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 and a plurality of natural amino acids,
• incorporating the unnatural amino acid and a plurality of natural amino acids at genetically-encoded positions in a target polypeptide, wherein incorporating comprises an incorporation efficiency, an incorporation specificity and a post-incorporation stability,
• generating between 5 and 500 picomoles of a target polypeptide comprising the unnatural amino acid and a plurality of natural amino acids incorporated at genetically-encoded target 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 target positions in the target polypeptide is at least 91%, as compared to the efficiency of incorporating non-target natural amino acid or the efficiency of terminating translation via peptide releases, wherein the incorporation specificity of the unnatural amino-acid incorporation events for the particular genetically-encoded target positions in the target polypeptide is at least 91%, as compared to incorporation of the target 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 91%, as compared to the level of inactivation of the reactive group;
• and wherein the combination such that when combined the incorporation efficiency, the incorporation specificity, and the post-incorporation stability renders target purification and concentration unnecessary for a purpose of achieving the labeling efficiencies and specificities required for single molecule screening applications;
• and
• modifying the reactive 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 and a labeling specificity.