Norikazu Ichihashi

Replication of Genetic Information with Self-Encoded Replicase in Liposomes

In all living systems, the genome is replicated by proteins that are encoded within the genome itself. This universal reaction is essential to allow the system to evolve. Here, we have constructed a simplified system involving encapsulated macromolecules termed a “self‐encoding system”, in which the genetic information is replicated by self‐encoded replicase in liposomes. That is, the universal reaction was reconstituted within a microcompartment bound by a lipid bilayer. The system was assembled by using one template RNA sequence as the information molecule and an in vitro translation system reconstituted from purified translation factors as the machinery for decoding the information. In this system, the catalytic subunit of Qβ replicase is synthesized from the template RNA that encodes the protein. The replicase then replicates the template RNA that was used for its production. This in‐liposome self‐encoding system is one of the simplest such systems available; it consists of only 144 gene products, while the information and the function for its replication are encoded on different molecules and are compartmentalized into the microenvironment for evolvability.

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.

Importance of Parasite RNA Species Repression for Prolonged Translation-Coupled RNA Self-Replication

Increasingly complex reactions are being constructed by bottom-up approaches with the aim of developing an artificial cell. We have been engaged in the construction of a translation-coupled replication system of genetic information from RNA and a reconstituted translation system. Here a mathematical model was established to gain a quantitative understanding of the complex reaction network. The sensitivity analysis predicted that the limiting factor for the present replication reaction was the appearance of parasitic replicators. We then confirmed experimentally that repression of such parasitic replicators by compartmentalization of the reaction in water-in-oil emulsions improved the duration of self-replication. We also found that the main source of the parasite was genomic RNA, probably by nonhomologous recombination. This result provided experimental evidence for the importance of parasite repression for the development of long-lasting genome replication systems.

Constructing Partial Models of Cells

Understanding the origin of life requires knowledge not only of the origin of biological molecules such as amino acids, nucleotides and their polymers, but also the manner in which those molecules are integrated into the organized systems that characterize cellular life. In this article, we introduce a constructive approach to understand how biological molecules can be arranged to achieve a higher-order biological function: replication of genetic information.

A controllable gene expression system in liposomes that includes a positive feedback loop

We introduced a positive feedback loop into a LacI-dependent gene expression system in lipid vesicles, producing a cell-like system that senses and responds to an external signal with a high signal-to-noise ratio. This fully reconstituted system will be a useful tool in future applications in in vitro synthetic biology.

Compartmentalization in a Water-in-Oil Emulsion Repressed the Spontaneous Amplification of RNA by Qβ Replicase

During RNA replication mediated by Qbeta replicase, self-replicating RNAs (RQ RNAs) are amplified without the addition of template RNA. This undesired amplification makes the study of target RNA replication difficult, especially for long RNA such as genomic RNA of Qbeta phage. This perhaps is one of the reasons why the precise rate of genomic RNA replication in the presence of host factor Hfq has not been reported in vitro. Here, we report a method to repress RQ RNA amplification by compartmentalization of the reaction using a water-in-oil emulsion but maintaining the activity of Qbeta replicase. This method allowed us to amplify the phage Qbeta genome RNA exponentially without detectable amplification of RQ RNA. Furthermore, we found that the rate constant of genome RNA replication in the exponential phase at the optimum Hfq concentration was approximately 4.6 times larger than that of a previous report, close to in vivo data. This result indicates that the replication rate in vivo is largely explained by the presence of Hfq. This easy method paves the way for the study of genomic RNA replication without special care for the undesired RQ RNA amplification.

