Electron paramagnetic resonance spectroscopy of spin-labeled RNA: an emerging tool for the elucidation of RNA structure and dynamics

Abstract

Electron paramagnetic resonance (EPR) spectroscopy was applied to the study of RNA structure and dynamics. A general and efficient method for the site-specific incorporation of nitroxide spin-labels into internal sites of RNA is described. The spin labeling reagent 4-isocyanato TEMPO reacts with 2'-amino modified RNA in >90% conversion to produce spin-labeled RNA. Several spin-labeled RNAs were shown to exhibit structure-dependent dynamics.The trans activation responsive (TAR) RNA of the human immunodeficiency virus (HIV) forms an essential interaction with the Tat protein during the HIV lifecycle. Four spin-labeled TAR RNAs were prepared, and their interactions with metal ions, derivatives of the Tat protein, and small molecules were studied by EPR spectroscopy. The spin-label was shown to have a minimal effect on RNA-peptide affinity. Argininamide and a mutant Tat peptide induced similar changes in TAR RNA internal dynamics upon binding. The wild-type Tat peptide was shown to have a dramatically different effect on the dynamics of U23 and U38 compared to the mutant peptide, despite similar binding affinities. Peptide sequence mutation and EPR spectroscopic analysis revealed that in addition to R52, modification at residues 53--57 affected TAR dynamics, indicating their role in forming a specific TAR-Tat complex. Small molecules which were known to bind similarly induced similar changes in TAR RNA internal dynamics, correlating RNA structure to RNA dynamics. Dynamic signatures were used to provide evidence that guanidinoneomycin binds to the TAR RNA in a manner similar to that of argininamide rather than neomycin.In addition to the ability to carry genetic information, RNA has been shown to catalyze chemical reactions. These "ribozymes" are important in many cellular processes and have implications for the origin of life. We studied the metal ion-dependent folding of the hammerhead ribozyme by EPR spectroscopy, specifically the two-step divalent metal ion-dependent pathway and the effect of high concentrations of monovalent metal ions. These results have implications for the role of metal ions in structure assembly of this well studied ribozyme.