title: Differential scanning fluorimetry for monitoring RNA stability.
abstract: Cellular RNA function is closely linked to RNA structure. It is therefore imperative to develop methods that report on structural stability of RNA and how it is modulated by binding of ions, other osmolytes, and RNA-binding ligands. Here, we present a novel method to analyze the stability of virtually any structured RNA in a highly parallel fashion. This method can easily determine the influence of various additives on RNA stability, and even characterize ligand-induced stabilization of riboswitch RNA. Current approaches to assess RNA stability include thermal melting profiles (absorption or circular dichroism) and differential scanning calorimetry. These techniques, however, require a substantial amount of material and cannot be significantly parallelized. Current fluorescence spectroscopic methods rely on intercalating dyes, which alter the stability of RNA. We employ the commercial fluorescent dye RiboGreen, which discriminates between single-stranded (or unstructured regions) and double-stranded RNA. Binding leads to an increase in fluorescence quantum yield, and thus reports structural changes by a change in fluorescence intensity.
authors: Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Max-von-Laue-Strasse 7, 60438 Frankfurt am Main (Germany); Current address: Department of Chemistry, Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139 (USA).
authors: Robert
authors: R
authors: Silvers
authors: Heiko
authors: H
authors: Keller
authors: Harald
authors: H
authors: Schwalbe
authors: Martin
authors: M
authors: Hengesbach
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry*; D005470:Fluorometry / Q000379:methods*; D012313:RNA / Q000737:chemistry*; D020871:RNA Stability
chemical_list: D005456:Fluorescent Dyes; D012313:RNA
keywords: RNA structures; differential scanning fluorimetry; fluorescent probes; melting curves; riboswitches