After a lengthy experiment of fascinating significance to the study of the origin of life, a research team from Japan reported that they had created a test tube world composed of molecules that spontaneously evolved complexity and surprisingly evolved cooperation**
Ryo mizuuchi, the first author of the study and a project assistant professor at the University of Tokyo, pointed out that in the process of hundreds of hours of replication, a single type of RNA evolved into five different molecular "species" or host and parasite lineages. They coexisted harmoniously, survived cooperatively, and began like a "molecular ecosystem".
Their experiments confirmed previous theoretical findings that molecules with replication means can spontaneously develop complexity through Darwinian evolution, "which is a key step in the emergence of life," the researchers wrote.
"We can provide direct evidence; we can see what actually happens when a replicated molecule is complicated in a test tube," mizuuchi said
Sijbren Otto, a professor of systems chemistry at Groningen University in the Netherlands, who was not involved in the study, said that this is the first and perhaps the most important step towards evolving a complex replicator network in the laboratory. "With what is shown here, the road to the future becomes clearer, people become more optimistic and think it can actually succeed."
Joana Xavier, a computational biologist from University College London, praised the work of mizuchi and his colleagues as a great proof of concept that shows how a minimal system can be complicated.
The baby of Spiegelman's monster
The roots of the new experiment can be traced back to the 1960s, when molecular biologist sol Spiegelman created what he called "little monsters" in his laboratory. Although the label has the shadow of Frankenstein, his little monster is not green, square browed, roaring - or even lifeless. It is a synthetic molecule that can fill a test tube with copies of itself.
Spiegelman's monster is an RNA mutation chain based on the virus genome. The biologist found that he could replicate it indefinitely as long as he heated and mixed it with a nucleotide component and a polymerase called replicase. However, he soon realized that his molecules became smaller and smaller over time. Copies without unnecessary genes replicate faster, which improves their chances of being collected in the sample and transferred to a new test tube for further replication. Like biological species, their molecules began to mutate and evolve under the pressure of natural selection, so that they could better survive in their glass world.
These studies are the world's first experiment to demonstrate Darwinian evolution at the molecular level - "survival of the fittest through the evolution of natural selection," said Eugene Koonin, an outstanding investigator at the National Biotechnology Information Center of the National Institutes of health. "Under those conditions, survival of the fittest only means the fastest replication."
Spiegelman's work has inspired decades of further research, most of which are basic research on the origin of life. In addition, it also provides fuel for the RNA world hypothesis that life originates from self replicating RNA molecules. But these studies do not answer a key question. Can a single molecular replicator evolve into a complex network composed of multiple replicators?
About a decade ago, when NORIKAZU Ichihashi was an associate professor of bioinformatics engineering at Osaka University in Japan, he began to understand the answer by adjusting Spiegelman's test tube world. "We're trying to make our system more realistic," Ichihashi said.
To this end, Ichihashi and his team developed an RNA molecule encoding replicase, which can make copies of RNA. But in order for molecules to translate their own code, scientists need to add something: ribosomes and other gene translation machines, which they borrowed from the common intestinal bacterium E. coli. They embedded these machines in droplets and added them to a mixture of RNA and raw materials. Then years of tedious mixing and waiting.
Their long-term experiments included incubating their replication system at 37 degrees Celsius, adding new droplets with a fresh translation system and stirring the mixture to induce replication. Every few days or so, they analyze the RNA concentration in the test tube, and every other week or so, they freeze the sample from the latest mixture. Every six months or so, they sequenced a large number of samples collected to see whether RNA had obtained new mutations and evolved into a new lineage.
Evolution in test tube
After 215 hours and 43 rounds of replication, researchers began to see interesting results, which they reported in the 2016 proceedings of the National Academy of Sciences. The original RNA has been replaced by two other RNA lineages. One is described by the researchers as a "host", which can replicate itself with its own replicase, just like the original molecule. Another lineage, a "parasite", needs to borrow the gene expression mechanism of the host.
When Ichihashi and his colleagues extended the experiment to 120 rounds of replication within 600 hours, they found that the host line had split into two independent host lines, and one host had evolved two different parasites. But not only has the number of lineages increased, but the complexity of their interactions has also increased. The host acquires mutations that interfere with the parasite's ability to hijack its replication resources -- but the parasite also develops mutations as a defense against these obstacles. Hosts and parasites seem to be coevolutionary.
