Biology subjects

Ichihashi, N.

Publications and source records attributed to Ichihashi, N..

16 recordsLinked to original sources

bioRxiv2026

Molecular communication enables cooperative genomic RNA replication in all-aqueous droplet colonies

Multicellular organization enables biological functions to be distributed among specialized cells and coordinated through intercellular communication. Integrating this organizational principle with genome replication would link functional division of labor to the propagation of genetic information. Here, we show that genomic RNAs with complementary functions can cooperatively replicate across communicating artificial compartments. We constructed multicell-like colonies from all-aqueous droplets formed by phase separation of two incompatible polymers and stabilized at their interfaces by liposomes and amyloid-like proteins. The droplets assembled spontaneously while remaining permeable to protein-sized macromolecules. Two genomic RNAs encoding a replication enzyme and a metabolic enzyme were distributed in distinct colony-forming droplets and cooperatively replicated through cell-free translation and reciprocal molecular communication. These findings establish that genome replication can be collectively supported by communicating artificial compartments and provide a route toward multicell-like systems that coordinate spatially distributed genetic functions.

synthetic biology↗

bioRxiv2026

Emergent primordial heritability in short random RNA pools

The emergence of heritable molecular information is a fundamental step in the origins of life. Prior to the evolution of defined self-reproducing RNAs in the RNA world hypothesis, mixtures of primordial oligonucleotides may have supported more rudimentary forms of information propagation at the population level. To explore this possibility, we investigated the long-term dynamics of fully random short RNA pools under serial transfer conditions. Across multiple populations undergoing iterative nonenzymatic recombination and ligation, overall sequence compositions changed dynamically; however, sequence and structural traits emerged and exhibited modest yet detectable persistence, indicative of a primordial form of heritability. These traits were largely associated with terminal RNA regions, which tend to be unstructured and accessible for subsequent templating reactions, consistent with the enrichment of complementary sequences. The role of complementarity in shaping sequence composition was further confirmed using designed RNAs. Together, these results suggest that fully random RNA mixtures can exhibit a measurable, albeit weak, capacity to propagate newly generated information in the absence of enzymes, potentially representing an early step toward the emergence of genetic systems.

biochemistry↗

bioRxiv2026

Theoretical framework and experimental demonstration of sustainable in vitro regeneration of major translation factors EF-Tu and IF3

The development of molecular systems capable of self-regeneration, much like living organisms, is a major goal in synthetic biology. Previous efforts to expand the repertoire of regenerating proteins have relied on empirical optimization without a theoretical foundation, fundamentally limiting the scalability of this approach. Here, we developed the first theoretical framework, based on measurable parameters of translational proteins, to rationally predict the dilution rate that enables sustainable regeneration and the steady-state translation level. Using this framework, we successfully demonstrated sustainable regeneration of EF-Tu, the most abundant translation factor, for up to 15 rounds of serial dilution. We further extended this framework to the co-regeneration of multiple translation factors and demonstrated sustainable co-regeneration of EF-Tu and IF3, another major essential translation protein, for up to 20 rounds. These results establish a rational methodology for systematically expanding the number of regenerating proteins, providing a clear path toward the realization of a fully self-regenerating system.

Synthetic Biology↗

bioRxiv2026

A two-step selection method for in vitro evolution of translational proteins

Improving the reconstituted translation system is a key requirement for bottom-up synthetic biology. Here, we developed a two-step in vitro evolutionary method that can be used for improving translational proteins. In this method, two distinct conditions were sequentially applied while maintaining genotype-phenotype linkage in water-in-oil droplets. Using this method, we performed in vitro evolution of four translation factors, IleRS, PheRS, EF-G, and EF-Tu, and identified mutations that modestly enhanced translation activity in in vitro expression assays. One of the EF-G mutations (P610S) increased activity per protein approximately 2-fold for the recombinant protein purified from E. coli. This selection method is useful for improving translational proteins for bottom-up synthetic biology.

