Engineering a Ribozyme with Aminoacyl-tRNA Synthetase Activity

Abstract A ribozyme that can charge a tRNA with amino acids and discriminate between cognate and non-cognate tRNAs is of interest because an RNA with this ability may have been critical for translation in the transition from the RNA world. In addition, a fully optimized ribozyme could provide a tool for incorporating non-canonical amino acids for biotechnology applications. Here, we rationally engineer a ribozyme by fusing a tRNA binding module derived from a T-box riboswitch with a catalytic module (a flexizyme) to generate a ribozyme that can aminoacylate a target tRNA. We demonstrate that this ribozyme can be readily redesigned to alter tRNA specificity. This ribozyme is compatible with an in vitro translation system and could be used to recode a protein sequence to site-specifically incorporate a non-canonical amino acid.