Abstract: Sense codon reassignment to unnatural amino acids (uAAs) represents a powerful approach for introducing novel properties into polypeptides. The main obstacle to this approach is competition between the native isoacceptor tRNA(s) and orthogonal tRNA(s) for the reassigned codon. While several chromatographic and enzymatic procedures for selective deactivation of tRNA isoacceptors in cell-free translation systems exist, they are complex and not scalable. The present invention provides oligonucleotides that hybridise to a tRNA of interest when said tRNA is in a folded state, thereby disrupting the function of the tRNA. The present invention also provides the use of these oligonucleotides in methods for sense codon reassignment and methods for incorporation of uAAs into proteins. The approach described herein represents a new direction in genetic code reassignment with numerous practical applications.

Abstract: A complement to tRNAs and a protein translation system are provided that enable the incorporation of non-natural moieties such as non-natural amino acids without compromising the ability to incorporate all of the twenty natural amino acids into the protein. This is achieved by reassigning one of the tRNA anticodons for amino acids that are normally decoded by at least two different tRNA anticodons to a non-natural moiety, wherein at least one codon can be uniquely recognized by the reassigned anticodon and at least one another codon from the same codon box cannot be recognized by the reassigned anticodon. Accordingly, an mRNA for translation is engineered to comprise one or more specific codons corresponding to the reassigned tRNA anticodons so that the non-natural moiety is incorporated into the translated protein at a selected position.