Scientists reported in the "eLife" in 2020 that the populations of parasites and hosts fluctuated greatly when competing for fields in the "evolutionary arms race". Each RNA lineage briefly rises to dominance and is then taken away by another lineage. "If one lineage is dominant, the other will decline," said Ichihashi, now a professor at the University of Tokyo
But the researchers continued the experiment until round 130, when another host had evolved. By round 160, one of the parasites disappeared; After a few rounds, another parasite appeared. By round 190, the researchers found a new surprise. The huge dynamic fluctuations of the population of each lineage have begun to give way to smaller fluctuations. This stability suggests that these lineages are no longer competitive copies. On the contrary, they have begun to interact as a network and cooperate in a quasi stable coexistence state.
Mizuuchi and Ichihashi (then a doctoral student at Ichihashi laboratory and now a researcher at the University of Tokyo) did these experiments together. They were shocked by these findings and reported in nature communications in March. Mizuuchi pointed out that they are just "simple molecules, which is very unexpected".
Cooperative parasites do their work
Koonin agrees that their findings are compelling. He said that their experimental device is more refined, more practical, and the results are more complex and rich, but it can be fully compatible with Spiegelman. The researchers looked at the replication and collection of mutations of a single type of molecule under natural selection - but then went further, allowing different molecules to evolve into a group under the influence of each other, just like a group of living cells, animals or humans. In this process, the researchers explored some rules that determine that this complex group should become a stable and lasting group.
Some of these results confirm early predictions and theoretical work on how complexity occurs in viruses, bacteria and eukaryotes. For example, a study by Koonin laboratory also shows that parasites are inevitable in the emergence of complexity.
"Without parasites, this degree of diversification may not be possible. The evolutionary pressure exerted by parasites and their hosts causes both sides to split into new lineages," mizuuchi said.
A more surprising basic principle that has emerged is the key role of cooperation. These five lineages belong to different small cooperative networks, and some lineages are more cooperative than others.
Xavier said that scientists have long focused on competition in evolution, so that the role of cooperation has been somewhat ignored. "I think cooperation will also begin to play an important role, especially in terms of origin, because there are so many things that must be combined in the right way."
In the system observed by Ichihashi, mizuuchi and their colleagues, the cooperation between RNAs is entirely focused on replication. But the researchers hope that by adjusting the natural selection criteria in the test tube, it will be possible to force RNA to evolve completely different functions, such as metabolic function.
Different destiny
"Scientists like to entertain each other, and the best entertainment is a surprise," said David deamer, a professor of biomolecular engineering at the University of California, Santa Cruz He thought it was a good paper, but he pointed out that what happened in the laboratory might not translate into what happened at the dawn of life.
In fact, the scenario in Ichihashi's lab cannot reflect the beginning of life, because the experiment depends on the translation machine of E. coli. "The typical question of the origin of life is how protein synthesis itself began," said Charlie Carter, a professor of Biochemistry and Biophysics at the University of North Carolina School of medicine.
However, Koonin believes that if researchers find a way to use real self replicating molecular systems to evolve complexity, they will see something very similar to the networks described in the paper. "They are at least a good illustration of the types of processes that may occur in the origin of life," Koonin said.
For Otto, this study shows that once the precise replication of molecules with this complexity is solved, they will be further complicated. He pointed out that the experiment did not show people how to get there, but once they got there at this stage, it did depict the future.
Ichihashi and his colleagues continued their research to see if they could recreate the same sustainable network in a separate experiment. For this purpose, they took samples of five departments. This time, however, they found that although four lineages continued to replicate and survive for at least 22 rounds, the fifth lineage disappeared. "I don't know why, it's a very strange point," Ichihashi said.
One possibility is that the system is even more complex than researchers thought. When these five lineages are separated, it misses the sixth lineage that is crucial to the survival of the disappeared lineage. Through the theoretical model, Ichihashi's team confirmed that the remaining four lineages can be replicated sustainably and interdependent, and knocking out any of these four lineages will lead to the extinction of at least one of the other lineages. In addition, their simulation also pointed to an abnormal finding that the elimination of one of the parasites would lead to the extinction of its host.
At the same time, the researchers continue their major test tube experiments and are waiting to see if their network will be further complicated. In addition, they have begun similar experiments. They use DNA instead of RNA.
"What we have observed is just the beginning of how these molecular replicator communities have evolved. I think they will have different destinies in the future - we can't predict what will happen," Ichihashi said