Construction of a Gene Screening System Using Giant Unilamellar Liposomes and a Fluorescence-Activated Cell Sorter

We have constructed a gene screening system composed of an in vitro transcription-translation system encapsulated within giant unilamellar liposomes and a fluorescence-activated cell sorter (FACS), which allows high-throughput screening of genes encoding proteins of interest. A mock gene library of β-glucuronidase (GUS) was compartmentalized into liposomes at the single-molecule level, and liposomes exhibiting green fluorescence derived from hydrolysis of the fluorogenic substrate by the synthesized enzyme were sorted using FACS. More than 10-fold enrichment of GUS gene with higher catalytic activity was obtained when a single copy of the GUS gene was encapsulated in each liposome. Quantitative analysis of the enrichment factors and their liposome size dependencies showed that experimentally obtained and theoretical values were in agreement. Using this method, genes encoding active GUS were then enriched from a gene library of randomly mutated GUS genes. Only three rounds of screening were required, which was also consistent with our theoretical estimation.

Kinetic Analysis of the Entire RNA Amplification Process by Qβ Replicase

The kinetics of the RNA replication reaction by Qβ replicase were investigated. Qβ replicase is an RNA-dependent RNA polymerase responsible for replicating the RNA genome of coliphage Qβ and plays a key role in the life cycle of the Qβ phage. Although the RNA replication reaction using this enzyme has long been studied, a kinetic model that can describe the entire RNA amplification process has yet to be determined. In this study, we propose a kinetic model that is able to account for the entire RNA amplification process. The key to our proposed kinetic model is the consideration of nonproductive binding (i.e. binding of an enzyme to the RNA where the enzyme cannot initiate the reaction). By considering nonproductive binding and the notable enzyme inactivation we observed, the previous observations that remained unresolved could also be explained. Moreover, based on the kinetic model and the experimental results, we determined rate and equilibrium constants using template RNAs of various lengths. The proposed model and the obtained constants provide important information both for understanding the basis of Qβ phage amplification and the applications using Qβ replicase.

Importance of Translation-Replication Balance for Efficient Replication by the Self-Encoded Replicase

In all living systems, the genetic information is replicated by the self‐encoded replicase (Rep); this can be said to be a self‐encoding system. Recently, we constructed a self‐encoding system in liposomes as an artificial cell model, consisting of a reconstituted translation system and an RNA encoding the catalytic subunit of Qβ Rep and the RNA was replicated by the self‐encoded Rep produced by the translation reaction. In this system, both the ribosome (Rib) and Rep bind to the same RNA for translation and replication, respectively. Thus, there could be a dilemma: effective RNA replication requires high levels of Rep translation, but excessive translation in turn inhibits replication. Herein, we actually observed the competition between the Rib and Rep, and evaluated the effect for RNA replication by constructing a kinetic model that quantitatively explained the behavior of the self‐encoding system. Both the experimental and theoretical results consistently indicated that the balance between translation and replication is critical for an efficient self‐encoded system, and we determined the optimum balance.

Kinetic Analysis of β-Galactosidase and β-Glucuronidase Tetramerization Coupled with Protein Translation

Both β-galactosidase (GAL) and β-glucuronidase (GUS) are tetrameric enzymes used widely as reporter proteins. However, little is known about the folding and assembly of these enzymes. Although the refolding kinetics of GAL from a denatured enzyme have been reported, it is not known how the kinetics differ when coupled with a protein translation reaction. Elucidating the assembly kinetics of GAL and GUS when coupled with protein translation will illustrate the differences between these two reporter proteins and also the assembly process under conditions more relevant to those in vivo. In this study, we used an in vitro translation/transcription system to synthesize GAL and GUS, measured the time development of the activity and oligomerization state of these enzymes, and determined the rate constants of the monomer to tetramer assembly process. We found that at similar concentrations, GAL assembles into tetramers faster than GUS. The rate constant of monomer to dimer assembly of GAL was 50-fold faster when coupled with protein translation than that of refolding from the denatured state. Furthermore, GAL synthesis was found to lack the rate-limiting step in the assembly process, whereas GUS has two rate-limiting steps: monomer to dimer assembly and dimer to tetramer assembly. The consequence of these differences when used as reporter proteins is discussed.