Kimihito Usui

Darwinian evolution in a translation-coupled RNA replication system within a cell-like compartment

The ability to evolve is a key characteristic that distinguishes living things from non-living chemical compounds. The construction of an evolvable cell-like system entirely from non-living molecules has been a major challenge. Here we construct an evolvable artificial cell model from an assembly of biochemical molecules. The artificial cell model contains artificial genomic RNA that replicates through the translation of its encoded RNA replicase. We perform a long-term (600-generation) replication experiment using this system, in which mutations are spontaneously introduced into the RNA by replication error, and highly replicable mutants dominate the population according to Darwinian principles. During evolution, the genomic RNA gradually reinforces its interaction with the translated replicase, thereby acquiring competitiveness against selfish (parasitic) RNAs. This study provides the first experimental evidence that replicating systems can be developed through Darwinian evolution in a cell-like compartment, even in the presence of parasitic replicators.

Kinetic model of double‐stranded RNA formation during long RNA replication by Qβ replicase

Qβ replicase is an RNA‐dependent RNA polymerase, which synthesizes the complementary RNA using a single‐stranded RNA as a template. The formation of non‐replicable double‐stranded RNA (dsRNA) by hybridization between newly synthesized RNA and the template RNA hinders the broader application of Qβ replicase. Here, we developed a kinetic model of Qβ RNA replication consisting of two reaction pathways of dsRNA formation, which quantitatively explains the dynamics of dsRNA formation of three template RNAs. We also found that part of the Qβ phage genomic RNA sequence including the central hairpin loop significantly decreases the rate of dsRNA formation.

A design principle for a single-stranded RNA genome that replicates with less double-strand formation

Abstract Single-stranded RNA (ssRNA) is the simplest form of genetic molecule and constitutes the genome in some viruses and presumably in primitive life-forms. However, an innate and unsolved problem regarding the ssRNA genome is formation of inactive double-stranded RNA (dsRNA) during replication. Here, we addressed this problem by focusing on the secondary structure. We systematically designed RNAs with various structures and observed dsRNA formation during replication using an RNA replicase (Qβ replicase). From the results, we extracted a simple rule regarding ssRNA genome replication with less dsRNA formation (less GC number in loops) and then designed an artificial RNA that encodes a domain of the β-galactosidase gene based on this rule. We also obtained evidence that this rule governs the natural genomes of all bacterial and most fungal viruses presently known. This study revealed one of the structural design principles of an ssRNA genome that replicates continuously with less dsRNA formation.

Adaptive evolution of an artificial RNA genome to a reduced ribosome environment.

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 genome 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.

Effects of ribosomes on the kinetics of Qβ replication

Bacteriophage Qβ utilizes some host cell translation factors during replication. Previously, we constructed a kinetic model that explains replication of long RNA molecules by Qβ replicase. Here, we expanded the previous kinetic model to include the effects of ribosome concentration on RNA replication. The expanded model quantitatively explained single‐ and double‐strand formation kinetics during replication with various ribosome concentrations for two artificial long RNAs. This expanded model and the knowledge obtained in this study provide useful frameworks to understand the precise replication mechanism of Qβ replicase with ribosomes and to design amplifiable RNA genomes in translation‐coupling systems.