SMINAFEL

Single molecule investigation of nucleic acids free energy landscapes: Bringing together computational models and laser tweezer experiments

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 Obiettivo del progetto (Objective)

'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.'

Introduzione (Teaser)

European scientists investigated the dynamic unfolding of DNA during replication by generating a tool that could subsequently be applied to screen helicase-targeting drugs for infection and oncologic applications.

Descrizione progetto (Article)

In order to study the mechanical unfolding and refolding of various molecules including proteins and nucleic acids, and determine misfolded states, special equipment and techniques are required. To this end, optical tweezers and atomic force microscopes are proving extremely versatile tools that facilitate access to the inner functioning of biomolecules at an unprecedented level of detail.

The EU-funded Sminafel project focused on the activity of the helicase enzymes that assist the replication-repair of DNA. By hydrolysing adenosine triphosphate (ATP), these proteins convert chemical energy to the unzipping of the DNA double helix.

Scientists developed and optimised an optical tweezer-related technology that enabled the investigation of helicase function. More specifically, a DNA hairpin was fixed onto coated beads between a micropipette and an optical trap, and fluxing of the helicase and ATP solutions was facilitated through a microfluidics system. Various parameters of the system, including the valves and the length of the DNA molecule were standardised to allow efficient opening of the DNA hairpin, allowing the measurement of helicase activity.

Experimental results showed that the amplitude of fluctuations in the helicase activity remained constant independently of ATP concentration. The only determinant factors proved to be the opening-closing fluctuations of the replication fork.

The Sminafel technology constituted a significant step towards understanding the functioning of molecular motors involved in the DNA molecular repair and duplication machinery. The developed system is envisioned to provide a unique tool for studying various biomolecules in detail.