuse G5AG7 sequence. Moreover, the existence of a guanine located 1 bp upstream promotes G4 formation. In all cases, parallel G4s are formed, but their topology changes from a less ordered to a highly ordered G4 when adding small amounts of potassium or sodium cations. Circular dichroism was used due to discriminate different conformations and topologies of nucleic acids and was complemented with gel electrophoresis and fluorescence spectroscopy studies.

A Surfactant Concentration Model for the Systematic Determination of the Critical Micellar Concentration and the Transition Width.

The critical micellar concentration (cmc) is a fundamental property of surfactant solutions. Many proposed methods for the definition and determination of the cmc from property-concentration plots yield values, which depend on the studied property, on the specific technique used for its analysis and in many cases on the subjective choice of the chosen type of plot and concentration interval. In this focus review, we revise the application of a surfactant concentration model we proposed earlier that defines the cmc directly based on the surfactant concentration. Known equations for the concentration-dependence of different surfactant properties can then be combined with this concentration model and fitted to experimental data. This modular concept makes it possible to determine the cmc and the transition width in a systematic and unambiguous way. We revise its use in the literature in different contexts: the determination of the cmc of surfactants and their mixtures from different properties (electrical conductivity, NMR chemical shift, self-diffusion, surface tension, UV-Vis absorption, fluorescence intensity and fluorescence correlation). We also revise the dependence of the width of the transition region on composition, detailed studies of the properties of fluorescent probes and the aggregation of non-surfactant systems, namely amyloid peptides.

Towards ratiometric sensing of amyloid fibrils in vitro.

The aggregation of amyloid-ß peptide and its accumulation in the human brain has an important role in the etiology of Alzheimer's disease. Thioflavin T has been widely used as a fluorescent marker for these amyloid aggregates. Nevertheless, its complex photophysical behavior, with strong wavelength dependencies of all its fluorescence properties, requires searching for new fluorescent probes. The use of 2-(2'-hydroxyphenyl)imidazo[4,5-b]pyridine (HPIP), which shows two emission bands and a rich excited-state behavior due to the existence of excited-state intramolecular processes of proton transfer and charge transfer, is proposed. These properties result in a high sensitivity of HPIP fluorescence to its microenvironment and cause a large differential fluorescence enhancement of the two bands upon binding to aggregates of the amyloid-ß peptide. Based on this behavior, a very sensitive ratiometric method is established for the detection and quantification of amyloid fibrils, which can be combined with the monitoring of fluorescence anisotropy. The binding selectivity of HPIP is discussed on the basis of the apparent binding equilibrium constants of this probe to amyloid-ß (1-42) fibrils and to the nonfibrillar protein bovine serum albumin. Finally, an exhaustive comparison between HPIP and thioflavin T is presented to discuss the sensitivity and specificity of these probes to amyloid aggregates and the significant advantages of the HPIP dye for quantitative determinations.

Fluorescence-labeled bis-benzamidines as fluorogenic DNA minor-groove binders: photophysics and binding dynamics.

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 properties.

Early Aggregation of Amyloid-ß(1-42) Studied by Fluorescence Correlation Spectroscopy.

Alzheimer's disease (AD) is a progressive neurodegenerative disease affecting cognitive and memory abilities and is believed to be linked to the formation and accumulation of neurotoxic aggregates of the Amyloid-ß peptide (Aß). In particular, it is the formation of soluble pre-fibrillar oligomers within the early stage of Aß aggregation which is thought to represent a key step in the development of AD, thus underlining the interest in characterizing the aggregation process and the nature of these aggregates. In this context, fluorescence correlation spectroscopy (FCS) has emerged as a valuable alternative for the study of these systems in solution. Indeed, the use of FCS to study terminally labelled Aß provides a means to detect changes in the size and concentration of initially monomeric Aß samples by monitoring these fluorescently labelled species freely diffusing in solution with single-molecule resolution. Herein, we show how to employ FCS to study the early aggregation process of Aß(1-42) and how this can be used to estimate the critical concentration for oligomer formation and to characterize the aggregates formed.

