Constructing an Artificial Self-replication System of Genetic Information from RNA and Proteins

Self-replication of genetic information is one of the central functions of living systems. This function enables the living system to reproduce itself, introduce mutations, and evolve. How could a self-replication system be constructed from non-living materials on the earth? What conditions are required? The answers to these questions are largely unknown. Here, we attempted to construct an artificial self-replication system of genetic information from biological materials, such as RNA and proteins, to identify the conditions necessary to establish self-replication and enable the system to evolve. Based on previous reports, we constructed a selfreplication system of genetic information from RNA (genetic information) encoding RNA replicase (Q replicase) and a cell-free translation system (PURE system). During the reaction, RNA replicase was translated from the RNA, and then bound to the original RNA and catalyzed its replication. These successive reactions are referred to here as self-replication of genetic information. This system consisted of more than 100 components, all of which were identified. Therefore, we can control all the components independently and quantitative analysis is possible. The reaction efficiency was markedly lower than expected from the activity of the replicase and the translation system. This poor efficiency suggests that there are as yet unknown conditions required for efficient self-replication. To clarify the problems, we analyzed the self-replication system by mathematical modeling, which indicated three limiting factors: 1) competition between translation and replication for RNA; 2) parasitic RNA amplification; and 3) inactive doublestranded RNA formation. Overcoming these problems will be necessary for realization of an in vitro self-replication system. To resolve the first problems, we measured the affinity of RNA with replicase and ribosome, and adjusted the ribosomal concentration to the optimum level. To resolve the second problem, we compartmentalized the reaction into a micrometer-sized water-in-oil emulsion. This was considered to confine the parasitic RNA to minor compartments, so that the other major compartments were free from parasite where self-replication continued. Although the third problem is now under investigation, the self-replication efficiency has improved significantly. These result demonstrated that establishment of an efficient self-replication system requires coordination of internal reactions and a mechanism for repression of parasitic replicator.