Force-spectroscopy of small ligands binding to nucleic acids

• Autores: Joan Camuñas Soler
• Directores de la Tesis: Felix Ritort Farran (dir. tes.)
• Lectura: En la Universitat de Barcelona ( España ) en 2015
• Idioma: inglés
• Tribunal Calificador de la Tesis: David Rueda (presid.), Gijs J. L. Wuite (secret.), Fernando Moreno Herrero (voc.)
• Materias:
  • Física
    • Mecánica
      • Mecánica de sólidos
    • Física molecular
      • Macromoléculas
      • Moléculas orgánicas
  • Química
    • Bioquímica
      • Péptidos
• Enlaces
  • Tesis en acceso abierto en: Dipòsit Digital de la UB
• Resumen

  • 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.