Fluorescence of covalently attached pyrene as a general RNA folding probe

Fluorescence techniques are commonly and powerfully applied to monitor biomolecular folding. In a limited fashion, the fluorescence emission intensity of covalently attached pyrene has been used as a reporter of RNA conformational changes. Here, we pursue two goals: we examine the relationship between tether identity and fluorescence response, and we determine the general utility of pyrene fluorescence to monitor RNA folding. The P4–P6 domain of the Tetrahymena group I intron RNA was systematically modified at multiple nucleotide positions with pyrene derivatives that provide a range of tether lengths and compositions between the RNA and chromophore. Certain tethers typically lead to a superior fluorescence signal upon RNA folding, as demonstrated by equilibrium titrations with Mg(2+). In addition, useful fluorescence responses were obtained with pyrene placed at several nucleotide positions dispersed throughout P4–P6. This suggests that monitoring of tertiary folding by fluorescence of covalently attached pyrene will be generally applicable to structured RNA molecules.