Unraveling the secrets of life using the ancient code of RNA

Salik scholars reveal RNA Capabilities that enable Darwinian evolution at the molecular level, and bring researchers closer to producing autonomous RNA life in the laboratory.

Charles Darwin described evolution as “descent with modification.” Genetic information in the form of DNA Sequences are copied and transmitted from generation to generation. But this process must also be somewhat flexible, allowing subtle variations in genes to emerge over time and new traits to be introduced into the population.

But how did it all start? In the origins of life, long before cells, proteins and DNA, could a similar kind of evolution have occurred on a simpler scale? Scientists in the 1960s, including Salk colleague Leslie Orgill, proposed that life began with the “RNA world,” a hypothetical era in which small, strand-bound RNA molecules ruled the early Earth and established the dynamics of Darwinian evolution.

Hammerhead sequences transcribed by low-fidelity polymerase drift away from the original RNA sequence (top) and lose their function over time. Hammerheads catalyzed by HD polymerase retain function and evolve more appropriate sequences (bottom). Credit: Salk Institute

Pioneering research on the role of RNA in early development

New research at the Salk Institute now provides new insights into the origins of life, providing compelling evidence supporting the RNA World hypothesis. The study published in Proceedings of the National Academy of Sciences (PNAS) On March 4, 2024, he unveils an RNA enzyme that can make exact copies of other functional RNA strands, while also allowing new variants of the molecule to emerge over time. These remarkable abilities suggest that the first forms of evolution may have occurred at the molecular level in RNA.

The results also bring scientists one step closer to recreating RNA-based life in the laboratory. By modeling these primitive environments in the laboratory, scientists can directly test hypotheses about how life began on Earth, or even other planets.

“We are chasing the dawn of evolution,” says senior author and Salk Foundation President Gerald Joyce. “By revealing these new capabilities of RNA, we are revealing the potential origins of life itself, and how simple molecules could have paved the way for the complexity and diversity of life we ​​see today.”

Scatterplots show the evolution of hammerhead populations over multiple rounds of evolution. Hammerheads transcribed by low-fidelity polymerase (52-2) drift away from the original RNA sequence (white lines) and lose their function. Hammerheads transcribed by a novel high-fidelity polymerase (71-89) retain function, with new functional sequences emerging over time. Credit: Salk Institute

The unique function of RNA and the pursuit of replication fidelity

Scientists can use DNA to trace the evolutionary history of modern plants and animals all the way back to the oldest single-celled organisms. But what happened before that remains unclear. Double-stranded DNA helices are great for storing genetic information. Many of these genes ultimately code for proteins, complex molecular machines that do all sorts of functions to keep cells alive. What makes RNA unique is that these molecules can do both. They are made of extended nucleotide sequences, similar to DNA, but they can also function as enzymes to facilitate reactions, such as proteins. So, could RNA be a precursor to life as we know it?

Scientists like Joyce have been exploring this idea for years, with a particular focus on RNA polymerase ribozymes — RNA molecules that can make copies of other RNA strands. Over the past decade, Joyce and his team have been developing RNA polymerase ribozymes in the laboratory, using a form of directed evolution to produce new versions capable of replicating larger molecules. But most of them suffer from a fatal flaw: they are unable to replicate sequences to a high enough degree Accuracy. Over many generations, so many errors are introduced into the sequence that the resulting RNA strands no longer resemble the original sequence and their function is completely lost.

So far. The latest laboratory-developed RNA polymerase ribozyme has a number of important mutations that allow it to copy a strand of RNA with much greater fidelity.

From left: David Horning, Gerald Joyce, and Nikolaos Papastavrou. Credit: Salk Institute

In these experiments, the strand of RNA being transcribed is the “hammerhead,” a small molecule that breaks other RNA molecules into pieces. The researchers were surprised to discover that not only did the RNA polymerase ribozyme precisely replicate functional hammerheads, but over time, new variations of the hammerheads began to appear. These new variants performed similarly, but their mutations made them easier to reproduce, which increased their evolutionary fitness and eventually led them to dominate hammerhead populations in the laboratory.

“We have long wondered how simple life was at its beginning, and when it acquired the ability to begin improving itself,” says first author Nikolaos Papastavrou, a research associate in Joyce's lab. “This study suggests that the dawn of evolution could have been very early and very simple. Something at the level of individual molecules could have powered Darwinian evolution, and this may have been the spark that allowed life to become more complex, moving from molecules to cells to organisms.” Multicellular.

The results highlight the critical importance of replication fidelity in making evolution possible. The fidelity of RNA polymerase transcription must exceed a critical threshold to maintain heritable information over multiple generations, and this threshold would have risen as the size and complexity of evolving RNA increased.

The future of RNA research and independent living

Joyce's team is recreating this process in laboratory test tubes, applying increasing selective pressure to the system to produce better-performing polymerases, with the goal of one day producing an RNA polymerase that can replicate itself. This would mark the beginning of autonomous RNA life in the laboratory, which researchers say could be achieved within the next decade.

Scientists are also interested in what might happen when the little “RNA world” gains more independence.

“We have seen that selection pressure can improve an RNA with an existing function, but if we allow the system to evolve longer with larger sets of RNA molecules, can new functions be invented?” says co-author David Horning, a scientist in Joyce's lab. “We are excited to answer how early life could have increased complexity, using tools developed here at Salk.”

The methods used in Joyce's lab also pave the way for future experiments that test other ideas about the origins of life, including what environmental conditions could best support RNA evolution, both on Earth and on other planets.

Reference: “Evolution of RNA-catalyzed RNA” by Nikolaos Papastavrou, David P. Horning, and Gerald F. Joyce, 4 March 2024, Proceedings of the National Academy of Sciences.
doi: 10.1073/pnas.2321592121

The work was previously supported NASA (80NSSC22K0973) and the Simons Foundation (287624).