Molecular interactions and metal binding in the theophylline-binding core of an RNA aptamer

An RNA aptamer containing a 15-nt binding site shows high affinity and spec ificity for the bronchodilator theophylline. A variety of base modification s or 2' deoxyribose substitutions in binding-site residues were tested for theophylline-binding affinity and the results were compared with the previo usly determined three-dimensional structure of the RNA-theophylline complex . The RNA-theophylline complex contains a U6-A28-U23 base triple, and disru ption of this A28-U23 Hoogsteen-pair by a 7-deaza, 2'-deoxy A28 mutant redu ces 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 s ubstitution of U24 also reduces theophylline binding by >90-fold. Several m utations outside the "conserved core" of the RNA aptamer showed reduced bin ding affinity, and these effects could be rationalized by comparison with t he 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 signific ant binding was observed for other divalent or lanthanide ions. A metal-bin ding site in the core of the complex was revealed by paramagnetic Mn2+-indu ced broadening of specific RNA resonances in the NMR spectra. When caffeine is added to the aptamer in tenfold excess, the NMR spectra show no evidenc e for binding in the conserved core and instead the drug stacks on the term inal helix. The lack of interaction between caffeine and the theophylline-b inding site emphasizes the extreme molecular discrimination of this RNA apt amer.