Screening for Unnatural Amino Acid Incorporation in Cellular Machinery

Optical imaging using fluorescence is limited by fluorophore size and unsuitability for in vivo analysis. Unnatural fluorescent amino acids (UFAAs) provide an alternative to bulkier fluorophores for cellular and protein imaging. Additional small, red-shifted, nontoxic, and cell-permeable fluorophores that can be incorporated into proteins in vivo would be a significant advancement in the field (1-4). In order to find these fluorophores, the viability of pairings of UFAAs and the aminoacyl tRNA synthetases (tRNA-aaRS) which attach them to tRNA molecules were tested. Our work has screened seven UFAAs with four tRNA-aaRS developed for specific UFAAs, looking for incorporation into green fluorescent protein (GFP) plasmids using the amber suppression method. A stop (TAG) codon in a GFP plasmid inserted into the host E. Coli causes incomplete formation of the barrel-shaped protein, which then does not fluoresce. If the stop codon, however, is suppressed by the incorporation of a UFAA, GFP fluorescence is expressed from the complete protein, and this fluorescence can be measured and is proportional to the amount of incorporation. Overall, positive and negative controls behave as expected, and other promising hits will be further examined. Miniaturization of the screening has allowed the process to be performed in a microtiter plate, reducing screening time from 3 days to 1.5 days and enabled analyzation by a single instrument. 1.Lang, Chin. Chemical Reviews 2014. 2.Young. Biochemistry 2011. 3.Miyake-Stoner. Biochemistry 2010. 4.Speight. JACS 2013