Simultaneous in vitro expression of minimal 21 transfer RNAs by tRNA array method

Transfer RNA (tRNA) plays a central role in translation. The simultaneous in vitro synthesis of minimal yet sufficient tRNA species (at least 21) poses a challenge for constructing a self-reproducible artificial cell. A key obstacle is the processing of the 5' and 3' ends, which requires a multi-step reaction in the natural cells. In this study, we develop a simplified processing method that allows simultaneous expression of all 21 tRNAs in a reconstituted transcription/translation system (PURE system). We test two methods for 5'-end processing (the leader and 5'-G variants methods) and two methods for 3'-end processing (the direct tRNA linkage and HDVR attachment methods). Finally, by combining the direct tRNA linkage and HDVR attachment methods (newly termed the tRNA array method), we succeed in simultaneously expressing all 21 tRNAs from a single polycistronic DNA template in the PURE system. The tRNA mixture produced by the tRNA array method supports a similar level of translation to the individually synthesized tRNA mixture for luciferase and GFP. This study represents a step toward the realization of self-reproducible artificial cells and also provides an easy method for preparing all tRNAs useful for genetic code engineering.