RESUMEN
Helicases are a broad family of enzymes that perform crucial functions in DNA replication and in the maintenance of DNA and RNA integrity. A detailed mechanical study of helicases on DNA and RNA is possible using single molecule manipulation methods. Among those, magnetic tweezers (or traps) present a convenient, moderate throughput assay (tens of enzymes can be monitored simultaneously) that allow for high resolution (single base-pair) studies of these enzymes in various conditions and on various substrates (double and single stranded DNA and RNA). Here we discuss various implementation of the basic assay relevant for these studies.
Asunto(s)
ADN Helicasas/química , ADN Cruciforme/química , Magnetismo/métodos , Pinzas Ópticas , ADN/química , ADN/genética , ADN Helicasas/genética , Replicación del ADN/genética , ADN Cruciforme/genética , ARN/química , ARN/genética , Imagen Individual de Molécula/métodosRESUMEN
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/noise ratio (SNR) in hopping experiments as compared to conventional long-handled constructs (â 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, as far as we are aware, 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.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Pinzas Ópticas , Secuencia de Bases , ADN/genética , Elasticidad , Cinética , Datos de Secuencia Molecular , TermodinámicaRESUMEN
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 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.
Asunto(s)
Modelos Moleculares , Conformación de Ácido Nucleico , ARN/química , Fenómenos Biomecánicos , Fenómenos Biofísicos , Entropía , Cinética , Modelos Químicos , TermodinámicaRESUMEN
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. 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 the longest handles and softest traps that allow detection of the folding/unfolding signal (handles approximately 5-10 Kbp and traps approximately 0.03 pN/nm) represent the best conditions to obtain the intrinsic molecular rates. The methodology and rationale presented here can be applied to other experimental setups and other molecules.
Asunto(s)
Artefactos , Micromanipulación/métodos , Modelos Químicos , Modelos Moleculares , Pinzas Ópticas , ARN/química , ARN/ultraestructura , Simulación por Computador , Elasticidad , Cinética , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Estrés MecánicoRESUMEN
We apply Kramers theory to investigate the dissociation of multiple bonds under mechanical force and interpret experimental results for the unfolding and 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.
Asunto(s)
Micromanipulación/métodos , Modelos Químicos , Modelos Moleculares , ARN/química , Simulación por Computador , Elasticidad , Conformación de Ácido Nucleico , ARN/ultraestructura , Estrés MecánicoRESUMEN
Recent single-molecule pulling experiments have shown how it is possible to manipulate RNA molecules using laser tweezers. In this article we investigate a minimal model for the experimental setup which includes an RNA molecule connected to two polymers (handles) and a bead trapped in the optical potential and attached to one of the handles. We start by considering the case of small single-domain RNA molecules, which unfold in a cooperative way. The model qualitatively reproduces the experimental results and allows us to investigate the influence of the bead and handles on the unfolding reaction. A main ingredient of the 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 experimental data. The kinetics of unfolding is also studied by introducing a dynamical model. Finally, we apply the model to the more general problem of a multidomain RNA molecule with Mg(2+) tertiary contacts that unfolds in a sequential way.