Fluorescence-based strategies to investigate the structure and dynamics of aptamer-ligand complexes

This work was funded by the Scottish Universities Physics Alliance (SUPA), the University of St Andrews, the Engineering and Physical Sciences ResearchCouncil (EPSRC), the Canadian Institutes of Health Research (CIHR). Funding for open access charge: University of St Andrews.In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labelling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labelled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these single-molecule FRET microscopy techniques.Publisher PDFPeer reviewe

Optimització d'una aplicacio bioinformàtica d'alineament de seqüències executada en processadors multi-core i many-core (GPUs)

Las aplicaciones de alineamiento de secuencias son una herramienta importante para la comunidad científica. Estas aplicaciones bioinformáticas son usadas en muchos campos distintos como pueden ser la medicina, la biología, la farmacología, la genética, etc. A día de hoy los algoritmos de alineamiento de secuencias tienen una complejidad elevada y cada día tienen que manejar un volumen de datos más grande. Por esta razón se deben buscar alternativas para que estas aplicaciones sean capaces de manejar el aumento de tamaño que los bancos de secuencias están sufriendo día a día. En este proyecto se estudian y se investigan mejoras en este tipo de aplicaciones como puede ser el uso de sistemas paralelos que pueden mejorar el rendimiento notablemente.Les aplicacions d'alineament de seqüències són una eina important per a la comunitat científica. Aquestes aplicacions bioinformàtiques són utilitzades en molts camps diferents com poden ser la medicina, la biologia, la farmacologia, la genètica, etc. A dia d'avui els algorismes d'alineament de seqüències tenen una complexitat elevada i cada dia han de gestionar un volum de dades més gran. Per això s'han de buscar alternatives per a que aquestes aplicacions siguin capaces de gestionar l'augment de mida que els bancs de seqüències estan patint dia a dia. En aquest projecte s'estudien i s'investiguen millores en aquest tipus d'aplicacions com pot ser l'ús de sistemes paral·leles que poden millorar el rendiment notablement.The sequence alignment applications are an important tool for the scientific community. These bioinformatics applications are used in many different fields such as medicine, biology, pharmacology, genetics, etc. Today the sequence alignment algorithms are highly complex and every day have to handle a large volume of data. For this reason we must find alternatives for these applications are able to handle the increased size of sequences that banks are suffering every day. In this project we study and investigate improvements in these applications such as the use of parallel systems that can improve performance significantly

Conformational Rearrangements Regulating the DNA Repair Protein APE1

Apurinic apyrimidinic endonuclease 1 (APE1) is a key enzyme of the Base Excision Repair (BER) pathway, which primarily manages oxidative lesions of DNA. Once the damaged base is removed, APE1 recognises the resulting abasic site and cleaves the phosphodiester backbone to allow for the correction by subsequent enzymes of the BER machinery. In spite of a wealth of information on APE1 structure and activity, its regulation mechanism still remains to be understood. Human APE1 consists of a globular catalytic domain preceded by a flexible N-terminal extension, which might be involved in the interaction with DNA. Moreover, the binding of the nuclear chaperone nucleophosmin (NPM1) to this region has been reported to impact APE1 catalysis. To evaluate intra- and inter-molecular conformational rearrangements upon DNA binding, incision, and interaction with NPM1, we used Förster resonance energy transfer (FRET), a fluorescence spectroscopy technique sensitive to molecular distances. Our results suggest that the N-terminus approaches the DNA at the downstream side of the abasic site and enables the building of a predictive model of the full-length APE1/DNA complex. Furthermore, the spatial configuration of the N-terminal tail is sensitive to NPM1, which could be related to the regulation of APE1.This research was funded by the University of the Basque Country (grant number GIU18/172). The APC was funded by the Basque Government (grant number IT1454-22)

Single molecule spectroscopy of polyfluorene chains reveals β-phase content and phase reversibility in organic solvents

Conjugated polymers are an important class of organic semiconductors that can be deposited from solution to make optoelectronic devices. Among them, poly(9,9′-dioctylfluorene) (PFO) has distinctive optical properties arising from its ability to adopt an ordered planar conformation (β phase) from a disordered glassy phase (α phase). The β phase has attractive optical properties, but the precise mechanism of its formation in solution remains unknown. Here, we have combined specifically tailored polymer synthesis and surface-passivation strategies to provide the first spectroscopic characterization of single PFO chains in solution at room temperature. By anchoring PFO molecules at one end on an anti-adherent surface, we show that isolated chains can adopt the β-phase conformation in a solvent-dependent manner. Furthermore, we find that individual PFO chains can reversibly switch multiple times between phases in response to solvent-exchange events. The methodology presented here for polymer synthesis and immobilization is widely applicable to investigate other luminescent polymers.PostprintPostprintPeer reviewe

Review: Zoran Kurelić (ed.). Violence, Art and Politics.

