New research suggests rainwater helped form early protocell walls

One of the biggest unanswered questions about the origin of life is how the droplets of RNA floating in the primordial soup evolved into the membrane-protected bundles of life we ​​call cells.

A new paper by engineers from the University of Chicago Pritzker School of Molecular Engineering (UChicago PME), the University of Houston Department of Chemical Engineering, and biologists from the UChicago Department of Chemistry has proposed a solution.

In the article published in Scientific progressUChicago PME postdoctoral researcher Aman Agrawal and his co-authors, including UChicago PME Dean Emeritus Matthew Tirrell and Nobel Prize-winning biologist Jack Szostak, show how rainwater may have helped create a mesh wall around protocells 3.8 billion years ago, a crucial step in the transition from tiny beads of RNA to every bacterium, plant, animal and human that has ever lived.

“This is a unique and novel observation,” Tirrell said.

The researchers were interested in “coacervate droplets,” naturally occurring compartments of complex molecules like proteins, lipids, and RNA. These droplets, which behave like drops of cooking oil in water, were long thought to be the first protocells. But there was a problem. It wasn’t that these droplets couldn’t exchange molecules with each other, a key evolutionary step; the problem was that they did it too well and too quickly.

Any droplet containing a new, potentially useful pre-life mutation of RNA would exchange that RNA with the other RNA droplets within minutes, meaning they would all quickly become identical. There would be no differentiation and no competition, meaning there would be no evolution.

And that means no life.

“If molecules are continuously exchanged between droplets or between cells, then all the cells after a short time will look the same and there will be no evolution because you will end up with identical clones,” Agrawal said.

Design a solution

Life is inherently interdisciplinary, which is why Szostak, director of UChicago’s Chicago Center for the Origins of Life, said it was a natural fit to collaborate with both UChicago PME, UChicago’s interdisciplinary School of Molecular Engineering and the University of Houston’s Department of Chemical Engineering.

“Engineers have been studying the physical chemistry of these types of complexes, and polymer chemistry more generally, for a long time. So it makes sense that engineering schools have expertise,” Szostak said. “When we’re studying a topic like the origin of life, it’s so complicated and there are so many parts that we need people with relevant experience.”

In the early 2000s, Szostak began studying RNA as the first biological material to develop, solving a problem that had long prevented researchers from studying DNA or proteins as the first molecules of life.

“It’s like the chicken and egg problem. Which came first?” Agrawal said. “DNA is the molecule that encodes information, but it can’t perform a function. Proteins are the molecules that perform functions, but they don’t encode any hereditary information.”

Researchers like Szostak have theorized that RNA came first, “taking care of everything,” in Agrawal’s words, with proteins and DNA slowly evolving from it.

“RNA is a molecule that, like DNA, can encode information, but it also folds like proteins so it can also perform functions such as catalysis,” Agrawal said.

RNA was a likely candidate for the first biological material. Coacervate droplets were likely candidates for the first protocells. Coacervate droplets containing early forms of RNA seemed like the natural next step.

Until Szostak threw a wrench in that theory, publishing a paper in 2014 showing that RNA in coacervate droplets was being exchanged too quickly.

“You can make all sorts of droplets of different types of coacervates, but they don’t retain their own identity. They tend to exchange their RNA content too quickly. This has been a long-standing problem,” Szostak said.

“What we’ve shown in this new paper is that you can overcome at least part of this problem by transferring these coacervate droplets into distilled water, like rainwater or any kind of fresh water, and they form a kind of tough skin around the droplets that prevents them from exchanging their RNA content.”

“A spontaneous combustion of ideas”

Agrawal began transferring coacervate droplets into distilled water during his doctoral research at the University of Houston, studying their behavior under an electric field. At that point, the research had nothing to do with the origin of life, but was limited to studying this fascinating material from a technical perspective.

“Engineers, especially those in chemistry and materials, have a good understanding of how to manipulate material properties such as interfacial tension, the role of charged polymers, salt, pH control, and so on,” said University of Houston Professor Alamgir Karim, Agrawal’s former thesis advisor and co-senior author of the new paper. “These are all key aspects of the world known as ‘complex fluids’ – think shampoo and liquid soap.”

Agrawal wanted to study other fundamental properties of coacervates during his PhD. This was not Karim’s field of study, but he had worked decades earlier at the University of Minnesota under one of the world’s leading experts, Tirrell, who later became founding dean of UChicago’s Pritzker School of Molecular Engineering.

Over lunch with Agrawal and Karim, Tirrell discussed the connection between research on the effects of distilled water on coacervate droplets and the origin of life on Earth. Tirrell asked where distilled water might have existed 3.8 billion years ago.

“I spontaneously said ‘rainwater!’ His eyes lit up and he was very enthusiastic about it,” Karim said. “So you could say it was a spontaneous combustion of ideas or ideation!”

Tirrell introduced Agrawal’s research on distilled water to Szostak, who had just joined the University of Chicago to lead what was then called the Origins of Life Initiative. He asked him the same question he had asked Karim.

“I asked him, ‘Where do you think distilled water might come from in a prebiotic world?’” Tirrell recalls. “And Jack said exactly what I was hoping he would say, which was rain.”

Working with Szostak’s RNA samples, Agrawal found that transferring coacervate droplets into distilled water increased the time for RNA exchange from minutes to days. This time frame was sufficient for mutation, competition, and evolution.

“If populations of protocells are unstable, they will exchange their genetic material with each other and become clones. There is no possibility of Darwinian evolution,” Agrawal said. “But if they stabilize themselves against exchange in such a way that they store their genetic information well enough, at least for several days, that mutations can occur in their genetic sequences, then a population can evolve.”

Rain, checked

Initially, Agrawal experimented with deionized water, purified under laboratory conditions. “That prompted the journal’s reviewers to wonder what would happen if prebiotic rainwater were highly acidic,” he said.

Commercial laboratory water is free of contaminants, contains no salt, and has a neutral pH that is perfectly balanced between base and acid. In short, it is as far from real-world conditions as a material can be. They needed to work with a material that was closer to real rain.

What’s more like rain than rain?

“We just collected rainwater in Houston and tested the stability of our droplets, just to make sure what we’re reporting is accurate,” Agrawal said.

In tests with real rainwater and with laboratory water modified to mimic the acidity of rainwater, they got the same results. The lattice walls formed, creating the conditions that could have supported life.

The chemical composition of the rain falling on Houston in the 2020s is not what would have fallen 750 million years after the Earth formed, and the same can be said of the model protocellular system Agrawal tested.

This new study proves that this approach of building a mesh wall around protocells is possible and can work together to compartmentalize the molecules of life, bringing researchers closer than ever to finding the right set of chemical and environmental conditions that allow protocells to evolve.

“The molecules we used to build these protocells are just templates until more suitable molecules are found as substitutes,” Agrawal said. “Even though the chemistry will be slightly different, the physics will remain the same.”