How biophysics shapes the origins of life: a 21st IUPAB/62nd BSJ joint congress symposium overview.

The origins of life remains an unsolved mystery for all of humankind which requires a uniquely interdisciplinary approach incorporating many different subfields from astronomy, to geology, to chemistry, to biology, and beyond. One of these subfields which contributes greatly to understanding of various aspects of the origins of life is biophysics. As part of the 21st IUPAB/62nd BSJ Joint Congress in Kyoto, Japan, a symposium on the Origin of Life was held on June 28, 2024, featuring invited and contributed presentations on various aspects of origins of life research from Japan and around the world.

Cell-free expression of RuBisCO for ATP production in the synthetic cells.

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 CO fixation, holds potential as an energy production system, but its feasibility in a cell-free expression system has not yet been tested.

Emergence of linkage between cooperative RNA replicators encoding replication and metabolic enzymes through 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 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.

Minimal RNA self-reproduction discovered from a random pool of oligomers.

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.

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. 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.

Experimental evidence for the correlation between RNA structural fluctuations and the frequency of beneficial mutations.

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.

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 perform long-term evolution experiments of RNA that replicates using a self-encoded RNA replicase.

Relaxed Substrate Specificity in Qβ Replicase through Long-Term In Vitro Evolution.

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.

Primitive Compartmentalization for the Sustainable Replication of Genetic Molecules.

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.

PURE mRNA display and cDNA display provide rapid detection of core epitope motif via high-throughput sequencing.

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.

Translation-coupled RNA replication and parasitic replicators in membrane-free compartments.

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.

Emergence and diversification of a host-parasite RNA ecosystem through Darwinian evolution.

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.

In vitro evolution of phi29 DNA polymerases through compartmentalized gene expression and rolling-circle replication.

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.

Structural transition of replicable RNAs during in vitro evolution with Qβ replicase.

Single-stranded RNAs (ssRNAs) are utilized as genomes in some viruses and also in experimental models of ancient life-forms, owing to their simplicity. One of the largest problems for ssRNA replication is the formation of double-stranded RNA (dsRNA), a dead-end product for ssRNA replication. A possible strategy to avoid dsRNA formation is to create strong intramolecular secondary structures of ssRNA.

A Fusion Method to Develop an Expanded Artificial Genomic RNA Replicable by Qβ Replicase.

RNA-based genomes are used to synthesize artificial cells that harbor genome replication systems. Previously, continuous replication of an artificial genomic RNA that encoded an RNA replicase was demonstrated. The next important challenge is to expand such genomes by increasing the number of encoded genes.

Limited Sequence Diversity Within a Population Supports Prebiotic RNA Reproduction.

The origins of life require the emergence of informational polymers capable of reproduction. In the RNA world on the primordial Earth, reproducible RNA molecules would have arisen from a mixture of compositionally biased, poorly available, short RNA sequences in prebiotic environments. However, it remains unclear what level of sequence diversity within a small subset of population is required to initiate RNA reproduction by prebiotic mechanisms.

Mineral surfaces select for longer RNA molecules.

We report empirically and theoretically that multiple prebiotic minerals can selectively accumulate longer RNAs, with selectivity enhanced at higher temperatures. We further demonstrate that surfaces can be combined with a catalytic RNA to form longer RNA polymers, supporting the potential of minerals to develop genetic information on the early Earth.

Spontaneous advent of genetic diversity in RNA populations through multiple recombination mechanisms.

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.

Sustainable replication and coevolution of cooperative RNAs in an artificial cell-like system.

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.

Simple rules for construction of a geometric nest structure by pufferfish.

A small (~10 cm) male pufferfish (Torquigener albomaculosus) builds a large (~2 m) sandy nest structure, resembling a mysterious crop circle, to attract females. The circle consists of radially arranged deep ditches in the outer ring region, and maze-like shallow ditches in the central region. The configuration is geometrical.

Adaptation and Diversification of an RNA Replication System under Initiation- or Termination-Impaired Translational Conditions.

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.

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.