Abstract: There is provided amino acid recognizers with improved binding properties, allowing for more structural information to be obtained from polypeptides based on the kinetics of on-off binding between recognizer and polypeptide. An amino acid recognizer may comprise an amino acid binding protein with an engineered binding pocket having one or more modifications relative to a homologous protein. The modified binding pocket may increase the number of interactions formed between the binding pocket and an amino acid ligand as compared to an unmodified binding pocket of the homologous protein, increase the number of types of amino acid ligands capable of being detectably bound as compared to an unmodified binding pocket of the homologous protein, and improve the kinetics of binding (e.g., KD, koff, kon) toward one or more types of amino acid ligands, which advantageously increases the amount of, or confidence in, structural information that may be derived from polypeptide analysis.

Abstract: Aspects of the disclosure provide methods and compositions for determining the identity of an analyte using molecular barcodes and single-molecule directed evolution of target biomolecules (e.g., proteins or aptamers).