Development of a high-throughput immunofluorescence assay platform using a DNA-encoded streptavidin library for the rapid evaluation of protein-catalyzed capture agents

Abstract

Protein Catalyzed Capture (PCC) Agents are an emergent class of macrocyclic peptides that selectively bind, with low NM affinities, to unique epitopes of specific proteins, such as oncoproteins and blood protein biomarkers. The development of a PCC binder is a robust process that involves screening a two million elements one-bead-one-compd. (OBOC) macrocyclic peptide library against a synthetic polypeptide representing the epitope of interest. A single generation screen is sufficient to identify candidate PCC ligands. Each candidate is tested via a series of binding assays to identify a PCC with optimal avidity characteristics. Such testing requires running multiple binding curve assays in series. We report on a high-throughput, microchip-based platform for the rapid development of PCCs. Individual PCC candidates are prepd. with a biotin label, coupled onto individual members of a DNA-encoded streptavidin library (DESL), and assembled, via DNA-hybridization, onto a micropatterned DNA barcode located within a microwell of a multi-well chip. The platform allows for the simultaneous evaluation of fifteen different PCC ligands in up to sixteen different assay conditions on a single microchip. Addnl., the barcode surface chem. reduces the non-specific background commonly seen in a sandwich ELISA (ELISA). We compare the barcode platform against traditional sandwich ELISA assays, and then utilize it to identify allosteric binders against the KRAS protein.

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