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Search Results: 1 - 10 of 71024 matches for " Maria Manosas "
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Thermodynamic and kinetic aspects of RNA pulling experiments
Maria Manosas,Felix Ritort
Quantitative Biology , 2004, DOI: 10.1529/biophysj.104.045344
Abstract: Recent single-molecule pulling experiments have shown how it is possible to manipulate RNA molecules using optical tweezers force microscopy. We investigate a minimal model for the experimental setup which includes a RNA molecule connected to two polymers (handles) and a bead, trapped in the optical potential, attached to one of the handles. Initially, we focus on small single-domain RNA molecules which unfold in a cooperative way. The model qualitatively reproduces the experimental results and allow us to investigate the influence of the bead and handles on the unfolding reaction. A main ingredient of our model is to consider the appropriate statistical ensemble and the corresponding thermodynamic potential describing thermal fluctuations in the system. We then investigate several questions relevant to extract thermodynamic information from the experimental data. Next, we study the kinetics using a dynamical model. Finally, we address the more general problem of a multidomain RNA molecule with Mg2+-tertiary contacts that unfolds in a sequential way and propose techniques to analyze the breakage force data in order to obtain the reliable kinetics parameters that characterize each domain.
Recovery of free energy branches in single molecule experiments
Ivan Junier,Alessandro Mossa,Maria Manosas,Felix Ritort
Physics , 2009, DOI: 10.1103/PhysRevLett.102.070602
Abstract: We present a method for determining the free energy of coexisting states from irreversible work measurements. Our approach is based on a fluctuation relation that is valid for dissipative transformations in partially equilibrated systems. To illustrate the validity and usefulness of the approach, we use optical tweezers to determine the free energy branches of the native and unfolded states of a two-state molecule as a function of the pulling control parameter. We determine, within 0.6 kT accuracy, the transition point where the free energies of the native and the unfolded states are equal.
Dynamic force spectroscopy of DNA hairpins. II. Irreversibility and dissipation
Maria Manosas,Alessandro Mossa,Nuria Forns,Josep Maria Huguet,Felix Ritort
Physics , 2009, DOI: 10.1088/1742-5468/2009/02/P02061
Abstract: We investigate irreversibility and dissipation in single molecules that cooperatively fold/unfold in a two state manner under the action of mechanical force. We apply path thermodynamics to derive analytical expressions for the average dissipated work and the average hopping number in two state systems. It is shown how these quantities only depend on two parameters that characterize the folding/unfolding kinetics of the molecule: the fragility and the coexistence hopping rate. The latter has to be rescaled to take into account the appropriate experimental setup. Finally we carry out pulling experiments with optical tweezers in a specifically designed DNA hairpin that shows two-state cooperative folding. We then use these experimental results to validate our theoretical predictions.
Dynamic force spectroscopy of DNA hairpins. I. Force kinetics and free energy landscapes
Alessandro Mossa,Maria Manosas,Nuria Forns,Josep Maria Huguet,Felix Ritort
Physics , 2009, DOI: 10.1088/1742-5468/2009/02/P02060
Abstract: We investigate the thermodynamics and kinetics of DNA hairpins that fold/unfold under the action of applied mechanical force. We introduce the concept of the molecular free energy landscape and derive simplified expressions for the force dependent Kramers-Bell rates. To test the theory we have designed a specific DNA hairpin sequence that shows two-state cooperative folding under mechanical tension and carried out pulling experiments using optical tweezers. We show how we can determine the parameters that characterize the molecular free energy landscape of such sequence from rupture force kinetic studies. Finally we combine such kinetic studies with experimental investigations of the Crooks fluctuation relation to derive the free energy of formation of the hairpin at zero force.
Force dependent fragility in RNA hairpins
M. Manosas,D. Collin,F. Ritort
Physics , 2006, DOI: 10.1103/PhysRevLett.96.218301
Abstract: We apply Kramers theory to investigate the dissociation of multiple bonds under mechanical force and interpret experimental results for the unfolding/refolding force distributions of an RNA hairpin pulled at different loading rates using laser tweezers. We identify two different kinetic regimes depending on the range of forces explored during the unfolding and refolding process. The present approach extends the range of validity of the two-states approximation by providing a theoretical framework to reconstruct free-energy landscapes and identify force-induced structural changes in molecular transition states using single molecule pulling experiments. The method should be applicable to RNA hairpins with multiple kinetic barriers.
Force-induced misfolding in RNA
M. Manosas,I. Junier,F. Ritort
Physics , 2009, DOI: 10.1103/PhysRevE.78.061925
Abstract: RNA folding is a kinetic process governed by the competition of a large number of structures stabilized by the transient formation of base pairs that may induce complex folding pathways and the formation of misfolded structures. Despite of its importance in modern biophysics, the current understanding of RNA folding kinetics is limited by the complex interplay between the weak base-pair interactions that stabilize the native structure and the disordering effect of thermal forces. The possibility of mechanically pulling individual molecules offers a new perspective to understand the folding of nucleic acids. Here we investigate the folding and misfolding mechanism in RNA secondary structures pulled by mechanical forces. We introduce a model based on the identification of the minimal set of structures that reproduce the patterns of force-extension curves obtained in single molecule experiments. The model requires only two fitting parameters: the attempt frequency at the level of individual base pairs and a parameter associated to a free energy correction that accounts for the configurational entropy of an exponentially large number of neglected secondary structures. We apply the model to interpret results recently obtained in pulling experiments in the three-helix junction S15 RNA molecule (RNAS15). We show that RNAS15 undergoes force-induced misfolding where force favors the formation of a stable non-native hairpin. The model reproduces the pattern of unfolding and refolding force-extension curves, the distribution of breakage forces and the misfolding probability obtained in the experiments.
