A Semiconserved Residue Inhibits Complex Formation by Stabilizing Interactions in the Free State of a Theophylline-Binding RNA

Abstract

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 (K_d ∼ 0.2 μM), 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 (K_d values > 4 μM). 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.