Insitugeneration of aminoacyltRNAs assisted by ribozymes in translation apparatus

Abstract

Flexizymes are artificial RNA catalysts that enable us to readily prepare aminoacyl-tRNAs with a variety of amino acid and tRNA kinds. On the other hand, because their flexibility feature lacking high specificities toward amino acids and tRNAs, the in situ aminoacylation in a translation apparatus have not been able to executed. We here present a novel strategy to overcome this specificity problem to tRNA using a cis-acting flexizyme-tRNA construct, called a catalytic precursor tRNA, combining with a naturally occurring ribozyme, ribonuclease P (RNase P). In this coupling system of two RNA enzymes, self-aminoacylation occurs on the catalytic precursor tRNA for specific charging of amino acids at the 3'end of the tRNA domain in the presence of the cognate amino acid substrates. Subsequently, the aminoacylated catalytic precursor tRNA is specifically cleaved at the 5'linker region of the tRNA domain, giving the mature aminoacyl-tRNA. Most importantly, the generated flexizyme does not function in trans to tRNAs present in the translation apparatus, indicating that this two-ribozyme coupling system would potentially act as an orthogonal aminoacylation system in the translation apparatus.