Single-molecule stochastic resonance
K. Hayashi,S. de Lorenzo,M. Manosas,J. M. Huguet,F. Ritort
Quantitative Biology , 2012, DOI: 10.1103/PhysRevX.2.031012
Abstract: Stochastic resonance (SR) is a well known phenomenon in dynamical systems. It consists of the amplification and optimization of the response of a system assisted by stochastic noise. Here we carry out the first experimental study of SR in single DNA hairpins which exhibit cooperatively folding/unfolding transitions under the action of an applied oscillating mechanical force with optical tweezers. By varying the frequency of the force oscillation, we investigated the folding/unfolding kinetics of DNA hairpins in a periodically driven bistable free-energy potential. We measured several SR quantifiers under varied conditions of the experimental setup such as trap stiffness and length of the molecular handles used for single-molecule manipulation. We find that the signal-to-noise ratio (SNR) of the spectral density of measured fluctuations in molecular extension of the DNA hairpins is a good quantifier of the SR. The frequency dependence of the SNR exhibits a peak at a frequency value given by the resonance matching condition. Finally, we carried out experiments in short hairpins that show how SR might be useful to enhance the detection of conformational molecular transitions of low SNR.
Improving signal-to-noise resolution in single molecule experiments using molecular constructs with short handles
N. Forns,S. de Lorenzo,M. Manosas,K. Hayashi,J. M. Huguet,F. Ritort
Quantitative Biology , 2011, DOI: 10.1016/j.bpj.2011.01.071
Abstract: We investigate unfolding/folding force kinetics in DNA hairpins exhibiting two and three states with newly designed short dsDNA handles (29 bp) using optical tweezers. We show how the higher stiffness of the molecular setup moderately enhances the signal-to-noise ratio (SNR) in hopping experiments as compared to conventional long handles constructs (approximately 700 bp). The shorter construct results in a signal of higher SNR and slower folding/unfolding kinetics, thereby facilitating the detection of otherwise fast structural transitions. A novel analysis of the elastic properties of the molecular setup, based on high-bandwidth measurements of force fluctuations along the folded branch, reveals that the highest SNR that can be achieved with short handles is potentially limited by the marked reduction of the effective persistence length and stretch modulus of the short linker complex.
Force unfolding kinetics of RNA using optical tweezers. I. Effects of experimental variables on measured results
J. -D. Wen,M. Manosas,P. T. X. Li,S. B. Smith,C. Bustamante,F. Ritort,I. Tinoco Jr
Physics , 2007, DOI: 10.1529/biophysj.106.094052
Abstract: Experimental variables of optical tweezers instrumentation that affect RNA folding/unfolding kinetics were investigated. A model RNA hairpin, P5ab, was attached to two micron-sized beads through hybrid RNA/DNA handles; one bead was trapped by dual-beam lasers and the other was held by a micropipette. Several experimental variables were changed while measuring the unfolding/refolding kinetics, including handle lengths, trap stiffness, and modes of force applied to the molecule. In constant-force mode where the tension applied to the RNA was maintained through feedback control, the measured rate coefficients varied within 40% when the handle lengths were changed by 10 fold (1.1 to 10.2 Kbp); they increased by two- to three-fold when the trap stiffness was lowered to one third (from 0.1 to 0.035 pN/nm). In the passive mode, without feedback control and where the force applied to the RNA varied in response to the end-to-end distance change of the tether, the RNA hopped between a high-force folded-state and a low-force unfolded-state. In this mode, the rates increased up to two-fold with longer handles or softer traps. Overall, the measured rates remained with the same order-of-magnitude over the wide range of conditions studied. In the companion paper (1), we analyze how the measured kinetics parameters differ from the intrinsic molecular rates of the RNA, and thus how to obtain the molecular rates.
Force unfolding kinetics of RNA using optical tweezers. II. Modeling experiments
M. Manosas,J. -D. Wen,P. T. X. Li,S. B. Smith,C. Bustamante,I. Tinoco, Jr.,F. Ritort
Physics , 2007, DOI: 10.1529/biophysj.106.094243
Abstract: By exerting mechanical force it is possible to unfold/refold RNA molecules one at a time. In a small range of forces, an RNA molecule can hop between the folded and the unfolded state with force-dependent kinetic rates. Here, we introduce a mesoscopic model to analyze the hopping kinetics of RNA hairpins in an optical tweezers setup. The model includes different elements of the experimental setup (beads, handles and RNA sequence) and limitations of the instrument (time lag of the force-feedback mechanism and finite bandwidth of data acquisition). We investigated the influence of the instrument on the measured hopping rates. Results from the model are in good agreement with the experiments reported in the companion article (1). The comparison between theory and experiments allowed us to infer the values of the intrinsic molecular rates of the RNA hairpin alone and to search for the optimal experimental conditions to do the measurements. We conclude that long handles and soft laser traps represent the best conditions to extract rate estimates that are closest to the intrinsic molecular rates. The methodology and rationale presented here can be applied to other experimental setups and other molecules.
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