Maintenance of a translation-coupled RNA replication in dynamic micro-sized compartments under continuous stirring and flow

Abstract

We maintained a translation-coupled RNA replication (TcRR) in water-in-oil emulsion in a continuous flow stirring-tank reactor (CSTR) configuration. In micro-sized water droplets, the genetic information encoded in RNA was translated into a RNA-dependent RNA polymerase, which in turn replicated the original RNA. We supplied the fresh substrate for the reaction and a cell-free translation system as the form of droplets into the stirring tank. Unlike the conventional CSTR, stirring was not just for mixing but for inducing the continuous coalescence and breakage dynamics of the droplets. We designed that the coalescence supplied the substrate to TcRR, and the breakage restored the sizes of the droplets after the coalescence. We showed that this dynamics sustain the TcRR through the supplement of the substrate and dilution of the resultant RNA. We also showed that, by tuning the activity of the TcRR, we were able to suppress the outbreak of parasitic amplified RNA, which had been reported as an inhibitor of TcRR. Our reactor demonstrated a new way to continuously maintain a replication reaction in micro-sized compartments like as CSTR does for cell cultures.