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 under standing the basic relationships between RNA structure and function. W e describe a series of experiments that define the functional requirem ents for an RNA motif that specifies high-affinity binding to the carb oxylation 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, electrostatica lly neutral ligand. In contrast to virtually all previously identified aptamers, unpaired nucleotides make up a small fraction of the bindin g motif. Instead, the identity of 14 nucleotides involved in base pair ing is highly conserved among functional clones and their substitution by nonidentical base pairs significantly reduces or eliminates bindin g. Chemical probing is consistent with the predicted pseudoknot motif and indicates that relatively little change in structure accompanies l igand binding, a strong contrast with results for other aptamers. Comp etition experiments suggest that the aptamer recognizes all parts of t he biotin ligand, including its thiophane ring and fatty acid tail. Tw o alternative modes of binding are suggested by a three-dimensional mo del of the pseudoknot, both of which entail significant interactions w ith base-paired nucleotides.