Selection scheme for sense suppression. (A) Translation reactions in rabbit reticulocyte lysate contain a template library and exogenous, orthogonal tRNAs. The amber suppressor THG73 was mutated at the anticodon position to Watson-Crick base pair to all 16 GNN codons. The pool of 16 tRNAs were then acylated in one batch with the unnatural amino acid biocytin. (B) Incorporation of the unnatural amino acid into an mRNA–peptide fusion is a competition between the endogenous tRNA population charged with natural amino acids (aa) and the exogenous tRNA pool charged with biocytin (B). The sequence present in the peptide is then encoded in the covalently-attached mRNA through its 3′ puromycin (P), allowing the sequence information in the protein to be read and recovered via the attached RNA. Selecting for streptavidin binding through biocytin incorporation after several rounds enriches the template library at the randomized position for GNN codons.
