To visualize the interplay of RNA structural interactions in a ligand binding site, we have determined the solution structure of a high affinity RNA-theophylline complex using NMR spectroscopy. The structure provides insight into the ability of this in vitro selected RNA to discriminate theophylline from the structurally similar molecule caffeine. Numerous RNA structural motifs combine to form a well-ordered binding pocket where an intricate network of hydrogen bonds and stacking interactions lock the theophylline into the complex. Two internal loops interact to form the binding site which consists of a sandwich of three base triples. The complex also contains novel base-zipper and 1-3-2 stacking motifs, in addition to an adenosine platform and a reversed sugar. An important feature of the RNA is that many of the conserved core residues participate in multiple overlapping tertiary interactions. This complex illustrates how interlocking structural motifs can be assembled into a highly specific ligand-binding site that possesses high levels of affinity and molecular discrimination.
An RNA aptamer containing a 15-nt binding site shows high affinity and specificity for the bronchodilator theophylline. A variety of base modifications or 29 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, 29-deoxy A28 mutant reduces theophylline binding .45-fold at 25 8C. U24 is part of a U-turn in the core of the RNA, and disruption of this U-turn motif by a 29-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 Mg 21 , Mn 21 , or Co 21 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 Mn 21 -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.
The theophylline-binding RNA aptamer contains a 15 nucleotide motif that is required for high-affinity ligand binding. One residue within this RNA motif is only semiconserved and can be an A or C. This residue, C27, was disordered in the previously determined three-dimensional structure of the complex, suggesting that it is dynamic in solution. 13C Relaxation measurements are reported here, demonstrating that C27 is highly dynamic in the otherwise well-ordered RNA-theophylline complex. A synthetic complex with an abasic residue at position 27 was found to exhibit wild-type binding affinity (Kd approximately 0.2 microM), indicating that the base of residue 27 is not directly involved with theophylline binding. Surprisingly, the U27 and G27 RNAs were found to bind theophylline with low affinity (Kd values > 4 microM). NMR spectroscopy on the U27 RNA revealed the presence of an A7-U27 base pair in the free RNA that prevents formation of a critical base-platform structural motif and therefore blocks theophylline binding. Similarly, a protonated A7H+-C27 base pair forms in the absence of theophylline at low pH, which explains the unusual pH dependence of theophylline binding of the C27 RNA aptamer. Thus the weak binding for various nucleotides at position 27 arises not from unfavorable interactions in the RNA-theophylline complex but instead from stable interactions in the free state of the RNA that inhibit theophylline binding.
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