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

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

Helicases are molecular motors that assist the DNA repair-replication machinery. By hydrolysing adenosine triphosphate (ATP), these proteins convert chemical energy in to useful work and unzip the DNA double helix. The study of these molecules is both biomedically relevant and theoretically compelling since a good understanding of molecular motors, being amenable to be analysed in terms of efficiency and entropy generation, represents a first step in the quest a thermodynamic description of life. The present project has allowed to develop the expertise needed to perform experiments on helicase function in optical tweezers. Previously such experiments had been performed mainly with magnetic tweezers. Those instruments however are characterised by a lower time and length resolution thus hindering a detailed study of the fluctuations of motor functioning. The following setup was devised: a DNA hairpin is held fixed between two streptavidin and antidigoxigenin coated beads in such a way that the biotin and degoxigenin labelled ends bind onto said beads. The two beads are then held fixed by a micropipette and the optical trap respectively. By fluxing a solution containing both the helicase and ATP the hairpin is unzipped thus allowing to measure the average rate and the fluctuations of helicase activity. Several hurdles had to be overcome in order to devise an experimental set-up suited to the optical tweezer. First of all the right microfluidic scheme had to be developed. It was determined that, in order to prevent ATP depletion from helicases and keep ATP concentration constant during the experiment, helicases and ATP cannot be mixed from the start but must be fluxed trough separated channels of a microfluidic system and mixed before the entrance in the main microfluidic chamber. For this purpose both custom made and commercial valves where tested determining that T shaped three-way valves are better suited to ensure mixing between the helicase and the ATP bearing solution and therefore grant maximal helicase activity at a given ATP concentration. Since helicase activity at a given helicase concentration is proportional to the substrate length, in order to ensure a satisfactory statistics one must work with sufficiently long DNA molecules. On the other hand working with large DNA constructs increases the uncertainty in the conversion between end-to-end data -what the instrument measures- in to contour length data that best describe the motor functioning. In order to overcome this problem a 'blocking oligo' technique was developed which helps maintaining the hairpin open at a desired position thus granting sufficient substrate for helicase binding while, at the same time, easing the conversion from end-to-end distance to contour length. The amplitude of fluctuations in the helicase activity is dominated constant independently on ATP concentration showing to be dominated by opening-closing fluctuations of the replication fork. Taking this into consideration allows to restrict the parameter space for the fitting of kinetics models of the motor functioning. Finally, the motor displacement was shown to obey a fluctuation theorem which is interpreted in terms of a kinetic binomial process. This project represents a step forward in the understanding of the functioning of molecular motors involved in DNA molecular repair and duplication machinery. In perspective, experimental set-ups very similar to the one developed might be used as in vitro assays for the screening of drugs targeted against viral helicases or for helicase inactivating drugs for oncologic applications.

Data: CORDIS, © European Union