Cooperation among independently replicating molecules is a key phenomenon that allowed the development of complexity during the early evolution of life. Generally, this process is vulnerable to parasitic or selfish entities, which can easily appear and destroy such cooperation. It remains unclear how this fragile cooperation process appeared and has been sustained through evolution. Theoretical studies have indicated that spatial structures, such as compartments, allow sustainable replication and the evolution of cooperative replication, although this has yet to be confirmed experimentally. In this study, we constructed a molecular cooperative replication system, in which two types of RNA, encoding replication or metabolic enzymes, cooperate for their replication in compartments, and we performed long-term replication experiments to examine the sustainability and evolution of the RNAs. We demonstrate that the cooperative relationship of the two RNAs could be sustained at a certain range of RNA concentrations, even when parasitic RNA appeared in the system. We also found that more efficient cooperative RNA replication evolved during long-term replication through seemingly selfish evolution of each RNA. Our results provide experimental evidence supporting the sustainability and robustness of molecular cooperation on an evolutionary timescale.

In prebiotic evolution, molecular self-replicators are considered to develop into diverse, complex living organisms. The appearance of parasitic replicators is believed inevitable in this process. However, the role of parasitic replicators in prebiotic evolution remains elusive. Here, we demonstrated experimental coevolution of RNA self-replicators (host RNAs) and emerging parasitic replicators (parasitic RNAs) using an RNA-protein replication system we developed. During a long-term replication experiment, a clonal population of the host RNA turned into an evolving host-parasite ecosystem through the continuous emergence of new types of host and parasitic RNAs produced by replication errors. The host and parasitic RNAs diversified into at least two and three different lineages, respectively, and they exhibited evolutionary arms-race dynamics. The parasitic RNA accumulated unique mutations, thus adding a new genetic variation to the whole replicator ensemble. These results provide the first experimental evidence that the coevolutionary interplay between host-parasite molecules plays a key role in generating diversity and complexity in prebiotic molecular evolution.

In prebiotic evolution, self-replicating molecules are believed to have evolved into complex living systems by expanding their information and functions open-endedly. Theoretically, such evolutionary complexification could occur through successive appearance of novel replicators that interact with one another to form replication networks. Here we perform long-term evolution experiments of RNA that replicates using a self-encoded RNA replicase. The RNA diversifies into multiple coexisting host and parasite lineages, whose frequencies in the population initially fluctuate and gradually stabilize. The final population, comprising five RNA lineages, forms a replicator network with diverse interactions, including cooperation to help the replication of all other members. These results support the capability of molecular replicators to spontaneously develop complexity through Darwinian evolution, a critical step for the emergence of life.

There are several plausible abiotic synthetic routes from prebiotic chemical materials to ribonucleotides and even short RNA oligomers. However, for refinement of the RNA World hypothesis to help explain the origins of life on the Earth, there needs to be a manner by which such oligomers can increase their length and expand their sequence diversity. Oligomers longer than at least 10-20 nucleotides would be needed for raw material for subsequent natural selection. Here, we explore spontaneous RNA-RNA recombination as a facile means by which such length and diversity enhancement could have been realized. Motivated by the discovery that RNA oligomers stored for long periods of time in the freezer expand their lengths, we systematically investigated RNA-RNA recombination processes. In addition to one known mechanism, we discovered at least three new mechanisms. In these, one RNA oligomer acts as a splint to catalyze the hybridization of two other oligomers and facilitates the attack of a 5 ′ ′ ′ ′ ′ -OH, a 3 ′ ′ ′ ′ ′ -OH, or a 2 ′ ′ ′ ′ ′ -OH nucleophile of one oligomer onto a target atom of another. This leads to the displacement of one RNA fragment and the production of new recombinant oligomers. We show that this process can explain the spontaneous emergence of sequence complexity, both in vitro and in silico.