PREPARATION OF CHEMICALLY MISACYLATED SEMISYNTHETIC NONSENSE SUPPRESSOR TRANSFER-RNAS EMPLOYED IN BIOSYNTHETIC INCORPORATION OF NONNATURAL RESIDUES INTO PROTEINS

Both run-off transcription and direct chemical synthesis were employed to prepare semisynthetic, nonhypermodified tRNA(Gly) nonsense suppressors acylated with non-natural residues. L-3-Iodotyrosyl-tRNA(CUA)Gly-dCA (3) was prepared by four separate methods, and each of the resultant suppressors was evaluated for biological activity during in vitro translation of mRNA containing a nonsense suppression site (e.g., a UAG termination codon). Direct comparison of the individual translation experiments clearly demonstrates that all four methods yield acylated tRNA products that are biologically equivalent for the their intended purpose: site-specific incorporation of non-natural residues into proteins.