Advances in genetic code reprogramming have allowed the site-specific incorporation of noncanonical functionalities into polypeptides and proteins, providing access to wide swaths of chemical space via in vitro translation techniques like mRNA display. Prior efforts have established that the translation machinery can tolerate amino acids with modifications to both the peptide backbone and side chains, greatly broadening the chemical space that can be interrogated in ligand discovery efforts. However, existing methods for confirming the translation yield of new amino acid building blocks for these technologies necessitate multistep workups and, more importantly, are not relevant for measuring translation within the context of a combinatorial library consisting of multiple noncanonical amino acids. In this study, we developed a luminescence-based assay to rapidly assess the relative translation yield of any noncanonical amino acid in real time. Among the 59 amino acids tested here, we found that many translate with high efficiency, but translational yield is not necessarily correlated to whether the amino acid is proteinogenic or has high tRNA acylation efficiency. Interestingly, we found that single-template translation data can inform the library-scale translation yield and that shorter peptide libraries are more tolerant of lower-efficiency amino acid monomers. Together our data show that the luminescence-based assay described herein is an essential tool in evaluating new building blocks and codon table designs within mRNA display toward the goal of developing druglike peptide-based libraries for drug discovery campaigns.

Genetic code expansion has proven invaluable to the elucidation of functions of defined protein modifications through the site-specific incorporation of noncanonical amino acids. The use of nonhydrolyzable derivatives of post-translational modifications can greatly increase site stoichiometry and half-life. Investigating acetyllysine reader domain (bromodomain) interactions with acetylated nonhistone proteins is challenging due to the limited tools available and dynamic nature of this posttranslational modification. Here, we demonstrate that bromodomains bind acetyllysine peptides and those substituted with an acetyllysine derivative, trifluoroacetyllysine, with similar affinity and selectivity. Importantly, both trifluoroacetyllysine and acetyllysine can be site-specifically incorporated into proteins expressed in bacterial and mammalian cells, and the strong electron-withdrawing trifluoro substituent makes the latter resistant to deacetylation by sirtuins (SIRTs). The controlled expression of SIRT-resistant, site-specifically acetylated transcription factors expands the set of available tools for determining the function of acetylation, and it serves as a template for investigating bromodomain interactions with acetylated transcription factors.