Abstract 1736 Unnatural amino acid incorporation during in vitro translation is improved by using a thioester linkage between tRNA and amino acid

The ribosome and associated translation machinery is capable of polymerizing amino acids into peptides with both high yield and specificity in a sequence defined manner. Because of these unique properties, the ribosome has been identified as a system that has the potential to be repurposed to generate new biomaterials with diverse therapeutic and chemical functionalities through sequence defined incorporation of unnatural monomers. Although ribosomes are known to incorporate diverse non-natural monomers into peptides, the efficiency of incorporation is highly dependent on the specific structure of the monomers. In some cases, incorporation of unnatural monomers during translation is kinetically slow due to suboptimal positioning in the peptidyl transferase center (PTC). This prompted us to explore methods of enhancing the rate of peptide bond formation in the PTC. To this end, we have developed a novel 3'-thioribose adenosine triphosphate (thio-ATP) that can be installed on the 3'-end of a tRNA using a CCA-adding enzyme. The resulting 3'-thio-tRNA can be aminoacylated either chemically, using a synthetase, or using a flexizyme, generating a thioester linkage rather than the canonical ester linkage. Employing either liquid-chromatography mass-spectrometry or bioluminescence assays, we have demonstrated that using this modified aminoacyl-tRNA substrate in translation can increase the incorporation efficiency of unnatural amino acids. This research was funded by the National Science Foundation (2002182).