synthetic biology↗

bioRxiv2026

Compositional memory matters for early molecular systems

The error catastrophe refers to the proliferation of non-functional molecules in conditions where molecular replication has low accuracy, which is likely to correspond to conditions present at the Origin of Life. This error catastrophe can be avoided thanks to transient compartmentalization, provided that the compartments are sufficiently tight to prevent molecular leakage. Typically, transient compartmentalization models assume that the content of the compartments is completely pooled periodically, resulting in the complete loss of the compositional memory of the compartment. Furthermore, previous models that include the possibility of ecological interactions between molecular parasites and replicators within compartments generally do not study the effect of transient compartmentalization on their coevolution. To address both issues, we develop a framework that accounts for the coevolution of molecular replicators and parasites, along with specific compartmentalization dynamics that are transient yet partial, allowing compositional memory to accumulate from one round of compartmentalization to the next. We benchmark our model with a serial dilution experiment that displays complex oscillatory dynamics among four well-characterized RNA replicators. We also perform experiments to quantify the level of mixing in compartments when stronger stirring tends to homogenize their composition. We then model stirring-induced mixing and show how stirring alters the dynamics of compartmentalized replicators. We conclude that compositional memory arising from transient compartmentalization plays a major role in the dynamics of early molecular systems.

biophysics↗

bioRxiv2026

Experimental verification of the error minimization theory using non-standard genetic codes constructed in vitro

All living systems use an almost identical genetic code, the standard genetic code, in which 20 amino acids are assigned to 61 codons non-randomly. According to the error minimization theory, amino acids are arranged to minimize the mutational effect on protein function, while experimental verification remains limited. In this study, we constructed 10 non-standard genetic codes in vitro by reassigning three amino acids (Ala, Ser, and Leu) in vacant codons of the minimal genetic code, which consists of 21 tRNAs. Most of these non-standard genetic codes have a higher cost of amino acid replacement than the standard genetic code, calculated based on three amino acid properties: polar requirement (PR), molecular volume (MV), and hydropathy index (HI). The protein function of three reporter genes expressed using these non-standard genetic codes decreased similarly when random mutations were introduced into the genes, implying that the effect of mutations was similar across all the non-standard genetic codes tested here. This result provides direct experimental evidence that mutational robustness does not significantly change in individual reporter protein activity when the genetic code is altered within the range of mutational cost tested in this study (CostPR: 5.29 - 5.77, CostMV: 1848 - 2348, and CostHI: 3.27 - 5.10), which covers approximately 18.4% (PR), 37.6% (MV), and 50.8% (HI) of possible cost range achievable among one million randomly-generated genetic codes.

biochemistry↗

bioRxiv2026

Enhanced tRNA array method version 2 for simultaneous in vitro synthesis of 21 tRNAs

Transfer RNAs (tRNAs) play an essential role in translation, and their simultaneous in vitro synthesis remains a key challenge in bottom-up synthetic biology. We previously developed the tRNA array method that enables the simultaneous in vitro synthesis of 21 tRNAs from a single DNA template; however, the translational activity was substantially lower than that achieved using individually prepared 21 tRNAs for some proteins. Here, we identify the tRNA groups (PIEN group) that limit translation in the tRNA array method and improve the translational activities through sequence modification and incorporation of a leader sequence into the array construct. The resulting tRNA array method version 2 produces a tRNA set that allows translation at levels similar to those achieved with individually prepared tRNAs for multiple reporter proteins under both translation-coupled and uncoupled conditions. This tRNA synthesis scheme provides an improved platform for constructing self-reproducible gene expression systems.

synthetic biology↗

bioRxiv2025

The relationship between passive ankle joint stiffness and the stiffness of muscles, nerve, and tendon

Passive joint stiffness reflects the stiffness of various soft tissues across a joint. However, no previous studies have investigated the relationship between passive joint stiffness and muscle, nerve, and tendon stiffness. This study aimed to clarify whether passive ankle joint stiffness is related to stiffness in the triceps surae muscles, tibial nerve, and Achilles tendon. Thirty-eight healthy adults participated in the study. The passive ankle joint stiffness (slope of angle-passive torque curve) and shear wave velocities, which indicate soft tissue stiffness, of the triceps surae muscles and tibial nerve were measured at 5{degrees} of ankle plantarflexion and 5{degrees}, 15{degrees}, and 25{degrees} of ankle dorsiflexion. The shear wave velocity of the Achilles tendon was measured only at 5{degrees} of plantarflexion. A multiple regression model (forced-entry method) was constructed at each angle, specifying the shear wave velocities as the independent variables and passive joint stiffness as the dependent variable. At 5{degrees} of plantarflexion, no shear wave velocities were significantly related to passive joint stiffness (all p [≥] 0.05). At 5{degrees} and 15{degrees} of dorsiflexion, only the shear wave velocities of the tibial nerve were significantly positively related to passive joint stiffness (p = 0.024 and 0.008, respectively). At 25{degrees} of dorsiflexion, the shear wave velocities of the lateral gastrocnemius muscle and tibial nerve were significantly positively related to passive joint stiffness (p = 0.002 and 0.001, respectively). It can be concluded that both triceps surae muscles stiffness and tibial nerve stiffness are related to passive ankle joint stiffness.

