In the last decade the increasing ability in the manipulation of small systems has led to a flourishing of single molecule studies. Optical tweezers and atomic force microscopes are proving extremely versatile tools and they are currently enabling us to access the inner functioning of biomolecules at an unprecedented level of detail. In particular, in the vast scenario of emerging experimental applications made possible by these techniques, experiments of mechanical unfolding and refolding of proteins or RNA's hold particular promise as tools for the detection of folding intermediates and misfolded states. These alternative configurations correspond to local minima of the free energy of the molecule and their characterization is fundamental in order to understand the folding process as well as all large conformational rearrangement in biomolecules. They are however difficult to detect by means of traditional experiments of thermal or chemical denaturation because of the unavoidable averaging effects inherent in bulk techniques. Single molecule experiments circumvent this limitation and provide and efficient tool to locate the most relevant free energy minima accessible to the molecule. The objective of this research is to explore the free energy landscape of large functional RNA's such as riboswitches and ribozymes by a combined use of computational tools and mechanical stretching experiments. Especially we aim at characterizing all intermediate and misfolded states both from the thermodynamical and structural point of view. Particular care will be devoted to the investigation of the microscopic details of the transition between competing metastable minima of the free energy, thus helping elucidate the mechanisms of conformational plasticity in functional RNA's.
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