Supramolecular association studied by Fluorescence correlation spectroscopy.

A comprehensive description of a supramolecular system involves a full understanding of its thermodynamic and dynamic properties, as well as detailed knowledge of its structure. Fluorescence Correlation Spectroscopy (FCS) constitutes a powerful technique to acquire this information. Fluorescence correlation curves show a characteristic diffusion term that is related to the binding equilibrium constant or other thermodynamic properties of the supramolecular system. The association and dissociation rate constants of the binding process can be determined in FCS when the relaxation time of the binding is faster than the observation time-a regime called fast-exchange dynamics - in opposition to the slow-exchange regime. In all cases, structural information can be inferred from the diffusional properties of the supramolecular complexes. A short overview of the use of FCS for the study of supramolecular systems is given with examples which belong to the fast and slow regime.

Role of electrostatic and hydrophobic forces in the interaction of ionic dyes with charged micelles.

The nature and strength of the interactions between a cationic fluorophore, Rhodamine 123 (R123), and surfactants of different head charge are investigated. Series of absorption and fluorescence emission spectra and of fluorescence decays are measured. R123 does not interact with the monomers of both nonionic and cationic surfactants but it presents affinity to their micelles. A partition equilibrium model was proposed and the corresponding equilibrium constants were obtained, as well as the photophysical properties of the dye bound to the micelles. In the case of the cationic surfactants, changes of the fluorescence properties were already observed below the critical micellar concentration (CMC) due to dynamic quenching caused by the free counterions. In the presence of anionic surfactants R123 shows a very different behaviour with dramatic spectral changes below the CMC. The observed variations are attributed to a first, strong interaction between R123 and the surfactant monomers, which yields ionic pairs of singular photophysical properties and dominates at low surfactant concentrations, followed by the association of R123 with the surfactant premicellar and micellar aggregates at higher surfactant concentrations near the CMC. This behaviour results from the competition between the strong electrostatic interactions of the cationic dye with the anionic surfactant head groups and the hydrophobic forces stabilizing the dye inside the micelles. The results of this work illustrate the complex physicochemical and photophysical behaviour of a charged dye in micellar systems, which resembles the expected situation in similar systems such as biological membranes.

Host-guest complexation studied by fluorescence correlation spectroscopy: adamantane-cyclodextrin inclusion.

The host-guest complexation between an Alexa 488 labelled adamantane derivative and beta-cyclodextrin is studied by Fluorescence Correlation Spectroscopy (FCS). A 1:1 complex stoichiometry and a high association equilibrium constant of K = 5.2 x 10(4) M(-1) are obtained in aqueous solution at 25 degrees C and pH = 6. The necessary experimental conditions are discussed. FCS proves to be an excellent method for the determination of stoichiometry and association equilibrium constant of this type of complexes, where both host and guest are nonfluorescent and which are therefore not easily amenable to standard fluorescence spectroscopic methods.

Host-assisted guest self-assembly: enhancement of the dimerization of pyronines Y and B by gamma-cyclodextrin.

Buckle up! The dimerization of small fluorescent guests is strongly enhanced in presence of a cyclodextrin host. The host cavity acts like a belt to assist the self-assembly of guests (see picture). Small variations in the guest structure have significant influence on the stability and geometry of the aggregates.The role of small variations in the structural properties of host and guest molecules on the stoichiometry and strength of supramolecular associations is analyzed. Earlier we found that a change in substituents from pyronine B to pyronine Y has a dramatic effect on both the stability and the dynamics of the association of these guests with beta-cyclodextrin as host. Now we study the association between these two pyronines and a cyclodextrin with a bigger cavity (gamma-cyclodextrin) using UV/Vis absorption and fluorescence spectroscopy. The absorption spectra of the pyronines show complex variations with cyclodextrin concentration indicating that pyronine dimerization is strongly enhanced inside the cavity of the cyclodextrin. A full model is proposed and the equilibrium constants of the involved processes and the absorption and emission spectra of the different species are estimated. The equilibrium constants of the formation of complexed dimers are much higher than those for free dimerization or for the inclusion of a single guest. The gamma-cyclodextrin host acts like a belt to assist the guest self-assembly. The differences in the stability of pyronine B and pyronine Y dimers are explained on the basis of their structure and geometry.

