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.