Hydrogel-Bead Particles Enable Activity-Based Identification of Nucleic Acid Aptamer Enzyme Inhibitors

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.