Neutral Networks: Tracking 100,000 RNA Ribozyme Mutants

Author: Okinawa Institute of Science and Technology (OIST) Graduate University
Published: 20 Sep 2022 - Updated: 31 Jul 2026
Publication Details: Peer-Reviewed | Anthropology News

Table of Contents:
Synopsis - Definition - Overview - FAQs - Insights, Updates - Related Content

Synopsis

This research offers the first experimental confirmation of a neutral network, a concept about mutation and evolution that had until now only been theoretically predicted. Working with a ligase ribozyme, a type of functional RNA that joins two RNA strands together and is tied to the origins of self-replication, the team used computational sequence design, an evolutionary algorithm, and deep learning to build and measure the fitness of more than 120,000 variants, raising the share of neutral variants in the designed population from roughly 10 percent to nearly 90 percent. The work matters to evolutionary biologists, geneticists, and origin-of-life researchers because a neutral network - a chain of sequences that each differ by a single base yet keep about the same fitness - is thought to be central to how populations build the genetic diversity needed to adapt to changing environments. The findings carry authority because they were peer reviewed and published in Nature Communications, and they hold broader interest for anyone curious about how the diversity of life, including the genetic variation underlying human health and disability, accumulates over millions of years through inherited mutations.*

At a Glance

• 1 - The full ribozyme has about 80 base pairs, but the study focused on a 35 base pair region.
• 2 - One chosen variant differed from the original ribozyme by 16 separate mutations across the sequence.
• 3 - Screening every one-step path across those 16 mutations produced 65,536 variants, and 60 percent of them stayed functional.
• 4 - Among the paths made only of functional mutations, 10 percent were accessible, a rate the team called unusually high compared with earlier experimental work.

Topic Definition

Neutral Network

A neutral network is a connected set of gene sequences in which each member differs from its neighbors by a single point mutation while keeping equivalent function or fitness. Pictured as a map, every node stands for one sequence and every line marks the mutation linking two of them, forming what amounts to a broad, flat plateau across a fitness landscape. Because movement along this plateau does not cost an organism its viability, genes can drift randomly from one sequence to the next, quietly building genetic diversity and shaping both the mutational robustness and the evolvability of biomolecules such as RNA. In practical terms, a neutral network is the pathway that lets living things accumulate variation without immediately paying a survival penalty, which is why it is regarded as important to how species adapt over long stretches of time.

Overview

Mutations and Evolution: Tracking a Network of 100,000 Mutants

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

What fundamentally sets a human being apart from every 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, mainly via random mutations-basically errors in how the DNA was copied. Most of these mutations negatively impact the organism and will likely die before it has a chance to reproduce. However, some will positively or neutrally impact the organism and spread through the population. These mistakes in DNA sequences have resulted in the diversity of life we see around the world today. But many aspects of how these mutations can increase fitness remain poorly understood.

"We've managed to experimentally show, for the first time, a concept about mutation and evolution that has previously only been theoretically predicated," said Prof. Yohei Yokobayashi, who leads to Nucleic Acid Chemistry and Engineering Unit at the Okinawa Institute of Science and Technology Graduate University (OIST). "It's called a neutral network, and it's thought vital for increasing diversity in a population." This research was published in Nature Communications.

Genes made up of DNA base pairs contain the instructions needed to create proteins and lead to the proper care and maintenance of a cell. For the instructions to be carried out, the 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'. Prof. 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 connected to the second one, connected to the third by just one single mutation. If these mutations maintain the same fitness, the organism might survive, and future generations might inherit the mutation. This increases the overall diversity, and diversity is essential for a species to adapt to environmental changes."

A neutral network is a series of base sequences just like this (albeit much longer), where each new sequence, differing by just one base, has roughly the same fitness as the one before and after. Scientists have suspected their existence for some time, but they're difficult to prove experimentally. Originally it was predicated that all RNA sequence spaces should have a possibility of harboring a large neutral network. Still, no one has ever found these large-scale neutral networks in practice.

Classic Phenotype Landscapes

Ligase Ribozyme

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

The entire ribozyme has around 80 base pairs, but the researchers focused on an area of 35 base pairs that is important for the function of the ribozyme and thus for measuring its overall fitness.

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

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

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

"This is pretty high," said PhD candidate and first author, Rachapun Rotrattanadumrong. "Other experimental work has never found such many accessible pathways between two variants."

The researchers emphasized that this experimental evidence of a neutral network has shown that neutral networks can form in an RNA sequence space, though not as extensively as the theoretical work would have them believe. Another interesting question may involve looking at what properties are needed for this kind of network to form.

"This work will allow people to experimentally test many hypotheses," continued Rotrattanadumrong. "We've provided the first experimental dataset of a neutral network. It will allow researchers to answer questions on how RNA evolved and continues to evolve."

Frequently Asked Questions

NOTE: Researched FAQs by Disabled World (DW)

What is a ligase ribozyme?

A ligase ribozyme is a type of functional RNA molecule that catalyzes the joining, or ligation, of two separate pieces of RNA together, a role that scientists connect to self-replication and the origin of life.

What are the four RNA bases?

The four standard bases in RNA are represented by the letters A, G, C, and U, and different orderings of these bases spell out the instructions carried within a sequence.

How is RNA related to DNA?

DNA must first be transcribed into RNA before its instructions can be carried out, so RNA acts as a working reflection of the DNA and helps guide the creation of proteins inside a cell.

What is a point mutation?

A point mutation is a change that affects a single base in a genetic sequence, and when many such single-base changes preserve fitness they can link sequences together into a neutral network.

What is a fitness landscape?

A fitness landscape is a conceptual map that plots genetic sequences against their fitness, where high peaks represent well-adapted sequences and a neutral network appears as a flat plateau of equally fit options.

Why does genetic diversity matter for a species?

Genetic diversity gives a population a wider range of traits to draw on, which improves its ability to adapt when environmental conditions change and raises its chances of long-term survival.

What is the difference between robustness and evolvability?

Robustness describes an organism's ability to keep its function despite mutations, while evolvability describes its capacity to generate new and useful variation, and neutral networks are thought to support both at once.

Insights, Analysis, and Developments

Editorial Note: The strength of this study lies in its scale and its patience, because proving a neutral network exists has long frustrated scientists who suspected such structures but could never catch one in practice. By deliberately engineering mutants rather than waiting on chance, the OIST team led by Prof. Yohei Yokobayashi, with first author Rachapun Rotrattanadumrong, converted a decades-old theoretical prediction into a real dataset that other laboratories can now probe. Their tempered conclusion is worth noting - neutral networks do form in RNA sequence space, yet not as sweepingly as early theory suggested - and that honesty about limits is exactly what makes the result credible and open to further testing on how RNA first arose and keeps evolving.*

Attribution/Source(s): This peer reviewed publication was selected for publishing by the editors of Disabled World (DW) due to its relevance to the disability community. Originally authored by Okinawa Institute of Science and Technology (OIST) Graduate University and published on 20 Sep 2022, this content may have been edited for style, clarity, or brevity.

* Editorial additions by Ian C. Langtree.