Tracking a network of 100,000 mutants

Phenotypic landscapes - classic

image: This image is a simplified representation of the classic fitness landscape. If a mutation occurs, the organism will normally fall from the top of the peak into the gap, losing its physical form, and will likely not survive or reproduce.
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Credit: OIST

  • Mutations are responsible for most of the diversity of life we ​​see in the world today, but many aspects of how mutations affect an organism’s fitness remain poorly understood.
  • Now researchers have shown experimentally, for the first time, a concept about mutations and evolution that was previously only theoretically sound.
  • Called the neutral network, it is believed to be vital for increasing the genetic diversity of a population.
  • A neutral lattice is an entire series of base sequences, where each new sequence, differing by only one base, has roughly the same fitness as the one before it and the one after it.
  • The researchers stressed that this will help scientists test many hypotheses, especially about how RNA evolved and continues to evolve.

What fundamentally distinguishes a human being from any other living creature comes down to differences in DNA sequences – a set of genetically inherited molecules found in every cell of every organism. These differences have accumulated over millions of years, mostly through random mutations – essentially errors in the way DNA has been copied. Most of these mutations negatively impact the organism and will likely kill it before it has had a chance to reproduce. However, some will have a positive or neutral impact on the organism and spread through the population. These errors in DNA sequences have resulted in the diversity of life we ​​see in the world today. But many aspects of how these mutations can increase fitness remain poorly understood.

“We have succeeded in showing experimentally, for the first time, a concept about mutation and evolution that was previously only theoretically based,” said Professor Yohei Yokobayashi, who heads the Chemistry and Engineering Unit. nucleic acids at the Okinawa Institute of Science. and the University of Advanced Technological Studies (OIST). “It’s called a neutral network and it’s believed to be vital for increasing the diversity of a population.” This research was published in Nature Communication.

Genes, made up of base pairs of DNA, contain the instructions needed to create proteins and lead to the proper care and maintenance of a cell. For instructions to be carried out, DNA must first be transcribed into RNA. Thus, RNA is like a reflection of DNA.

There are four standard base pairs for RNA and DNA. For RNA, these are ‘A’, ‘G’, ‘C’ and ‘U’. Professor Yokobayashi explained the concept of a neutral network by giving an example of a simplified sequence of RNA bases.

“Say, the RNA sequence AAAAAAA mutates to AAAUAAA, which then mutates to GAAUAAA. The first variant is linked to the second, which is linked to the third by a single and unique mutation. organism could survive and the mutation could be inherited by future generations.This increases overall diversity, and diversity is essential for a species to adapt to changes in the environment.

A neutral lattice is an entire series of base sequences like this (albeit much longer), where each new sequence, differing by only one base, has roughly the same fitness as the one before it and the one after it. follows. Scientists have suspected their existence for some time, but they are very difficult to prove experimentally. Originally, it was assumed that all RNA sequence spaces should have the possibility of hosting a large neutral network, but no one has ever found such large-scale neutral networks in practice.

In this study, the researchers looked at a type of RNA called ribozyme ligase, which had been synthesized before but was not known to form a neutral network. They chose this ribozyme because its function is to connect or ligate two pieces of RNA together. This role has important implications for the origin of life as it is vital for self-replication.

The entire ribozyme is about 80 base pairs long, but the researchers focused on a 35 base pair area that is important for the function of the ribozyme and therefore for measuring its overall fitness.

Through computational sequence design aided by an evolutionary algorithm and deep learning, scientists have engineered numerous mutants of this ribozyme and experimentally determined their fitness. They ended up testing over 120,000 variants and were able to increase the fraction of neutral variants in the engineered population from around 10% to almost 90%.

They then chose one of the variants they had identified. This differed from the original ribozyme by 16 mutations. They looked at all the different paths that could have been taken to jump from the original ribozyme to the new variant, one mutation at a time for 16 steps, and found 65,536 different variants of which 60% were functional.

The researchers then looked at the pathways that contained only functional mutations and found that 10% of the pathways were accessible.

“That’s quite high,” said PhD candidate and first author Rachapun Rotrattanadumrong. “Other experimental work has never found such a large number of accessible paths between two variants.”

The researchers pointed out that this experimental proof of a neutral network showed that it is possible for neutral networks to form in RNA sequence space, but not as widely as the theoretical work would suggest. Another interesting question may be to consider what properties are needed for this type of network to form.

“This work will allow people to experimentally test many hypotheses,” Rotrattanadumrong continued. “We have provided the first experimental dataset of a neutral network. This will allow researchers to answer questions about how RNA evolved and continues to evolve.


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