Repetitive temperature cycles with DMSO sustain RNA replicating liposome compartments via fusion mediated content...

Compartmentalization by phospholipid membranes was a crucial step in the origin of life, enabling primitive cells to evolve through gene replication and diversification. Periodic freeze-thaw cycles are known to induce fusion and fission of phospholipid compartments; however, these processes typically involve membrane disruption followed by reassembly. Here, we report a dimethyl sulfoxide (DMSO)-mediated fusion mechanism that enables liposome fusion at -20 °C without freezing either the internal or external aqueous phases, thereby preserving membrane integrity. Using this approach, we achieved repetitive liposome fusion coupled with RNA replication for at least seven consecutive cycles at -20 °C, under non-freezing conditions. We further show that membrane lipid exchange occurs even at 4 °C, whereas efficient internal content mixing depends on lipid phase behavior, indicating a decoupling between membrane exchange and content mixing. This system enables redistribution of molecular substrates and genetic material across compartments, supporting recursive replication under fluctuating conditions.