In Vitro Transcription/Translation-Coupled DNA Replication through Partial Regeneration of 20 Aminoacyl-tRNA Synthetases

The in vitro reconstruction of life-like self-reproducing systems is a major challenge in in vitro synthetic biology. Self-reproduction requires regeneration of all molecules involved in DNA replication, transcription, and translation. This study demonstrated the continuous DNA replication and partial regeneration of major translation factors, 20 aminoacyl-tRNA synthetases (aaRS), in a reconstituted transcription/translation system (PURE system) for the first time. First, we replicated each DNA that encodes one of the 20 aaRSs through aaRS expression from the DNA by serial transfer experiments. Thereafter, we successively increased the number of aaRS genes and achieved simultaneous, continuous replication of DNA that encodes all 20 aaRSs, which comprised approximately half the number of protein factors in the PURE system, except for ribosomes, by employing dialyzed reaction and sequence optimization. This study provides a step-by-step methodology for continuous DNA replication with an increasing number of self-regenerative genes toward self-reproducing artificial systems.