Import of amber and ochre suppressor tRNAs into mammalian cells: A generalapproach to site-specific insertion of amino acid analogues into proteins

A general approach to site-specific insertion of amino acid analogues into proteins in vivo would be the import into cells of a suppressor tRNA aminoa cylated with the analogue of choice. The analogue would be inserted at any site in the protein specified by a stop codon in the mRNA. The only require ment is that the suppressor tRNA must not be a substrate for any of the cel lular aminoacyl-tRNA synthetases. Here, we describe conditions for the impo rt of amber and ochre suppressor tRNAs derived from Escherichia coli initia tor tRNA into mammalian COS1 cells, and we present evidence for their activ ity in the specific suppression of amber (UAG) and ochre (UAA) codons, resp ectively. We show that an aminoacylated amber suppressor tRNA (supF) derive d from the E. coli tyrosine tRNA can be imported into COS1 cells and acts a s a suppressor of amber codons, whereas the same suppressor tRNA imported w ithout prior aminoacylation does not, suggesting that the supF tRNA is not a substrate for any mammalian aminoacyl-tRNA synthetase. These results open the possibility of using the supF tRNA aminoacylated with an amino acid an alogue as a general approach for the site-specific insertion of amino acid analogues into proteins in mammalian cells. We discuss the possibility furt her of importing a mixture of amber and ochre suppressor tRNAs for the inse rtion of two different amino acid analogues into a protein and the potentia l use of suppressor tRNA import for treatment of some of the human genetic diseases caused by nonsense mutations.