Unveiling the multi-step solubilization mechanism of sub-micron size vesicles by detergents.

The solubilization of membranes by detergents is critical for many technological applications and has become widely used in biochemistry research to induce cell rupture, extract cell constituents, and to purify, reconstitute and crystallize membrane proteins. The thermodynamic details of solubilization have been extensively investigated, but the kinetic aspects remain poorly understood. Here we used a combination of single-vesicle Förster resonance energy transfer (svFRET), fluorescence correlation spectroscopy and quartz-crystal microbalance with dissipation monitoring to access the real-time kinetics and elementary solubilization steps of sub-micron sized vesicles, which are inaccessible by conventional diffraction-limited optical methods. Real-time injection of a non-ionic detergent, Triton X, induced biphasic solubilization kinetics of surface-immobilized vesicles labelled with the Dil/DiD FRET pair. The nanoscale sensitivity accessible by svFRET allowed us to unambiguously assign each kinetic step to distortions of the vesicle structure comprising an initial fast vesicle-swelling event followed by slow lipid loss and micellization. We expect the svFRET platform to be applicable beyond the sub-micron sizes studied here and become a unique tool to unravel the complex kinetics of detergent-lipid interactions.

Dynamics of supramolecular association monitored by fluorescence correlation spectroscopy.

Supramolecular binding is a key process in many biological systems and in newly developed supramolecular assemblies. Most of the scientific work on these systems is focused on their structural properties and on the thermodynamics of the association process. However, the underlying dynamics are usually much less known, in spite of the great importance they have during the binding process in these highly dynamic systems. Understanding supramolecular binding in biological systems and controlling the functionality of new synthetic supramolecular systems can only be achieved through knowledge of the structure-dynamics relationship. There is a strong need for suitable techniques which cover the typically wide time interval of the association dynamics and which do not need a perturbation of the system. We briefly review high-resolution fluorescence correlation spectroscopy (FCS) as a technique to monitor supramolecular dynamics and to give information on how structure determines the dynamics of host-guest association. The comparison of hosts and guests with different structures shows that geometrical and orientational requirements determine the association rate constant, whereas the dissociation is defined by the strength of specific interactions. As model hosts cyclodextrins and micelles are studied.

Critical aggregation concentration for the formation of early Amyloid-ß (1-42) oligomers.

The oligomers formed during the early steps of amyloid aggregation are thought to be responsible for the neurotoxic damage associated with Alzheimer's disease. It is therefore of great interest to characterize this early aggregation process and the aggregates formed, especially for the most significant peptide in amyloid fibrils, Amyloid-ß(1-42) (Aß42). For this purpose, we directly monitored the changes in size and concentration of initially monomeric Aß42 samples, using Fluorescence Correlation Spectroscopy. We found that Aß42 undergoes aggregation only when the amount of amyloid monomers exceeds the critical aggregation concentration (cac) of about 90 nM. This spontaneous, cooperative process resembles surfactants self-assembly and yields stable micelle-like oligomers whose size (≈50 monomers, R h ≈ 7-11 nm) and elongated shape are independent of incubation time and peptide concentration. These findings reveal essential features of in vitro amyloid aggregation, which may illuminate the complex in vivo process.

Dye-exchange dynamics in micellar solutions studied by fluorescence correlation spectroscopy.

