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

Abstract

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.

Prevalence of syn nucleobases in the active sites of functional RNAs

“…We also note that syn nucleobases occur in nonfunctional regions of this structure too, including U23 and G25 in the UUCG tetraloop as expected from NMR studies on this tetraloop (Cheong et al 1990). While most aptamers have syn nucleobases in their binding motifs, some do not, with the most notable examples being the theophylline and caffeine aptamers (Zimmermann et al 2000), although the possibility of transient syn conformations remains possible (see Discussion).…”

An artificial riboswitch for controlling pre-mRNA splicing

“…This may explain why none of the 15 residues (Fig. 1A, nucleotides shown in the rectangular box) required for high-affinity theophylline binding resides in the lower stem (Jenison et al 1994;Zimmermann et al 2000).…”

Use of a Riboswitch-controlled Conditional Hypomorphic Mutation to Uncover a Role for the Essential csrA Gene in Bacterial Autoaggregation

“…Such riboswitches may be composed of a theophylline aptamer and a short nucleotide sequence, which, together with the RBS region of the essential gene of interest, would form multiple conformations at equilibrium (after transcription). The sequence of the theopylline aptamer would be conserved (12,13), whereas the composition of short nucleotide sequence would be gene-specific. This short sequence should be designed such that the RBS of the target gene is blocked in the RNA conformations adopted in the absence of theophylline, thus repressing gene translation, but is "freed up" after theophylline binding, facilitating gene expression.…”