Abstract: The present inventors used aminoacylated tRNAs prepared by ligating a pCpA-amino acid to a tRNA lacking the 3?-terminal CA sequence using a ligase, and thereby succeeded in discrimination between the NNA and NNG codons in specific codon boxes, which had been difficult to achieve due to the presence of wobble base pairing. Furthermore, the inventors assigned another amino acid to the NNU or NNC codon in the same codon box, and performed translation of a sequence containing the NNU, NNA, and NNG codons, or the NNC, NNA, and NNG codons to evaluate the codon discrimination ability. As a result, the codons of interest were specifically translated into only the corresponding amino acids, achieving accurate discrimination successfully. Furthermore, the inventors confirmed that similar effects can also be obtained even when the nucleotide sequence of the tRNA body is altered.

Abstract: In some embodiments, the present disclosure relates to mutated tRNAs in which the first letter of the anticodon has been substituted to lysidine or agmatidine, and translation systems containing the mutated tRNAs. In a specific embodiment, the present disclosure provides mutated tRNAs capable of selectively translating codon NNA. In another embodiment, the present disclosure provides translation systems capable of translating two or three types of amino acids from a single codon box. In still another embodiment, the present disclosure provides novel methods for synthesizing lysidine diphosphate, agmatidine diphosphate, and derivatives thereof.