Site-directed selection of oligonucleotide antagonists by competitive elution

Oligonucleotide ligands that bind a protein or a small molecule of interest are readily isolated by in vitro selection and amplification of rare seque nces from combinatorial libraries of sequence-randomized oligonucleotides ( Gold et al., 1995), Classic systematic evolution of ligands by exponential enrichment (SELEX) protocols are affinity based (Tuerk and Gold, 1990), but because many problems and applications require antagonists, protocols for selecting inhibitors are both desirable and valuable. A widely applicable a pproach for isolating inhibitors is competitive elution with a molecule tha t binds the targeted molecule's active or binding site. We have used this a pproach to isolate antagonists of wheat germ agglutinin (WGA) from a librar y of 2'NH2-pyrimidine, 2'OH-purine oligonucleotides by elution with N N' N" -triacetylchitotriose, (GlcNAc)(3). The highest affinity aptamers have equi librium dissociation constants of 1 nM-20 nM for WGA, a 10(3)-10(4)-fold im provement relative to (GlcNAc)(3), and unlike the carbohydrate, are highly specific In addition to competiting for binding with (GlcNAc)(3), aptamers inhibit WGA-mediated agglutination of sheep erythrocytes, demonstrating tha t they are able to compete with natural ligands presented on the surfaces o f cells. These results illustrate the feasibility of isolating high-affinit y, high-specificity antagonists by competitive elution with low molecular w eight, relatively low-affinity, and low-specificity small molecules.