Continuous Fluorescence Assay for In Vitro Translation Compatible with Noncanonical Amino Acids

The tolerance of the translation apparatus toward noncanonical amino acids (ncAAs) has enabled the creation of diverse natural-product-like peptide libraries using mRNA display for use in drug discovery. Typical experiments testing for ribosomal ncAA incorporation involve radioactive end point assays to measure yield alongside mass spectrometry experiments to validate incorporation. These end point assays require significant postexperimental manipulation for analysis and prevent higher throughput analysis and optimization experiments. Continuous assays for in vitro translation involve the synthesis of fluorescent proteins which require the full complement of canonical AAs for function and are therefore of limited utility for testing of ncAAs. Here, we describe a new, continuous fluorescence assay for in vitro translation based on detection of a short peptide tag using an affinity clamp protein, which exhibits changes in its fluorescent properties upon binding. Using this assay in a 384-well format, we were able to validate the incorporation of a variety of ncAAs and also quickly test for the codon reading specificities of a variety of Escherichia coli tRNAs. This assay enables rapid assessment of ncAAs and optimization of translation components and is therefore expected to advance the engineering of the translation apparatus for drug discovery and synthetic biology.