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 natural cells. In this study, we developed a simplified processing method that allows simultaneous expression of all 21 tRNAs in a reconstituted transcription/translation system (PURE system). We tested three available methods (leader, 5-G variants, and HDVR attachment methods) and one new method (direct tRNA linkage method). Using these methods, we succeeded in simultaneous expression of six non-G-start tRNA from monocistronic six DNA templates in the PURE system. Furthermore, we developed a method that combines the direct tRNA linkage and HDVR attachment methods (termed tRNA array method). Using this method, we succeeded in simultaneous expression of all 21 tRNAs from a single polycistronic DNA template in the PURE system. The tRNA mixture produced by the tRNA array method supported a similar level of translation to the individually synthesized tRNA mixture. Additionally, we demonstrated that the minimal tRNA sets prepared by the tRNA array method can be used for genetic code engineering. 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.