Molecular interactions and metal binding in the theophylline-binding core of an RNA aptamer
| Autor: | Catherine L. Wick, Thomas P. Shields, Arthur Pardi, Robert D. Jenison, Grant R. Zimmermann |
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| Rok vydání: | 2000 |
| Předmět: |
Models
Molecular Magnetic Resonance Spectroscopy Stereochemistry Aptamer In Vitro Techniques Biology Divalent chemistry.chemical_compound Theophylline Caffeine medicine Humans Binding site Molecular Biology chemistry.chemical_classification Binding Sites Base Sequence RNA Nuclear magnetic resonance spectroscopy Bronchodilator Agents NMR spectra database Deoxyribose chemistry Biochemistry Metals Mutation Nucleic Acid Conformation Polyribonucleotides Research Article medicine.drug |
| Zdroj: | RNA. 6:659-667 |
| ISSN: | 1355-8382 |
| Popis: | An RNA aptamer containing a 15-nt binding site shows high affinity and specificity for the bronchodilator theophylline. A variety of base modifications or 2' deoxyribose substitutions in binding-site residues were tested for theophyllinebinding affinity and the results were compared with the previously determined three-dimensional structure of the RNA-theophylline complex. The RNA-theophylline complex contains a U6-A28-U23 base triple, and disruption of this A28-U23 Hoogsteen-pair by a 7-deaza, 2'-deoxy A28 mutant reduces theophylline binding >45-fold at 25 degrees C. U24 is part of a U-turn in the core of the RNA, and disruption of this U-turn motif by a 2'-deoxy substitution of U24 also reduces theophylline binding by >90-fold. Several mutations outside the "conserved core" of the RNA aptamer showed reduced binding affinity, and these effects could be rationalized by comparison with the three-dimensional structure of the complex. Divalent ions are absolutely required for high-affinity theophylline binding. High-affinity binding was observed with 5 mM Mg2+, Mn2+, or Co2+ ions, whereas little or no significant binding was observed for other divalent or lanthanide ions. A metal-binding site in the core of the complex was revealed by paramagnetic Mn2+-induced broadening of specific RNA resonances in the NMR spectra. When caffeine is added to the aptamer in tenfold excess, the NMR spectra show no evidence for binding in the conserved core and instead the drug stacks on the terminal helix. The lack of interaction between caffeine and the theophylline-binding site emphasizes the extreme molecular discrimination of this RNA aptamer. |
| Databáze: | OpenAIRE |
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