biophysics↗

bioRxiv2025

Experimental evolution toward extinction in a molecular host-parasite system

Theoretical studies have proposed that coevolution with parasitic replicators plays a critical role in the evolution of primitive life; however, experimental verification of the potential outcomes of such coevolutionary dynamics remains limited. We previously conducted a co-evolutionary experiment using an RNA-protein replication system that resulted in the spontaneous diversification of host and parasitic RNAs into five distinct lineages with robust co-replication. Here, we report contrasting evolutionary outcomes from a second long-term co-evolutionary experiment. Using a droplet flow reactor system with increased dilution frequency over 5000 h (1,600 generations), we observed reduced diversity and frequent extinctions in later experimental stages. Co-replication assays of RNA clones revealed that the primary cause of this diversity loss was the shortened reaction time resulting from frequent dilution. Further analysis of RNA clones that emerged during evolution suggested that the frequent extinctions resulted from the appearance of highly competitive parasite species and the dominance of host species that exhibited reduced replication ability. These findings demonstrate that co-evolution between host and parasitic replicators can result in diversity loss and frequent extinctions depending on dilution conditions, highlighting the critical role of environmental parameters, such as dilution ratio and frequency, in enabling primitive replicators to evolve sustainably toward the emergence of life.

evolutionary biology↗

bioRxiv2025

Tibial nerve stiffness is related to maximum angle of ankle dorsiflexion

The maximum angle of ankle dorsiflexion is affected by the triceps surae muscle stiffness and stretch tolerance, which may be strongly reflected by the tibial nerve stiffness. However, no study has evaluated the triceps surae muscle and tibial nerve stiffness simultaneously or clarified their association with the maximum angle. The purpose of this study was to investigate the association between the maximum angle and both the stiffness. Forty-one healthy adults participated. The shear wave velocities of the triceps surae muscles and tibial nerve were measured at 5{degrees}, 15{degrees}, and 25{degrees} ankle dorsiflexion. Multiple linear regression analysis was performed using the forced entry method, specifying the shear wave velocities of the four tissues as the independent variables and the maximum angle as the dependent variable. This analysis was performed at each angle where the shear wave velocity was measured. Multiple linear regression analysis was also performed using the stepwise method, specifying the shear wave velocities of the tibial nerve at the three angles as the independent variables and the maximum angle as the dependent variable. Using the forced entry method, the shear wave velocity of the tibial nerve at each angle was significantly negatively associated with the maximum angle, whereas those of the muscles were not. Using the stepwise method, only the shear wave velocity of the tibial nerve at 25{degrees} was significantly negatively associated with the maximum angle. These results suggest that the tibial nerve stiffness in a greatly lengthened position determines the maximum angle of ankle dorsiflexion.

physiology↗

bioRxiv2025

Simultaneous in vitro expression of minimal 21 transfer RNAs by tRNA array method

