Journal
NUCLEIC ACIDS RESEARCH
Volume 34, Issue 1, Pages 152-166Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkj420
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Funding
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM065966] Funding Source: NIH RePORTER
- NIGMS NIH HHS [GM-65966, R01 GM065966] Funding Source: Medline
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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.
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