AN ENGINEERED TETRAHYMENA TRNA(GLN) FOR IN-VIVO INCORPORATION OF UNNATURAL AMINO-ACIDS INTO PROTEINS BY NONSENSE SUPPRESSION

A new tRNA, THG73, has been designed and evaluated as a vehicle for in corporating unnatural amino acids site-specifically into proteins expr essed in vivo using the stop codon suppression technique, The construc t is a modification of tRNA(Gln)(CUA) from Tetrahymena thermophila, wh ich naturally recognizes the stop codon UAG. Using electrophysiologica l studies of mutations at several sites of the nicotinic acetylcholine receptor, it is established that THG73 represents a major improvement over previous nonsense suppressors both in terms of efficiency and fi delity of unnatural amino acid incorporation, Compared with a previous tRNA used for in vivo suppression, THG73 is as much as 100-fold less likely to be acylated by endogenous synthetases of the Xenopus oocyte. This effectively eliminates a major concern of the in vivo suppressio n methodology, the undesirable incorporation of natural amino acids at the suppression site. In addition, THG73 is 4-10-fold more efficient at incorporating unnatural amino acids in the oocyte system, Taken tog ether, these two advances should greatly expand the range of applicabi lity of the in vivo nonsense suppression methodology.