Force-spectroscopy of small ligands binding to nucleic acids

Abstract

Single-molecule techniques allow to following biomolecular reactions with unprecedented resolution. Particularly, optical tweezers can be used to manipulate and apply forces to individual molecules tethered between plastic beads that are optically-trapped. Optical trapping is achieved by using highly focused laser beams that exert a gradient force onto the micrometer-sized dielectric particles that become confined close to the focal position of the laser. By specifically attaching the ends of the molecule under study to two optically-trapped beads, it is possible to manipulate and apply forces to an individual molecule. Typical experiments with optical tweezers consist in manipulating nucleic acids (DNA, RNA) or proteins one at a time. For instance DNA molecules can be stretched to measure its elastic properties, or unzipped to measure their base-pairing energies. Many small anticancer drugs target nucleic acids to exert their cytotoxic activity against cancer cells. To understand their mechanism of action it is important to know in which positions, how strong, and how fast do they bind to different specific sites in DNA. Single-molecule optical tweezers experiments can be used to unravel the binding thermodynamics and kinetics of many of these ligands, especially those difficult to characterize with bulk techniques. Thiocoraline is one of such drugs, and binds DNA through bis-intercalation. Experiments with optical tweezers show that the kinetics of intercalation are very slow (hours) and strongly force-dependent: force facilitates binding but slows down unbinding. Experiments performed in different conditions also reveal that the binding pathway proceeds through a mono-intercalated intermediate that causes the observed slow kinetics. In this sense, we present a three-state model that offers a theoretical framework from which the kinetic rates of the reaction can be extracted, and that could be useful to characterize other bis-intercalators. We also show that DNA unzipping experiments can be used to determine the preferred binding sequences of Thiocoraline, finding that it preferentially clamps CpG steps. This methodology is potentially very useful as it provides direct access to the preferred binding sites of small ligands due to its thermodynamic stability with one base pair resolution and without the requirement of restriction enzymes or radioactive labeling. This single-molecule footprinting technique is also adapted to a magnetic tweezers instrument in order to perform parallelized measurements. The fact that bis-intercalation does not modify the persistence length of dsDNA is also found in the pulling experiments. From the elasticity measurements, we also extract equilibrium quantitates of the interaction by using classic statistical models. This combination of DNA stretching and unzipping assays can also be used to follow how the anticancer agent Kahalalide F self-assembles and compacts DNA. Kahalalide F forms nanometric particles that are positively charged able to bind and condense DNA. The binding reaction shows to phases: an initial compaction of electrostatic origin, and its subsequent stiffening due to the hydrophobic collapse of the complex. The combination of quantitative force-spectroscopy measurements with AFM images of the complexes and other bulk tech- niques (DLS, EM) provides a consistent picture of the compaction and aggregation process. Modeling of the experiments provides the thermodynamic parameters of the interaction that are complemented with kinetic measurements. A simple technique to study ssDNA with optical tweezers is also presented and used to study how the stiffness of the polyanion affects the compaction process. We exploit this methodology to understand how the stiffness of the polyanion affects the compaction kinetics, and later on, we also show its utility to study the elasticity of ssDNA under varying ionic conditions. Finally, the utility of this methodology to study self-assembly and aggregation is explored with amyloidogenic peptides involved in neurodegenerative disorders.

Les tècniques de molècula individual permeten seguir les reaccions biomoleculars amb una resolució sense precedents, proveint als científics d’una sèrie d’instruments per a mesurar magnituds físiques i investigar sistemes experimentals difícilment accessibles amb les tradi­cionals mesures en volum (és a dir a on les mesures es realitzen amb mols de reactiu). Particularment, les pinces òptiques permeten manipular i aplicar forces a molècules individuals i determinar-ne així les seves propietats elàstiques i termodinàmiques.

L’atrapament òptic es basa en l’ús d’un feix làser focalitzat per exercir una força òptica a les microesferes (diàmetre ~ 3 µm), que queden confinades a prop del punt focal a causa de la conservació del moment lineal. Els experiments de micromanipulació es realitzen unint els extrems de la molècula que es vol estudiar a la superfícies de dues microesferes diferents, podent així aplicar forces a la molècula quan desplacem una microesfera respecte de l’altra. Per ancorar les molècules a la superfície de les microesferes s’utilitzen unions moleculars que tenen una alta a.nitat (p.ex. enllaç biotina-estreptavidina). Típicament els experiments amb pinces òptiques consisteixen en la micromanipulació d’àcids nucleics (ADN, ARN) o proteïnes de forma individual. Per exemple, una molècula d’ADN pot ésser estirada per a estudiar-ne les propietats elàstiques, o oberta mecànicament (separant les dues cadenes que formen la doble hèlix) per a mesurar les energies lliures d’aparellament entre bases.

Un gran nombre d’agents anticancerígens tenen com a diana els àcids nucleics, a on s’hi uneixen afi i efecte de dur a terme la seva acció citotòxica (p. ex. interferint amb processos cel·lulars essencials com són la replicació, la transcripció o els mecanismes de reparació). Per entendre el seu mecanisme d’acció és important conéixer en quines posicions, amb quina a.nitat, i amb quina cinètica s’uneixen a diferents seqüències d’ADN. Els experiments de molècula individual amb pinces òptiques permeten determinar la termodinàmica i cinètica d’unió de molts d’aquests lligands, especialment aquells difícils de caracteritzar mitjançant mesures en volum. És per això que un dels objectius principals d’aquesta tesi ha estat aprofitar les potencialitats de les mesures de molècula individual per a caracteritzar pèptids anti­cancerígens poc solubles i difícils d’estudiar amb tècniques alternatives: des de la cinètica i termodinàmica d’unió, a l’especi.citat en seqüència i la cinètica d’autoacoblament.