posted on 2024-01-09, 19:35authored byGianna
N. Kerestesy, Kara K. Dods, Clinton A. L. McFeely, Matthew C. T. Hartman
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.