Noise-correlation force spectroscopy in molecules and cells

Abstract

In this thesis, we employ noise-correlation force spectroscopy measurements using LOT to extract valuable information about the passive and active fluctuations of DNA molecules and red blood cells. We have chosen the laser optical tweezers (LOT) as the experimental setup because of its high resolution in both displacement (1nm) and force (0.1pN), and its versatility that allows us to measure DNA molecules and red blood cells (RBC).

In the first part, we study the mechanical deformability and active processes of RBC. The results demonstrate that RBC exhibits three well-separated timescales spanning three orders of magnitude with a novel kind of stress and strain relaxational behavior that we call discrete-stretched exponential behavior. This new relaxation response is intermediate between the pure exponential relaxation observed in linear and two- level systems and the more complex stretched-exponential relaxation observed in polymers and glassy matter. In addition, we have introduced a soft-glass rheology model (the disordered blobs model) that reproduces the experimental RBC pulling and force relaxation curves measured with LOT. Moreover, the worm-like chain model describes the force generated by the RBC and the stress behavior in continuous strain deformation (pulling) and strain jump deformation (relaxation) experiments. From the equipartition theorem, we develop two methods to quantify the non-equilibrium effects and activity of the RBCs. In the first method, we charactezed the RBC viscoelasticity at each time window by changing the trap stiffness. In the second method, we compute the effective temperature of the RBC, assuming a constant RBC stiffness obtained from the pulling experiments. Both methods present deviations as trap stiffness decreases indicating the presence of active processes.

In the second part, we derive a new variance relation derived from the FDT, that holds for systems of N particles at non-equilibrium steady states subjected to arbitrary potentials. Moreover, this relation introduces two upper bounds to the entropy production rate. We also present a stochastic switching trap (SST) solvable model that returns an exact expression to quantify the entropy production rate. From an experimental point of view, we validate the variance sum rule in three different systems: a single bead optically trapped with and without flow, a CD4 hairpin in its folded and unfolded states, and a single bead optically trapped in a NESS. From the single bead in the optical trap case, we observe how the variance sum rule holds with and without flow as expected. Moreover, we recover compatible values for the trap stiffness and the friction coefficient of the bead with respect to the ones obtained by the FPS analysis. In the molecular case, we also obtain realistic values for the system stiffness and the friction coefficient. However, we observe a discrepancy for large times that we associate with the micropipette drift. In the SST protocol, the results show a deviation from equilibrium as expected, which we can quantify by computing the entropy production rate.

In the third part, we study the elastic properties of dsDNA molecules of different molecular lengths by measuring the fluctuations of the force. By measuring the force power spectrum, we observe a significant deviation between our experimental data and the extensible worm-like chain model predicted values. This discrepancy introduces a novel-length dependence feature on the theoretical model. We interpret this disagreement by introducing a length dependence in the Young modulus. By letting the Young modulus change, we have observed it decreases for shorter molecules. We have proposed an analytical formula for the length dependence of the Young modulus following the one reported for the persistence length. This expression is in excellent agreement with our experimental data and other independent experimental studies using LOT and magnetic tweezers.

Aquesta tesis tracta de l'estudi experimental i teòric de les fluctuacions en molècules d'ADN de cadena doble (dsDNA) i de glòbuls vermells (GV). La tesi introdueix l'espectroscopia de mesures de soroll utilitzant pinces òptiques, com una metodologia experimental per caracteritzar processos biofísics en sistemes moleculars i cel·lulars. La primera part de la tesis es concentra en les propietats mecàniques i els processos de fora de l’equilibri dels GV. A través de mesures actives (aplicant perturbacions externes) i passives (espectres de potències en condicions estacionàries), s'estudien els processos de relaxació i dissipatius dels GV. Les dades experimentals de mesures actives són reproduïdes modelitzant el GV a través d'un model de reologia de sistemes tous. La segona part de la tesi consisteix en la demostració experimental d'una nova relació de fluctuacions, que a diferència del teorema de fluctuació-dissipació, es calcula a través de mesures únicament passives. Aquesta relació es verifica per a diferents sistemes i aporta una nova metodologia per calcular la producció d'entropia de sistemes fora de l'equilibri. La tercera part de la tesi estudia l’acoblament torsió-deformació de molècules de dsDNA a través de mesures passives d'espectroscopia de forces. Aquest estudi es basa en la caracterització de l'espectre de potències interpretat a través del model de barra elàstica del qual el model cucoide (worm-like chain en anglés) és un cas particular.