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.
Dipolar coupling interactions represent an extremely valuable source of long-range distance and angle information that was previously not available for solution structure determinations of macromolecules. This is because observation of these dipolar coupling data requires creating an anisotropic environment for the macromolecule. Here we introduce a new method for generating tunable degrees of alignment of macromolecules by addition of magnetically aligned Pf1 filamentous bacteriophage as a cosolute. This phage-induced alignment technique has been used to study 1H-1H, 1H-13C, and 1H-15N dipolar coupling interactions in a DNA duplex, an RNA hairpin and several proteins including thioredoxin and apo-calmodulin. The phage allow alignment of macromolecules over a wide range of temperature and solution conditions and thus represent a stable versatile method for generating partially aligned macromolecules in solution.
The most frequently occurring RNA hairpins in 16S and 23S ribosomal RNA contain a tetranucleotide loop that has a GNRA consensus sequence. The solution structures of the GCAA and GAAA hairpins have been determined by nuclear magnetic resonance spectroscopy. Both loops contain an unusual G-A base pair between the first and last residue in the loop, a hydrogen bond between a G base and a phosphate, extensive base stacking, and a hydrogen bond between a sugar 2'-end OH and a base. These interactions explain the high stability of these hairpins and the sequence requirements for the variant and invariant nucleotides in the GNRA tetranucleotide loop family.
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