Nanoluc synthesized by the PURE system with the tRNA array method

Transfer RNA (tRNA) plays a central role in translation. Simultaneously synthesizing the minimal yet sufficient number of tRNA species (at least 21) in vitro poses a challenge to constructing a self-reproducible artificial cell. In this study, we developed a simplified processing method that enables the simultaneous expression of all 21 tRNAs in a reconstituted transcription/translation system (PURE system). By combining direct tRNA linkage and HDVR attachment methods, termed the tRNA array method, we achieved simultaneous expression of all 21 tRNAs from a single polycistronic DNA template in the PURE system.
This method allows for the easy customization of the genetic code by introducing additional tRNAs. To demonstrate genetic code reassignment, we replaced five Leu residues (CUU) in Nanoluc with ACG, which is assigned to Thr in the native codon table. To confirm amino acid reassignment, we performed LC-MS/MS analysis of translated Nanoluc proteins. The results confirmed the incorporation of threonine at the reassigned ACG codon in the presence of tRNA^Thr_CGU, and leucine incorporation when using tRNA^Leu_CGU.