We investigated the dye-exchange dynamics between rhodamine 123 (R123), a mitochondrial fluorescent dye, and micelles as membrane mimetic systems. In the presence of neutral micelles (Triton X-100 and Brij 35) R123 partitions between the aqueous solution and the micellar pseudo-phase, undergoing red shift of the absorption and the emission spectra. Fluorescence correlation spectroscopy (FCS) was used to study the dynamics of these systems over an extremely wide time range and at the single-molecule level, yielding information in one and the same experiment about the diffusional dynamics of free and bound rhodamine and about the dye-exchange dynamics as well as several photophysical properties of the rhodamine bound to the micelles. It was found that the entry rate constants are diffusion-controlled, indicating that there are no geometric or orientational requirements for the association of the dye with the micelle. With respect to the dye-exchange dynamics, micelles are found to behave as soft supramolecular cages in contrast to other rigid supramolecular cavities, such as cyclodextrins. The exit rate constants depend on the surfactant and determine the stability of the binding. Single-molecule multiparameter fluorescence detection (MFD) was used to examine the fluorescence properties of individual molecules in comparison to ensembles of molecules. The MFD histograms confirm the fast dye-exchange dynamics observed by FCS and yield mean values of fluorescence lifetimes and anisotropies in agreement with those obtained in bulk measurements.

Specific interactions in the inclusion complexes of pyronines Y and B with beta-cyclodextrin.

The aim of this work is to analyze the role of specific interactions in host-guest association processes. The formation of inclusion complexes between pyronines Y and B and beta-cyclodextrin and the nature of the interactions involved have been studied using absorption, steady-state fluorescence, and time-resolved fluorescence spectroscopies. The two pyronines form 1:1 complexes with beta-cyclodextrin, with the association equilibrium constant being much higher in the case of pyronine B. Complexation causes a slight red shift of the emission spectra of the pyronines but decreases significantly their fluorescence quantum yields and lifetimes. To explain this atypical behavior, the photophysical properties of the pyronines in different solvents were determined and compared with those of the complexes. The similarities observed between the pyronines in dioxane and in the interior of the cyclodextrin cavity suggest that there are important specific interactions of the pyronines with the electron-rich oxygens present in these media. A possible explanation for the increase in the nonradiative rate constants in these media involves the existence of a charge-transfer excited state with the location of the positive charge at the xanthene moiety, which would be stabilized by the mentioned interactions. The observed differences between pyronine Y and B can be understood on the basis of these specific interactions.

Fluorescence emission of pyrene in surfactant solutions.

The systematic description of the complex photophysical behaviour of pyrene in surfactant solutions in combination with a quantitative model for the surfactant concentrations reproduces with high accuracy the steady-state and the time resolved fluorescence intensity of pyrene in surfactant solutions near the cmc, both in the monomer and in the excimer emission bands. We present concise model equations that can be used for the analysis of the pyrene fluorescence intensity in order to estimate fundamental parameters of the pyrene-surfactant system, such as the binding equilibrium constant K of pyrene to a given surfactant micelle, the rate constant of excimer formation in micelles, and the equilibrium constant of pyrene-surfactant quenching. The values of the binding equilibrium constant K(TX100)=3300·10³ M⁻¹ and K(SDS)=190·10³ M⁻¹ for Triton X-100 (TX100) and SDS micelles, respectively, show that the partition of pyrene between bulk water and micelles cannot be ignored, even at relatively high surfactant concentrations above the cmc. We apply the model to the determination of the cmc from the pyrene fluorescence intensity, especially from the intensity ratio at two vibronic bands in the monomer emission or from the ratio of excimer to monomer emission intensity. We relate the finite width of the transition region below and above the cmc with the observed changes in the pyrene fluorescence in this region.

Determination of second-order association constants by global analysis of 1H and 13C NMR chemical shifts. Application to the complexation of sodium fusidate and potassium helvolate by beta- and gamma-cyclodextrin.

