Gold-tagged RNA - A probe for macromolecular assemblies

Ribonucleic acids (RNAs) play a key role in many fundamental life processes . These polymers are often found complexed with proteins in extremely large particles whose molecular mass may reach several millions of daltons (e.g. ribosomes, spliceosomes, and viruses). Structural studies of such RNA-prot ein complexes should help elucidate their mode of action. For the structura l analyses of many macromolecular assemblies, electron microscopy (EM) has served an instrumental role. However, localization by EM of RNA within biol ogical complexes is not yet a straightforward undertaking. Here we describe a methodology for the covalent tagging of RNA molecules with gold clusters , thereby enabling their direct visualization by microscopical methods. Our strategy involves transcription in vitro of RNAs that carry free thiol gro ups, using ribonucleoside triphosphate analogs containing a substituent wit h a terminal thiol group on their heterocyclic ring. This synthesis is foll owed by coupling of gold clusters to the thiolated transcript through a mal eimido group. Visualization of such gold-tagged RNAs by transmission electr on microscopy showed spots of gold clusters, with a diameter of 1-2 nm, arr anged at nearly regular distances on an imaginary curve that presumably cor responds to the RNA chain. This assignment was corroborated by atomic force microscopy that exhibited images of RNA chains in which knoblike structure s, whose height corresponds to the diameter of the gold clusters, were clea rly seen. This study demonstrates the potential use of nucleic acids that a re covalently labeled with gold clusters for the structural characterizatio n of protein-RNA complexes, (C) 1999 Academic Press.