Screening Cellular Machinery for New Unnatural Amino Acids

Orthogonal tRNA/aminoacyl tRNA synthetase pairs allow the incorporation of unnatural amino acids into specific locations within a protein structure in vivo. Current screening methods to determine whether or not tRNA/aaRS pairs incorporate a specific unnatural amino acid fail to de-select pairs that can incorporate multiple unnatural amino acids beyond the one for which they are designed. A recently developed method allows for this screening of promiscuous unnatural amino acid incorporation using tRNA/aaRS pairs in conjunction with a green fluorescent protein (GFP) with a stop codon mutation at a central, non-essential position. We are recreating this GFP based screening system in order to test for the incorporation of fluorescent unnatural amino acids into proteins using these promiscuous tRNA/aaRS pairs. Thus far, we have tested the fluorescence and solvatochromic properties of the fluorescent unnatural amino acids dansylalanine, p-cyanophenylalanine, and natural tryptophan. We also constructed the screening system with several tRNA/aaRS pair plasmids and one GFP plasmid mutated with a centrally located stop codon. We are currently testing these fluorescent unnatural amino acids (dansylalanine and p-cyanophenylalanine) in our designed GFP based screening system, in order to see if they are incorporated into protein via the poly-specificity of the tRNA/aaRS pairs