Raimundo Gargallo

Experimental Methods for Studying the Interactions between G-Quadruplex Structures and Ligands

The present paper reviews the recent advances in and applications of experimental techniques used to study interactions between G-quadruplex structures and ligands that are potentially of pharmaceutical interest. Several instrumental techniques are used to study such interactions. The application of spectroscopic techniques such as molecular absorption, circular dichroism, molecular fluorescence, mass spectrometry and nuclear magnetic resonance are reviewed and we discuss the type of information (qualitative or quantitative) that can be obtained from the use of each technique. Additionally, the application of complementary techniques such as surface plasmon resonance, isothermal titration calorimetry and different methods based on biochemistry is considered. For each technique, the main applications are presented and they are classified according to the family of the ligand and the type of G-quadruplex forming sequence (human telomeric or promoter region of oncogenes) considered.

Validation of alternating least-squares multivariate curve resolution for chromatographic resolution and quantitation

Abstract The application of multivariate curve resolution using the alternating least-squares (ALS) method for the resolution and quantitation of mixtures of two co-eluted compounds is validated for hydrocortisone (main compound) and prednisone (impurity) in liquid chromatography with diode array detection. The recovery of the spectra of the co-eluted compounds and their quantitation are discussed in relation to the initial estimates provided by evolving factor analysis and SIMPLISMA methods. Quantitation errors below 1.5% have been obtained for the main compound for resolution values of 0.4 or above, and impurity concentrations in the range 0.5–20%.

Fundamental aspects of the nucleic acid i-motif structures

The i-motif structure is formed in cytosine-rich sequences, its building block being the cytosine·cytosine + base pair. This structure is particularly stable at pH values below physiological (∼7.4) and, because of that, it has not attracted as much biological interest as other non-canonical structures such as the G-quadruplex. Nowadays, the proposal of potential roles in vivo, as well as nanotechnological applications, has produced an increasing interest in its study. In this context, the present work provides an overall picture of the i-motif structure. Those aspects related to formation and stability, such as chemical modifications or the interaction with ligands, are discussed. Special attention has been made to the i-motif structures that could have a hypothetical role in vivo, such as those present near the promoter region of several oncogenes.

Study of the intercalation equilibrium between the polynucleotide poly(adenylic)-poly(uridylic) acid and the ethidium bromide dye by means of Multivariate Curve Resolution and the multivariate extension of the continuous variation and mole ratio methods

The intercalation equilibrium between the polynucleotide poly(adenylic)-poly(uridylic) acid and the dye ethidium bromide was studied using molecular absorption spectrophotometry, spectrofluorimetry, and circular dichroism. The presence of an intercalation complex, its pure spectrum, and its concentration profile were clearly deduced using Multivariate Curve Resolution and a multivariate extension of the classical continuous variation and mole-ratio methods. The value of the polynucleotide/dye ratio (r(poly/dye)) in the intercalation complex and of its apparent formation log constant were estimated to be 4 and 6.2, respectively. The proposed multivariate methodology allowed an accurate qualitative and quantitative description of the intercalation process.

Application of a multivariate curve resolution procedure to the analysis of second-order melting data of synthetic and natural polynucleotides.

The melting behavior of several synthetic polynucleotides and a mixture of natural tRNAs was studied by monitoring the changes in the whole UV absorbance spectrum at several pH values. The second-order absorbance data were analyzed with a soft-modeling multivariate curve resolution procedure that allows the determination of the number of different species or conformations present along the melting experiment and the calculation of the melting profile and the pure spectrum for each chemical species or conformation. The melting temperature, T(m), for each thermal transition can be calculated from the melting profiles, and structural information on the different species or conformations can be obtained from their pure spectra. The multistranded species formed at certain pH conditions show several sharp thermal transitions related to the loss of the initial highly ordered structure. For these transitions, the mixture of species obtained in the denaturing process can be resolved when several data matrices, each giving additional information, are analyzed simultaneously with the mathematical procedure proposed.

