Abstract
Nucleic acid aptamers bridge the gap between small molecules and antibodies as potentially nonimmunogenic, stable, selective, and potent ligands. Aptamer discovery starts with massively parallel affinity selection-driven directed evolution, which only identifies sequences based on target binding. We have developed an activity-based aptamer screening system using magnetic beads templated with aptamer-encoding DNAs and encapsulated in polyacrylamide hydrogel droplets. Magnetic bead-bound DNAs are copied to the oligonucleotide-functionalized gel periphery via transcription-reverse transcription amplification. A model enzyme (trypsin) in-gel activity assay was developed wherein tryptic hydrolysis of a gel-immobilized N-terminal-Cy5-labeled trypsin substrate peptide is inhibited in the presence of gel-immobilized trypsin inhibitor aptamers, leaving the bead with high Cy5 fluorescence for sorting. Proof-of-concept screens enriched positive controls 100,000-fold in multiround screens. Though this study used DNA, the technology could also be applied to non-natural nucleic acids and other encoded library formats.