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 specificity for the bronchodilator theophylline. A variety of base modifications or 2' 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, 2'-deoxy A28 mutant reduces 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 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 Mg2+, Mn2+, or Co2+ 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 Mn2+-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.

A high throughput platform for systematic evolution of ligands by exponential enrichment (SELEX).

The systematic evolution of ligands by exponential enrichment (SELEX) process is a combinatorial chemistry method for the isolation of nucleic acid ligands (aptamers) that bind to a desired target molecule with high affinity. In order to increase throughput via automation, we have adapted the SELEX process for protein targets to a robotics-compatible microtiter plate format. A remarkable feature of the platform is that targets are immobilized by hydrophobic adsorption onto the plate surface. Hydrophobic immobilization procedures are simple and require no specialized modification of the protein target. This format was tested by manually performing four independent SELEX experiments. All were concluded within 8 rounds of selection and yielded aptamers that bind in solution to their respective protein target, calf intestinal alkaline phosphatase, human alpha-thrombin or human platelet derived growth factor, with equilibrium dissociation constants below 3 x 10-10 M.

Oligonucleotide inhibitors of P-selectin-dependent neutrophil-platelet adhesion.

P-selectin, an inducible cell adhesion molecule, mediates rolling of neutrophils on activated vascular endothelium. Because rolling is an early event of the inflammatory response, therapeutic applications of selectin antagonists have been of broad interest. There are, however, no truly satisfactory therapeutic candidates among known inhibitors. Consequently, we have used Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology, a process based on oligonucleotide combinatorial chemistry and in vitro selection, to develop aptamer antagonists of P-selectin. Equilibrium dissociation constants for aptamer/P-selectin binding range from 16 to 710 pM, a 10(5)-10(6)-fold improvement compared with the minimal carbohydrate ligand, sialyl Lewis X (sLeX). Aptamer binding is divalent cation dependent and, unlike sLeX, is specific for P-selectin. The selectivity for human P-selectin relative to human E-selectin or human L-selectin is 10(4)-10(5). In vitro, aptamers bind with subnanomolar affinities to P-selectin expressed on thrombin-activated platelets, inhibit the binding of P-selectin-IgG chimera to sLeX and to neutrophils, and block the binding activated platelets to neutrophils in flow cytometry and in hydrodynamic assays. Extrapolating from their in vitro characteristics, these novel P-selectin-specific antagonists may be suitable candidates for therapeutic development.

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 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.

Interlocking structural motifs mediate molecular discrimination by a theophylline-binding RNA.

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.

High-resolution molecular discrimination by RNA.

Species of RNA that bind with high affinity and specificity to the bronchodilator theophylline were identified by selection from an oligonucleotide library. One RNA molecule binds to theophylline with a dissociation constant Kd of 0.1 microM. This binding affinity is 10,000-fold greater than the RNA molecule's affinity for caffeine, which differs from theophylline only by a methyl group at nitrogen atom N-7. Analysis by nuclear magnetic resonance indicates that this RNA molecule undergoes a significant change in its conformation or dynamics upon theophylline binding. Binding studies of compounds chemically related to theophylline have revealed structural features required for the observed binding specificity. These results demonstrate the ability of RNA molecules to exhibit an extremely high degree of ligand recognition and discrimination.

Pseudo--half-knot formation with RNA.

A pseudo--half-knot can be formed by binding an oligonucleotide asymmetrically to an RNA hairpin loop. This binding motif was used to target the human immunodeficiency virus TAR element, an important viral RNA structure that is the receptor for Tat, the major viral transactivator protein. Oligonucleotides complementary to different halves of the TAR structure bound with greater affinity than molecules designed to bind symmetrically around the hairpin. The pseudo--half-knot--forming oligonucleotides altered the TAR structure so that specific recognition and binding of a Tat-derived peptide was disrupted. This general binding motif may be used to disrupt the structure of regulatory RNA hairpins.