Recent advances in bottom-up synthetic biology have made it possible to reconstitute cellular systems from non-living components, yielding artificial cells with potential applications in industry, medicine and basic research. Although a variety of cellular functions and components have been reconstituted in previous studies, sustained biological energy production remains a challenge. ATP synthesis via ribulose-1,5-diphosphate carboxylase/oxygenase (RuBisCO), a central enzyme in biological CO2 fixation, holds potential as an energy production system, but its feasibility in a cell-free expression system has not yet been tested. In this study, we test RuBisCO expression and its activity-mediated ATP synthesis in a reconstituted Escherichia coli-based cell-free translation system. We then construct a system in which ATP is synthesized by RuBisCO activity in giant vesicles and used as energy for translation reactions. These results represent an advance toward independent energy production in artificial cells. Graphical Abstract.

The integration of individually replicating genes into a primitive chromosome is a key evolutionary transition in the development of life, allowing the simultaneous inheritance of genes. However, how this transition occurred is unclear because the extended size of primitive chromosomes replicate slower than unlinked genes. Theoretical studies have suggested that a primitive chromosome can evolve in the presence of cell-like compartments, as the physical linkage prevents the stochastic loss of essential genes upon division, but experimental support for this is lacking. Here, we demonstrate the evolution of a chromosome-like RNA from two cooperative RNA replicators encoding replication and metabolic enzymes. Through their long-term replication in cell-like compartments, linked RNAs emerged with the two cooperative RNAs connected end-to-end. The linked RNAs had different mutation patterns than the two unlinked RNAs, suggesting that they were maintained as partially distinct lineages in the population. Our results provide experimental evidence supporting the plausibility of the evolution of a primitive chromosome from unlinked gene fragments, an important step in the emergence of complex biological systems.

The emergence of RNA self-reproduction from prebiotic components would have been crucial in developing a genetic system during the origins of life. However, all known self-reproducing RNA molecules are complex ribozymes, and how they could have arisen from abiotic materials remains unclear. Therefore, it has been proposed that the first self-reproducing RNA may have been short oligomers that assemble their components as templates. Here, we sought such minimal RNA self-reproduction in prebiotically accessible short random RNA pools that undergo spontaneous ligation and recombination. By examining enriched RNA families with common motifs, we identified a 20-nucleotide (nt) RNA variant that self-reproduces via template-directed ligation of two 10 nt oligonucleotides. The RNA oligomer contains a 2'-5' phosphodiester bond, which typically forms during prebiotically plausible RNA synthesis. This non-canonical linkage helps prevent the formation of inactive complexes between self-complementary oligomers while decreasing the ligation efficiency. The system appears to possess an autocatalytic property consistent with exponential self-reproduction despite the limitation of forming a ternary complex of the template and two substrates, similar to the behavior of a much larger ligase ribozyme. Such a minimal, ribozyme-independent RNA self-reproduction may represent the first step in the emergence of an RNA-based genetic system from primordial components. Simultaneously, our examination of random RNA pools highlights the likelihood that complex species interactions were necessary to initiate RNA reproduction.

How the complexity of primitive self-replication molecules develops through Darwinian evolution remains a mystery with regards to the origin of life. Theoretical studies have proposed that coevolution with parasitic replicators increases network complexity by inducing inter-dependent replication. Particularly, Takeuchi and Hogeweg proposed a complexification process of replicator networks by successive appearance of a parasitic replicator followed by the addition of a new host replicator that is resistant to the parasitic replicator. However, the feasibility of such complexification with biologically relevant molecules is still unknown owing to the lack of an experimental model. Here, we investigated the plausible complexification pathway of host-parasite replicators using both an experimental host-parasite RNA replication system and a theoretical model based on the experimental system. We first analyzed the parameter space that allows for sustainable replication in various replication networks ranging from a single molecule to three-member networks using computer simulation. The analysis shows that the most plausible complexification pathway from a single host replicator is the addition of a parasitic replicator, followed by the addition of a new host replicator that is resistant to the parasite, consistent with the previous study by Takeuchi and Hogeweg. We also provide evidence that the pathway actually occurred in our previous evolutionary experiment. These results provide experimental evidence that a population of a single replicator spontaneously evolves into multi-replicator networks through coevolution with parasitic replicators.

RNA has been used as a model molecule to understand the adaptive evolution process owing to the simple relationship between the structure (i.e., phenotype) and sequence (i.e., genotype). RNA usually forms multiple substructures with similar thermodynamic stabilities, called structural fluctuations. Ancel and Fontana theoretically proposed that structural fluctuation is directly related to the ease of change in structures by mutations and thus works as a source of adaptive evolution; however, experimental verification is limited. Here, we analyzed 76 RNA genotypes that appeared in our previous in vitro evolution to examine whether 1) RNA fluctuation decreases as adaptive evolution proceeds and 2) RNAs that have larger fluctuations tend to have higher frequencies of beneficial mutations. We first computationally estimated the structural fluctuations of all RNAs and observed that they tended to decrease as their fitness increased. We next measured the frequency of beneficial mutations for 10 RNA genotypes and observed that the total number of beneficial mutations was correlated with the size of the structural fluctuations. These results consistently support the idea that the structural fluctuation of RNA, at least those evaluated in our study, works as a source of adaptive 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.