This work was supported by the Engineering and Physical Sciences Research Council UK (EPSRC) (project EP/N009886/1). F.T.-C. thanks EPSRC (Grant EP/L015110/1). S.F. acknowledges support from the National Science Foundation (Grant DMR 1411240). I.D.W.S. acknowledges support from a Royal Society Wolfson research merit award.Conjugated polymers (CPs) are an important class of organic semiconductors that combine novel optoelectronic properties with simple processing from organic solvents. It is important to study CP conformation in solution to understand the physics of these materials and because it affects the properties of solution-processed films. Single-molecule techniques are unique in their ability to extract information on a chain-to-chain basis; however, in the context of CPs, technical challenges have limited their general application to host matrices or semiliquid environments that constrain the conformational dynamics of the polymer. We introduce a conceptually different methodology that enables measurements in organic solvents using the single-end anchoring of polymer chains to avoid diffusion while preserving polymer flexibility. We explore the effect of organic solvents and show that, in addition to chain-to-chain conformational heterogeneity, collapsed and extended polymer segments can coexist within the same chain. The technique enables real-time solvent-exchange measurements, which show that anchored CP chains respond to sudden changes in solvent conditions on a subsecond time scale. Our results give an unprecedented glimpse into the mechanism of solvent-induced reorganization of CPs and can be expected to lead to a new range of techniques to investigate and conformationally manipulate CPs.Publisher PDFPeer reviewe

Building a Practice Research Network of Young Therapists and Early Career Researchers in Spanish speaking countries: Why, Who & How

La Red para el desarrollo de jóvenes psicoterapeutas e investigadores es una Red de Investigación Orientada por la Práctica (RIOP) conformada por clínicos e investigadores jóvenes, en sus primeros años de desarrollo profesional. Siguiendo los principios de la Investigación Orientada por la Práctica, el presente trabajo tiene como objetivo describir el proceso de construcción de esta RIOP proporcionando una explicación general de las razones que motivaron su desarrollo, así como las características actuales de los psicoterapeutas jóvenes. Para introducir el contexto en el que se basa esta RIOP, se proporcionan datos empíricos sobre la composición de los psicoterapeutas en Argentina y España. Se presentan dos estudios: el primero describe el desarrollo de un cuestionario que explora las actitudes de los psicoterapeutas y el uso de los resultados de la investigación en la práctica clínica, las dificultades que encuentran para acceder a los resultados científicos y para llevar a cabo una investigación. Además, se estudiaron las propiedades psicométricas del cuestionario, obteniéndose una solución de tres factores. También se observó que los clínicos españoles informan mayores dificultades de acceso a la investigación que los argentinos. El segundo estudio explora cómo los terapeutas toman decisiones clínicas y éstas impactan en el proceso de tratamiento y su resultado. Además, se observaron diferentes procesos de toma de decisión clínica al comparar un clínico puro (más orientado a la experiencia profesional y a los resultados de la investigación) con un clínico que realiza también tareas de investigación (más propenso a guiarse por la intuición y la supervisión). La alianza terapéutica y el curso clínico de los pacientes se asociaron con estos procesos de toma de decisiones. Se describen las implicaciones clínicas de los resultados y se presentan los planes futuros de la RIOP.Red para el desarrollo de jóvenes psicoterapeutas e investigadores (Network for the Development of Young Psychotherapists and Researchers) is a Practice Research Network (PRN) conformed by early career clinicians and researchers. Following Practice-Oriented Research principles, the present study aims to describe the building process of this PRN by providing an overall explanation of the reasons that motivate its development, as well as to characterize the current situation of early career psychotherapists. To introduce the context in which our PRN is built, empirical data on the composition of early career psychotherapists in Argentina and Spain will be provided. Two studies are presented. The first study describes the development of a questionnaire that explores psychotherapists’ attitudes and use of research outcomes in the clinical practice, as well as difficulties in accessing to scientific results and barriers to conducting research. The second study explores how therapists make clinical decisions and how these decisions impact on treatment process and outcome. In study 1, the psychometric properties of the questionnaire were explored and a three-factor solution was obtained. We also observed that Spanish clinicians reported greater research accessibility difficulties than Argentines. Study 2 revealed different clinical decision-making processes when comparing pure clinicians (more oriented towards professional experience and research outcomes) and research clinicians (more likely to be guided by intuition and supervision). The therapeutic alliance and the clinical course of patients were associated with these decision-making processes. Clinical implications of the results are described and future plans of the PRN are presented.Fil: Farfallini, Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Red de jóvenes para el desarrollo de la psicoterapia; Argentina. Fundación Aiglé; ArgentinaFil: Molinari, Guadalupe. Red de jóvenes para el desarrollo de la psicoterapia; Argentina. Fundación Aiglé; Argentina. Universitat Jaume I; EspañaFil: Espinoza, Macarena. Red de jóvenes para el desarrollo de la psicoterapia; Argentina. Universidad de Valencia; EspañaFil: Suso Ribera, Carlos. Universitat Jaume I; EspañaFil: Gómez Penedo, Juan Martín. Red de jóvenes para el desarrollo de la psicoterapia; Argentina. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernández Álvarez, Javier. Università Cattolica del Sacro Cuore; Italia. Red de jóvenes para el desarrollo de la psicoterapia; Argentin

Towards Ratiometric Sensing of Amyloid Fibrils In Vitro

This is the peer-reviewed version of the following article: Chemistry - A European Journal 2015, 21, 3425–3434, DOI: 10.1002/chem.201406110. The final form has been published at https://onlinelibrary.wiley.com/doi/abs/10.1002/chem.201406110. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsThe aggregation of amyloid‐β 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‐β 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‐β (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 determinationsXunta de Galicia European Regional Development Fund Ministerio de Ciencia e Innovación Xunta de Galicia. Grant Numbers: CTQ2010‐21369, CTQ2010‐17835, GPC2013/052, R2014/051 RS MacDonald Charitable TrustS

Unprecedented tunability of riboswitch structure and regulatory function by sub-millimolar variations in physiological Mg2+

Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer–ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.Publisher PDFPeer reviewe

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

Funding: EPSRC (EP/P030017/1).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.Publisher PDFPeer reviewe