Improving unnatural amino acid mutagensis efficiency and selectivity in mammalian cell

Abstract: Genetically encoded, site-specific incorporation of unnatural amino acids (UAA)into proteins through selective recoding of an amber stop codon provides apowerful route for expressing synthetic proteins in living cells. Recoding of theamber stop codon is achieved by introducing an amber suppressortRNA/synthetase pair orthogonal to the endogenous tRNA complement intocells. Methanosarcina is a methane producing archaea with the unusualcapability of suppressing the stop codon (specifically the amber codon). Bysuppressing the amber codon Methanosarcina facilitate the incorporation of thenon-canonical amino acid pyrrolysine (pyl). The suppressing mechanismoriginates from a evolutionary unique Pyrrolysyl-tRNA synthetase (PylRS) and itsmatching tRNApyl. The PylRS has been further evolved and modified to allowincorporation of a wide range of UAAs. Amber suppression is today used tocontrol and study protein function in living cells. By making a series of wellcontrolledexperiments with HEK293T cells we aimed to develop this techniqueinto a robust and general tool for mammalian cell biology. Specifically we weretesting the incorporation of the unnatural amino acid bicyclononyne (BCN) by aset of known PylRS mutants. Our results suggest the mutant aaRS PylRS “AF” isthe most robust and efficient synthetase for BCN. We have improved ambersuppression by determining which factors leads to a more efficient method andsimultaneously decreasing the cost of the method.