A system for multiplexed selection of aptamers with exquisite specificity without counterselection

Significance Aptamers have the capacity to discriminate between structurally similar small molecules. However, generating such highly specific aptamers has proven challenging using conventional processes based on counterselection against nontarget molecules. In this work, we describe a high-throughput screening platform that can characterize the specificity of millions of aptamers toward a group of structurally related molecules in a single experiment and generate exquisitely specific aptamers without any counterselection. As exemplars, we generated aptamers with high affinity and specificity toward three structurally related kynurenine metabolites using our platform. Our platform can be readily adapted to other small-molecule targets and should therefore accelerate the development of aptamer reagents with exquisite specificity.