Fig. 7: Simultaneous in vitro transcription of 21 tRNAs by the tRNA array method and an application for genetic code reassignment.

A Scheme of the simplified in vitro 21 tRNAs synthesis method. The single 21 tRNAs template (25 nM) was incubated with T7 RNA polymerase (1.0 μ/μL) and T4 PNK (0.1 μ/μL) at 37 °C for 12 h in in vitro transcription mixture. The transcript was purified using a spin column and incubated at 12 μM in buffer R with RNase P (4 μM of M1 RNA and 6 μM of C5 protein) at 37 °C for 16 h. The resulting 21 tRNAs were purified again before use in the translation assay. B PAGE analysis of the tRNA mixture prepared using the method described in (A). E. coli native tRNA mixture (Roche) was used as a control for quantification. The cleaved HDVR with T7 terminator is indicated by a green arrow (HDVR + term). C Translation assay using the 21 tRNAs mixture prepared by the tRNA array method (Array) and the conventional individually prepared method (Conv). The reaction mixture containing the 21 tRNAs mixture (600 ng/μL), Nanoluc template (1 nM), T7 RNA polymerase (0.42 μ/μL), and the tfPURE system (composition A). D Design of genetic-code reassignment. The Nanoluc gene was modified to replace five out of 16 UCC (Leu) codons with ACG codons (Thr in the native codon table). Translation with native tRNA^Thr_CGU produced a Nanoluc protein with reduced activity due to Thr substitutions at the five Leu residues. In contrast, translation with an anticodon-modified tRNA^Leu_CGU produced a normal Nanoluc protein, maintaining Nanoluc activity. E Reassigned genetic code. The vacant ACG codon (Thr in the native codon table) was reassigned to Leu, expanding the previously constructed minimal codon table⁴². F Translation experiment using the reassigned genetic code. The reaction mixture containing the 21 tRNAs mixture prepared by our method (600 ng/μL), tRNA^Thr_CGU or tRNA^Leu_CGU prepared by IVT (12 ng/μL), Nanoluc_ACG template (1 nM), T7 RNA polymerase (0.42 μ/μL), and the tfPURE system (composition A). Reactions in (C) and (F) were incubated at 30 °C for 16 h before luminescence measurement. Data in (C) and (F) are presented as mean values, each data point represents three independent experiments, and error bars indicate standard deviations.