Resolution of a structural competition involving dimeric G-quadruplex and its C-rich complementary strand

The resolution of the dimeric intermolecular G-quadruplex/duplex competition of the telomeric DNA sequence 5′-TAG GGT TAG GGT-3′ and of its complementary 5′ ACC CTA ACC CTA-3′ is reported. To achieve this goal, melting experiments of both sequences and of the mixtures of these sequences were monitored by molecular absorption, molecular fluorescence and circular dichroism spectroscopies. Molecular fluorescence measurements were carried out using molecular beacons technology, in which the 5′-TAG GGT TAG GGT-3′ sequence was labelled with a fluorophore and a quencher at the ends of the strand. Mathematical analysis of experimental spectroscopic data was performed by means of multivariate curve resolution, allowing the calculation of concentration profiles and pure spectra of all resolved structures (dimeric antiparallel and parallel G-quadruplexes, Watson–Crick duplex and single strands) present in solution. Our results show that parallel G-quadruplex is more stable than antiparallel G-quadruplex. When the complementary C-rich strand is present, a mixture of both G-quadruplex structures and Watson–Crick duplex is observed, the duplex being the major species. In addition to melting temperatures, equilibrium constants for the parallel/antiparallel G-quadruplex equilibrium and for the G-quadruplex/duplex equilibrium were determined from the concentration profiles.

pH-Modulated Watson-Crick duplex-quadruplex equilibria of guanine-rich and cytosine-rich DNA sequences 140 base pairs upstream of the c-kit transcription initiation site.

Guanine-rich regions of DNA are sequences capable of forming G-quadruplex structures. The formation of a G-quadruplex structure in a region 140 base pairs (bp) upstream of the c-kit transcription initiation site was recently proposed (Fernando et al., Biochemistry, 2006, 45, 7854). In the present study, the acid-base equilibria and the thermally induced unfolding of the structures formed by a guanine-rich region and by its complementary cytosine-rich strand in c-kit were studied by means of circular dichroism and molecular absorption spectroscopies. In addition, competition between the Watson-Crick duplex and the isolated structures was studied as a function of pH value and temperature. Multivariate data analysis methods based on both hard and soft modeling were used to allow accurate quantification of the various acid-base species present in the mixtures. Results showed that the G-quadruplex and i-motif coexist with the Watson-Crick duplex over the pH range from 3.0 to 6.5, approximately, under the experimental conditions tested in this study. At pH 7.0, the duplex is practically the only species present.

Application of an evolving factor analysis-based procedure to speciation analysis in the copper(II)-polyuridylic acid system

Abstract The acid-base properties and the copper(II)-complexing behaviour of the polynucleotide polyuridylic acid were studied by means of potentiometric, spectrophotometric and electron spin resonance titrations in a working aqueous medium of 0.15 M ionic strength and at 37°C. Spectrophotometric data were treated with an evolving factor analysis-based procedure that allows different sets of spectrometric titrations of the same multi-equilibria system to be analysed simultaneously. A dimeric macromolecular complex species between copper(II) ions and polyuridylic acid was detected in the system and its formation constant was evaluated.

Molecular dynamics simulation of highly charged proteins: Comparison of the particle-particle particle-mesh and reaction field methods for the calculation of electrostatic interactions

Molecular dynamics (MD) simulations of the activation domain of porcine procarboxypeptidase B (ADBp) were performed to examine the effect of using the particle‐particle particle‐mesh (P3M) or the reaction field (RF) method for calculating electrostatic interactions in simulations of highly charged proteins. Several structural, thermodynamic, and dynamic observables were derived from the MD trajectories, including estimated entropies and solvation free energies and essential dynamics (ED). The P3M method leads to slightly higher atomic positional fluctuations and deviations from the crystallographic structure, along with somewhat lower values of the total energy and solvation free energy. However, the ED analysis of the system leads to nearly identical results for both simulations. Because of the strong similarity between the results, both methods appear well suited for the simulation of highly charged globular proteins in explicit solvent. However, the lower computational demand of the RF method in the present implementation represents a clear advantage over the P3M method.

Application of a new multivariate curve resolution procedure to the simultaneous analysis of several spectroscopic titrations of the copper(II)—polyinosinic acid system

Abstract The acid-base properties and the copper(II)-complexing behaviour of the polynucleotide polyinosinic acid have been studied by means of potentiometric and spectrophotometric titrations. Spectrophotometric data have been treated with a new multivariate curve resolution procedure that allows to analyze simultaneously different sets of spectroscopic titrations of the same multi-equilibria system. The results obtained give a real proof of the advantages of the proposed procedure over the single titration analysis: intensity ambiguities are eliminated, as well as rotational ambiguities if the information yielded by at least one of the titrations is selective.