Site-specific co-translational incorporation of unnatural amino acids into the polypeptide chain gives invaluable tools for high-throughput peptide/protein engineering allowing production of constrained peptides and protein domains for the drug discovery and development as well as studies on structure, dynamics and kinetics of individual proteins and protein complexes by single molecule spectroscopy. Many attempts were made to harness the power of the ribosome for site- selective protein labeling. Reassignment of termination or the decoding of quadruplet codons is often not effective due to a high competition with release factors or a high rate of frameshift events respectively. While the redundancy of genetic code provides a large number of potentially reassignable codons this direction has received much less attention due to the inevitable competition with endogenous aminoacyl-tRNAs. In this work we assumed that the selective depletion of individual tRNA-isoacceptor(s) for amino acids encoded either by mixed codon families or by the codon families with high wobble restrictions would “open up” the respective codon(s) for unnatural amino acid while the remaining isoacceptor(s) would be decoding the native amino acid in the rest of the polypeptide chain. Small anti-sense RNA hairpins (“kissing loops”) targeted against a tRNA anticodon loop were used for selective depletion of target isoacceptors. Following this approach we demonstrated that we could significantly deplete individual isoacceptors for Ser, Gly and Arg from the cell-free extract of E. coli and use the resulting “freed” codon “slots” for the protein labeling with the fluorescent amino acid.