A SEMICONSERVED RESIDUE INHIBITS COMPLEX-FORMATION BY STABILIZING INTERACTIONS IN THE FREE STATE OF A THEOPHYLLINE-BINDING RNA

The theophylline-binding RNA aptamer contains a 15 nucleotide motif th at is required for high-affinity ligand binding. One residue within th is RNA motif is only semiconserved and can be an A or C. This residue, C27, was disordered in the previously determined three-dimensional st ructure of the complex, suggesting that it is dynamic in solution. C-1 3 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 (K-d similar to 0.2 mu M), indic ating that the base of residue 27 is not directly involved with theoph ylline binding. Surprisingly, the U27 and G27 RNAs were found to bind theophylline with low affinity (K-d values > 4 mu M). NMR spectroscopy on the U27 RNA revealed the presence of an A7-U27 base pair in the fr ee RNA that prevents formation of a critical base-platform structural motif and therefore blocks theophylline binding, Similarly, a protonat ed 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-theoph ylline complex but instead from stable interactions in the free state of the RNA that inhibit theophylline binding.