Functional requirements for specific ligand recognition by a biotin-binding RNA pseudoknot.
Ligand-binding RNAs and DNAs (aptamers) isolated by in vitro selection from random sequence pools provide convenient model systems for understanding the basic relationships between RNA structure and function. We describe a series of experiments that define the functional requirements for an RNA motif that specifies high-affinity binding to the carboxylation cofactor biotin. A simple pseudoknot containing an adenosine-rich loop accounts for binding in all independently derived aptamers selected to bind biotin, suggesting that it alone represents a global optimum for recognition of this particular nonaromatic, electrostatically neutral ligand. In contrast to virtually all previously identified aptamers, unpaired nucleotides make up a small fraction of the binding motif. Instead, the identity of 14 nucleotides involved in base pairing is highly conserved among functional clones and their substitution by nonidentical base pairs significantly reduces or eliminates binding. Chemical probing is consistent with the predicted pseudoknot motif and indicates that relatively little change in structure accompanies ligand binding, a strong contrast with results for other aptamers. Competition experiments suggest that the aptamer recognizes all parts of the biotin ligand, including its thiophane ring and fatty acid tail. Two alternative modes of binding are suggested by a three-dimensional model of the pseudoknot, both of which entail significant interactions with base-paired nucleotides.
Wilson
C
C
Department of Biology, Center for the Molecular Biology of RNA, University of California at Santa Cruz 95064, USA.
Nix
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J
Szostak
J
J
D020029:Base Pairing; D001483:Base Sequence; D001667:Binding, Competitive / Q000235:genetics; D001710:Biotin / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding / Q000235:genetics; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*
D008024:Ligands; D012313:RNA; D001710:Biotin