Engineered initiator tRNAs can effectively start translation at non-AUG start codons and diversify N-terminal amino...

mRNA display is an effective tool to identify high-affinity macrocyclic binders for challenging protein targets. The success of an mRNA display selection is dependent on generating highly diverse libraries with trillions of peptides. While translation elongation can canonically accommodate the 61 proteinogenic triplet codons, translation initiation is restricted to the native start codon AUG. Here, we investigate the ability of the Escherichia coli ribosome to initiate translation for 31 initiator tRNA (tRNAini) anticodon mutants at their respective cognate start codon using a NanoBiT translation assay. We show that the ability of those anticodon mutant tRNAsini to initiate translation is highly variable and is, in part, inhibited by tRNA misfolding induced by the anticodon mutations. We demonstrate based on two distinct misfolding patterns that translation efficiency can be effectively restored by introducing additional mutations that restore the active tRNA fold. We then used 10 of the engineered tRNAsini in a mutational analysis experiment for three reported macrocyclic peptides binding to Ubiquitin Specific Protease 7 (USP7). The observed enrichment of peptides correlates strongly with dissociation constants measured by surface plasmon resonance, and provides insights into the structure-activity relationship of the N-terminal amino acid without the requirement for peptide synthesis.