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 Mg2+. 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.
Smalley
Mary K
MK
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
Silverman
Scott K
SK
D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011721:Pyrenes / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D015347:RNA Probes / Q000737:chemistry*
D005456:Fluorescent Dyes; D011721:Pyrenes; D015347:RNA Probes; D012313:RNA; C030984:pyrene; D008274:Magnesium