Nonsense suppression approach

Most of the aforementioned efforts are based on the nonsense suppression methodology as the method for peptide altering [100]. In this approach, a nonsense codon is introduced into the enzyme-coding mRNA in the site that has to be altered. Simultaneously, the tRNA-noncoded amino acid hybrids are prepared with nonsense anticodons. Finally, the translation of modified mRNA is performed in vivo [101] or in vitro [102]. 

J. Schack, U.L. RajBhandary, A possible approach to site-specific insertion of two different unnatural amino acids into proteins in mammalian cells via nonsense suppression, Chem. Biol. 2003, 10, 1095-1102. 

Nonsense suppression is a biosynthetic approach enabling a large variety of unnatural amino acid to be incorporated site-specificaUy into proteins (Cornish and Schultz, 1994 Hohsaka et al., 2(X)1). The fundamental approach to nonsense (stop codon) suppression is outlined in Eigure 16.10. 

Several strategies exist for incorporating noncanonical amino acids into proteins.One powerfiil approach uses stop, or nonsense, codons that normally terminate protein translation. Suppressor tRNAs recognize stop codons and suppress translational termination. By linking a noncanonical amino acid to a suppressor tRNA by chemical misacylation, one can insert the new amino acid in vitro in response to a stop codon. " To accomplish the same objective in vivo, however, an aminoacyl-tRNA synthetase that recognizes and couples the suppressor and the noncanonical amino acid must be developed. This method enables insertion of a single noncanonical amino acid at a specified position in a protein. 

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