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.

Keywords: RNA structures; differential scanning fluorimetry; fluorescent probes; melting curves; riboswitches.