Transfer RNA (tRNA) plays a central role in translation. The simultaneous in vitro synthesis of minimal yet sufficient tRNA species (at least 21) poses a challenge for constructing a self-reproducible artificial cell. A key obstacle is the processing of the 5 and 3 ends, which requires a multi-step reaction in natural cells. In this study, we developed a simplified processing method that allows simultaneous expression of all 21 tRNAs in a reconstituted transcription/translation system (PURE system). We tested three available methods (leader, 5-G variants, and HDVR attachment methods) and one new method (direct tRNA linkage method). Using these methods, we succeeded in simultaneous expression of six non-G-start tRNA from monocistronic six DNA templates in the PURE system. Furthermore, we developed a method that combines the direct tRNA linkage and HDVR attachment methods (termed tRNA array method). Using this method, we succeeded in simultaneous expression of all 21 tRNAs from a single polycistronic DNA template in the PURE system. The tRNA mixture produced by the tRNA array method supported a similar level of translation to the individually synthesized tRNA mixture. Additionally, we demonstrated that the minimal tRNA sets prepared by the tRNA array method can be used for genetic code engineering. This study represents a step toward the realization of self-reproducible artificial cells and also provides an easy method for preparing all tRNAs useful for genetic code engineering.

synthetic biology↗

bioRxiv2024

Sustainable Regeneration of 20 Aminoacyl-tRNA Synthetases in a Reconstituted System Toward Self-Synthesizing Artificial Systems

In vitro construction of self-reproducible artificial systems is a major challenge in bottom-up synthetic biology. Here, we developed a reconstituted system capable of sustainably regenerating all 20 aminoacyl-tRNA synthetases (aaRSs), which are major components of the translation system. To achieve this, we needed five types of improvements: 1) optimization of aaRS sequences for efficient translation, 2) optimization of the composition of the translation system to enhance translation, 3) employment of another bacterial AlaRS and SerRS to improve each aminoacylation activity, 4) diminishing the translational inhibition caused by certain aaRS sequences by codon optimization and EF-P addition, and 5) balancing the DNA concentrations of 20 aaRSs to match each requirement. After these improvements, we succeeded in the sustainable regeneration of all 20 aaRSs for up to 20 cycles of 2.5-fold serial dilutions. These methodologies and results provide a substantial advancement toward the realization of self-reproducible artificial systems.

synthetic biology↗

bioRxiv2022

Emergence of linkage between cooperative RNA replicators encoding replication and metabolic enzymes thorough experimental evolution

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 of the extended size of primitive chromosomes, which 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. Author SummaryThe integration of genes into a chromosome is a fundamental genetic organization in all extant life. The assembly of unlinked genes during prebiotic evolution was likely a major evolutionary transition toward the development of a complex cell. Decades of theoretical studies have suggested a plausible evolutionary pathway to a primitive chromosome from replicating RNA molecules that harbor cooperative genes within a protocell structure. However, demonstrating the evolution of a primitive chromosome in an experimental setup is challenging. We previously developed a cooperative RNA replication system in which two types of RNAs co-replicate using their self-encoded replication and metabolic enzymes. Using this system, in the present study, we demonstrate the evolution of a linkage between the two cooperative RNA replicators in compartments. An evolved "linked" RNA harbored the entire region of both genes, accumulated distinct mutations, and retained the ability to replicate using the two proteins translated from itself. These experimental findings support a prebiotic evolutionary scenario, in which unlinked genes assembled into a single genomic structure.

evolutionary biology↗

bioRxiv2022

Plausible pathway for a host-parasite molecular replication network to increase its complexity through Darwinian evolution

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. 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. We also provide evidence that the pathway actually occurred in our previous evolutionary experiment. These results provide both a theoretical basis and experimental evidence that a single replicator spontaneously develops into multi-replicator networks through coevolution with parasitic replicators, which might be the first complexification step toward to the emergence of life.

evolutionary biology↗

bioRxiv2021

Evolutionary transition from a single RNA replicator to a multiple replicator network

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 performed long-term evolution experiments using an RNA that replicates by a self-encoded RNA replicase. The RNA diversified into multiple coexisting host and parasite lineages, whose frequencies in the population initially fluctuated and gradually stabilized. 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.

evolutionary biology↗

bioRxiv2019

Evolutionary arms-races between host and parasitic RNA replicators in an artificial cell-like system

In the 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 on prebiotic evolution remains elusive. Here, we demonstrated experimental coevolution of RNA self-replicators (host RNAs) and emerging parasitic replicators (parasitic RNAs) for the first time by using an RNA-protein replication system we had 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 diversified host and parasitic RNAs exhibited evolutionary arms-race dynamics. The parasitic RNA accumulated unique mutations that the host RNA had never acquired, 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 play a key role in generating diversity and complexity in prebiotic molecular evolution.

evolutionary biology↗