Multiple incorporation of non-natural amino acids into a single protein using tRNAs with non-standard structures

Abstract 

The ability to introduce non-natural amino acids into proteins opens up new vistas for the study of protein structure and function. This approach requires suppressor tRNAs that deliver the non-natural amino acid to a ribosome associated with an mRNA containing an expanded codon. The suppressor tRNAs must be absolutely protected from aminoacylation by any of the aminoacyl-tRNA synthetases in the protein synthesizing system, or a natural amino acid will be incorporated instead of the non-natural amino acid. Here, we found that some tRNAs with non-standard structures could work as efficient four-base suppressors fulfilling the above orthogonal conditions. Using these tRNAs, we successfully demonstrated incorporation of three different non-natural amino acids into a single protein.

Abbreviations: ntrPhe, l-p-nitrophenylalanine, acdAla, l-2-acridonylalanine, 2napAla, l-2-naphthylalanine, 2antAla, l-2-anthrylalanine, Xaa, non-natural amino acid, ARS, aminoacyl-tRNA synthetase, mt, mitochondrial, Sec, selenocysteine, Pyl, pyrrolysine, EF-Tu, elongation factor Tu

Keywords: tRNA, Non-natural amino acid, Mitochondrial tRNA, Four-base codon, Orthogonal tRNA