Abstract
Aptamers have proven to be valuable tools for the detection of small molecules due to their remarkable ability to specifically discriminate between structurally similar molecules. Most aptamer selection efforts have relied on counterselection to eliminate aptamers that exhibit unwanted cross-reactivity to interferents or structurally similar relatives to the target of interest. However, because the affinity and specificity characteristics of an aptamer library are fundamentally unknowable a priori, it is not possible to determine the optimal counterselection parameters. As a result, counterselection experiments require trial-and-error approaches that are inherently inefficient and may not result in aptamers with the best combination of affinity and specificity. In this work, we describe a high-throughput screening process for generating high-specificity aptamers to multiple targets in parallel while also eliminating the need for counterselection. We employ a platform based on a modified benchtop sequencer to conduct a massively parallel aptamer screening process that enables the selection of highly specific aptamers against multiple structurally similar molecules in a single experiment, without any counterselection. As a demonstration, we have selected aptamers with high affinity and exquisite specificity for three structurally similar kynurenine metabolites that differ by a single hydroxyl group in a single selection experiment. This process can easily be adapted to other small-molecule analytes and should greatly accelerate the development of aptamer reagents that achieve exquisite specificity for their target analytes.