Fluorescence quenching and dequenching analysis of RNA interactions in vitro and in vivo

This paper describes the use of fluorescence quenching and dequenching to analyze molecular interactions of RNA in vitro and in vivo. Fluorescein-labeled ribonucleotide was incorporated into an RNA substrate by in vitro transcription. The fluorescence quantum yield of the intact RNA was reduced by intramolecular quenching. When the RNA was degraded by ribonuclease digestion, the quantum yield increased by approximately 50%, reflecting dequenching due to separation of proximate fluorophores. Dequenching was dependent on the concentration of enzyme and substrate and was inhibited by the ribonuclease inhibitor RNasin. Comparable rates of dequenching were observed with RNase A and RNase T1. Dequenching provides a sensitive, quantitative, and convenient assay for RNA degradation. When fluorescent RNA was microinjected into cells in culture the intracellular fluorescence declined gradually with time after injection reflecting "superquenching: due to intermolecular interactions between the injected RNA and intracellular components. Capped RNA exhibited greater superquenching than uncapped RNA. Superquenching provides a sensitive, quantitative, and specific assay with subcellular resolution for intermolecular interactions of RNA in vivo. When RNase was injected into the same cells, fluorescence increased by approximately 50%, indicating that fluorescence dequenching due to RNA degradation can be measured in vivo as well as in vitro.