The host-guest interaction between the steroid antibiotics sodium fusidate and potassium helvolate as guests and the hosts beta- and gamma-cyclodextrin was studied by 13C and 1H NMR techniques. The analysis of chemical shifts of individual nuclei leads to inconsistent values of the association constants and fails generally in the case of mixtures of 1:1 and 1:2 stoichiometries. The problem of parameter correlation is identified and the global analysis of two or more nuclei is proposed as a very effective method for the detection of complexes of higher stoichiometries and for the precise determination of the involved association constants. A matrix formulation of global analysis and the determination of confidence intervals is described. An analytical solution of the cubic equation, necessary for the description of higher order complexes, is presented in detail and its use together with commercial fitting software is compared with dedicated implementations. gamma-Cyclodextrin forms with both studied steroids, sodium fusidate and potassium helvolate, 1:1 complexes with high values of the association constants, K(1)=(60+/-24)x10(3)lmol(-1), and K(2)=(22+/-9)x10(3)lmol(-1), respectively. To the contrary, beta-cyclodextrin forms 1:1 and 1:2 (guest:host) complexes with both steroids, with moderate K(1) and low K(2) values (K(1)=(0.74+/-0.13)x10(3)lmol(-1), K(2)=(0.210+/-0.075)x10(3)lmol(-1)), and (K(1)=(2.42+/-0.87)x10(3)lmol(-1), K(2)=(0.06+/-0.09)x10(3)lmol(-1)), respectively.

Spectra and structure of complexes formed by sodium fusidate and potassium helvolate with beta- and gamma-cyclodextrin.

The complexation of two steroid antibiotics of the fusidane family, sodium fusidate and potassium helvolate, by beta-CD and gamma-CD has been studied by using 1D and 2D-NMR techniques. Both guests form 1:1 complexes with gamma-CD and 1:2 (guest:cyclodextrin) complexes with beta-CD. Thus, both antibiotics behave as monotopic and ditopic guests when they are complexed by gamma-CD and beta-CD, respectively. Both steroids enter into the cavity of the gamma-CD by the side chain, reaching the central region of the steroid (rings C and D), whereas the A and B (partially) rings remain outside. For beta-CD complexes, ROESY spectra show a remarkable absence of interactions of the protons of the C and D rings, whereas clear interactions corresponding to the side chain, and A and B rings are observed. The obtained equilibrium constants (see previous paper) are discussed in terms of the structures proposed for the complexes. NMR spectra of sodium fusidate are revised, and a full assignment of the 1H and 13C NMR spectra is presented for potassium helvolate.

Complexation of several benzimidazole-type fungicides with alpha- and beta-cyclodextrins.

The potential increase in water solubility of three benzimidazole-type fungicides (thiabendazole, carbendazim, and fuberidazole) due to complexation with alpha- and beta-cyclodextrins was investigated. Fluorescence emission spectra of the fungicides in the presence of different concentrations of the cyclodextrins were measured. Analysis of these spectra by the method of principal components global analysis (PCGA) yielded precise values for the association constants and the emission spectra of the fungicide-cyclodextrin inclusion complexes. Phase-solubility diagrams confirmed the formation of inclusion complexes between each of the fungicides and beta-cyclodextrin and showed significant increases of their solubilities due to complexation.

Complexation of several fungicides with beta-cyclodextrin: determination of the association constants and isolation of the solid complexes.

The formation of inclusion complexes with beta-cyclodextrin was studied for several popular fungicides of different types: prochloraz, 2-phenylphenol, thiophanate methyl, 8-hydroxyquinoline, and benalaxyl. Phase solubility diagrams showed that in all cases complexation takes place, leading to an important increase of water solubility in prochloraz and benalaxyl. Equilibrium association constants could be determined from the phase solubility data and from NMR titrations in the case of 2-phenylphenol. Because of the low solubility of the complex formed between 8-hydroxyquinoline and beta-cyclodextrin, the corresponding association constant could not be determined. The solid complexes of fungicide-cyclodextrin were prepared and isolated by different methods. The isolation of real complexes and not physical mixtures was confirmed in the cases of prochloraz, 2-phenylphenol, and benalaxyl by differential scanning calorimetry.