A change from RNA- to DNA-based genetic systems is hypothesized as a major transition in the evolution of early life forms. One of the possible requirements for this transition is a change in the substrate specificity of the replication enzyme. It is largely unknown how such changes would have occurred during early evolutionary history. In this study, we present evidence that an RNA replication enzyme that has evolved in the absence of deoxyribonucleotide triphosphates (dNTPs) relaxes its substrate specificity and incorporates labeled dNTPs. This result implies that ancient replication enzymes, which probably evolved in the absence of dNTPs, could have incorporated dNTPs to synthesize DNA soon after dNTPs became available. The transition from RNA to DNA, therefore, might have been easier than previously thought.

Sustainable replication and evolution of genetic molecules such as RNA are likely requisites for the emergence of life; however, these processes are easily affected by the appearance of parasitic molecules that replicate by relying on the function of other molecules, while not contributing to their replication. A possible mechanism to repress parasite amplification is compartmentalization that segregates parasitic molecules and limits their access to functional genetic molecules. Although extent cells encapsulate genomes within lipid-based membranes, more primitive materials or simple geological processes could have provided compartmentalization on early Earth. In this review, we summarize the current understanding of the types and roles of primitive compartmentalization regarding sustainable replication of genetic molecules, especially from the perspective of the prevention of parasite replication. In addition, we also describe the ability of several environments to selectively accumulate longer genetic molecules, which could also have helped select functional genetic molecules rather than fast-replicating short parasitic molecules.

The reconstructed in vitro translation system known as the PURE system has been used in a variety of cell-free experiments such as the expression of native and de novo proteins as well as various display methods to select for functional polypeptides. We developed a refined PURE-based display method for the preparation of stable messenger RNA (mRNA) and complementary DNA (cDNA)-peptide conjugates and validated its utility for in vitro selection. Our conjugate formation efficiency exceeded 40%, followed by gel purification to allow minimum carry-over of components from the translation system to the downstream assay enabling clean and efficient random peptide sequence screening. We chose the commercially available anti-FLAG M2 antibody as a target molecule for validation. Starting from approximately 1.7 × 1012 random sequences, a round-by-round high-throughput sequencing showed clear enrichment of the FLAG epitope DYKDDD as well as revealing consensus FLAG epitope motif DYK(D/L/N)(L/Y/D/N/F)D. Enrichment of core FLAG motifs lacking one of the four key residues (DYKxxD) indicates that Tyr (Y) and Lys (K) appear as the two key residues essential for binding. Furthermore, the comparison between mRNA display and cDNA display method resulted in overall similar performance with slightly higher enrichment for mRNA display. We also show that gel purification steps in the refined PURE-based display method improve conjugate formation efficiency and enhance the enrichment rate of FLAG epitope motifs in later rounds of selection especially for mRNA display. Overall, the generalized procedure and consistent performance of two different display methods achieved by the commercially available PURE system will be useful for future studies to explore the sequence and functional space of diverse polypeptides.

We report RNA self-replication through the translation of its encoded protein within membrane-free compartments generated by liquid-liquid phase separation. The aqueous droplets support RNA self-replication by concentrating a genomic RNA and translation proteins, facilitating the uptake of small substrates, and preventing the replication of parasitic RNAs through compartmentalization.

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.

Phi29 DNA polymerase is widely used for DNA amplification through rolling-circle replication or multiple displacement amplification. Here, we performed completely in vitro artificial evolution of phi29 DNA polymerase by combining the in vitro compartmentalization and the gene expression-coupled rolling-circle replication of a circular DNA encoding the polymerase. We conducted the experiments in six different conditions composed of three different levels of inhibitor concentrations with two different DNA labeling methods. One of the experiments was performed in our previous study and the other five experiments were newly conducted in this study. Under all conditions, we found several mutations that enhance the rolling-circle amplification by the polymerase when it was expressed in the reconstituted gene expression system. Especially, a combinatorial mutant polymerase (K555T/D570N) exhibits significantly higher rolling-circle activity than the wild type. These highly active mutant polymerases would be useful for various applications.

Adaptation to various environments is a remarkable characteristic of life. Is this limited to extant complex living organisms, or is it also possible for a simpler self-replication system to adapt? In this study, we addressed this question by using a translation-coupled RNA replication system that comprised a reconstituted translation system and an RNA genome that encoded a replicase gene. We performed RNA replication reactions under four conditions, under which different components of translation were partly inhibited. We found that replication efficiency increased with the number of rounds of replication under all the tested conditions. The types of dominant mutations differed depending on the condition, thus indicating that this simple system adapted to different environments in different ways. This suggests that even a primitive self-replication system composed of a small number of genes on the early earth could have had the ability to adapt to various environments.

The reconstitution of an artificial system that has the same evolutionary ability as a living thing is a major challenge in the in vitro synthetic biology. In this study, we tested the adaptive evolutionary ability of an artificial RNA genome replication system, termed the translation coupled RNA replication (TcRR) system. In a previous work, we performed a study of the long-term evolution of the genome with an excess amount of ribosome. In this study, we continued the evolution experiment in a reduced ribosome environment and observed that the mutant genorne compensated for the reduced ribosome concentration. This result demonstrated the ability of the TcRR system to adapt and may be a step toward generating living things with evolutionary ability.