Biology subjects

Miyachi, R.

Publications and source records attributed to Miyachi, R..

3 recordsLinked to original sources

bioRxiv2026

Experimental verification of the error minimization theory using non-standard genetic codes constructed in vitro

All living systems use an almost identical genetic code, the standard genetic code, in which 20 amino acids are assigned to 61 codons non-randomly. According to the error minimization theory, amino acids are arranged to minimize the mutational effect on protein function, while experimental verification remains limited. In this study, we constructed 10 non-standard genetic codes in vitro by reassigning three amino acids (Ala, Ser, and Leu) in vacant codons of the minimal genetic code, which consists of 21 tRNAs. Most of these non-standard genetic codes have a higher cost of amino acid replacement than the standard genetic code, calculated based on three amino acid properties: polar requirement (PR), molecular volume (MV), and hydropathy index (HI). The protein function of three reporter genes expressed using these non-standard genetic codes decreased similarly when random mutations were introduced into the genes, implying that the effect of mutations was similar across all the non-standard genetic codes tested here. This result provides direct experimental evidence that mutational robustness does not significantly change in individual reporter protein activity when the genetic code is altered within the range of mutational cost tested in this study (CostPR: 5.29 - 5.77, CostMV: 1848 - 2348, and CostHI: 3.27 - 5.10), which covers approximately 18.4% (PR), 37.6% (MV), and 50.8% (HI) of possible cost range achievable among one million randomly-generated genetic codes.

biochemistry↗

bioRxiv2026

Enhanced tRNA array method version 2 for simultaneous in vitro synthesis of 21 tRNAs

Transfer RNAs (tRNAs) play an essential role in translation, and their simultaneous in vitro synthesis remains a key challenge in bottom-up synthetic biology. We previously developed the tRNA array method that enables the simultaneous in vitro synthesis of 21 tRNAs from a single DNA template; however, the translational activity was substantially lower than that achieved using individually prepared 21 tRNAs for some proteins. Here, we identify the tRNA groups (PIEN group) that limit translation in the tRNA array method and improve the translational activities through sequence modification and incorporation of a leader sequence into the array construct. The resulting tRNA array method version 2 produces a tRNA set that allows translation at levels similar to those achieved with individually prepared tRNAs for multiple reporter proteins under both translation-coupled and uncoupled conditions. This tRNA synthesis scheme provides an improved platform for constructing self-reproducible gene expression systems.

synthetic biology↗

bioRxiv2025

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.

synthetic biology↗