Single-Molecule Phenotype Analysis

A method of producing modified polypeptides suitable for structural and functional single molecule screening applications, the method comprising:

• carrying out in vitro cell-free translation of a polynucleotide in the presence of an unnatural amino acid,
• incorporating the unnatural amino acid at genetically-encoded positions in the target polypedtide, wherein incorporating comprises an incorporation efficiency, an incorporation specificity and a post-incorporation stability,
• generating between 5 and 500 picomoles of a polypeptide comprising the unnatural amino acid incorporated at genetically-encoded positions in the target polypeptide, wherein the unnatural amino acid comprises a reactive group, wherein the incorporation efficiency of the unnatural amino acid incorporation events at each of the genetically-encoded positions in the target polypeptide is at least about 10:1, as compared to incorporation of a natural an amino acid into a target position in the target polypeptide, wherein the incorporation specificity of the unnatural amino-acid incorporation events for the particular genetically-encoded positions in the target polypeptide is at least about 10:1, as compared to incorporation of the unnatural amino acid into a non-target polypeptide or a non-target position in the target polypeptide, wherein the post-incorporation stability of the unnatural amino acid reactive group is at least about 10:1, as the combination of the incorporation efficiency, the incorporation specificity, and the post-incorporation stability renders target purification unnecessary for single molecule screening applications;
• and
• modifying the unnatural amino acid by cycloaddition of a heterologous moiety thereby producing a modified polypeptide comprising the heterologous moiety, wherein modifying is quantified using a labeling efficiency.