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-protein complexes should help elucidate their mode of action. For the structural analyses of many macromolecular assemblies, electron microscopy (EM) has served an instrumental role. However, localization by EM of RNA within biological 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 groups, using ribonucleoside triphosphate analogs containing a substituent with a terminal thiol group on their heterocyclic ring. This synthesis is followed by coupling of gold clusters to the thiolated transcript through a maleimido group. Visualization of such gold-tagged RNAs by transmission electron microscopy showed spots of gold clusters, with a diameter of 1-2 nm, arranged at nearly regular distances on an imaginary curve that presumably corresponds to the RNA chain. This assignment was corroborated by atomic force microscopy that exhibited images of RNA chains in which knob-like structures, whose height corresponds to the diameter of the gold clusters, were clearly seen. This study demonstrates the potential use of nucleic acids that are covalently labeled with gold clusters for the structural characterization of protein-RNA complexes.
Medalia
O
O
Department of Chemistry, The Weizmann Institute of Science, Rehovot, 76100, Israel.
Heim
M
M
Guckenberger
R
R
Sperling
R
R
Sperling
J
J
D005914:Globins / Q000235:genetics; D006046:Gold / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D046911:Macromolecular Substances; D018625:Microscopy, Atomic Force / Q000379:methods; D008854:Microscopy, Electron / Q000379:methods; D015335:Molecular Probes / Q000032:analysis* / Q000138:chemical synthesis; D050607:Organogold Compounds; D009942:Organometallic Compounds / Q000737:chemistry; D012313:RNA / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry*
D046911:Macromolecular Substances; D015335:Molecular Probes; D050607:Organogold Compounds; D009942:Organometallic Compounds; D012313:RNA; C035797:undecagold; D006046:Gold; D005914:Globins