Impacts of the Extreme Conditions of Environments on RNA's structure and function from the Avocado Sunblotch Viroid : Application of NIR-Raman Spectroscopy and ad hoc Baro-Bio-Reactor

Abstract

The typical environment for biomolecules in vivo is highly crowded. Molecular crowding effects can affect radically RNA folding mechanism as well as stability and function. To mimic in dilute solution, some of these effects, a combination of biophysical techniques and extreme physical-chemical parameters changes is used to probe the structural flexibility and function of RNA from Avocado Sunblotch viroids. Such viroids are non encapsilated RNA plant pathogens which possess a catalytic hammerhead ribozyme activity. To date, little is known regarding the conformation of ribonucleic acids (RNA), and the ways by which such viroid induces diseases. NIR Raman spectroscopy study gives several markers which are sensitive to RNA folding and structural changes. A typical « A-type » RNA conformation with ordered double helical content and a C3’-endo/anti sugar pucker configuration are found which are destabilized by high temperature. Deuteration not only decreases RNA self-cleavage activity but induces a new secondary structure. Surprisingly, the self-cleaved RNA Raman vibrational modes, exhibit noticeable frequency downshifts suggesting that phosphodiester and phosphodioxy structures are modified. A designed baro bio-reactor has been developped which allows at constant pressure, rapid injection of activators and sampling out of products. Bell-shape temperature-profiles of RNA’s cleavage activity are obtained which is independent of the pressure. However pressure decreases the rates in a non-linear manner with an initial activation volume ΔV‡ = + 20.5 ml/mole, and an activation isothermal compressibility Δβ‡ = 8x10-6 MPa-1 suggesting that the active site is controlled by complex multi-conformers. However, study on a model full-length ribozyme (54), shows clearly two distinct pressure-sensitive activities indicating the participation of a fast cleaving and a slow cleaving conformers.