Life from a drop of rain: New research suggests rainwater helped form the first protocell walls

One of the major unanswered questions about the origin of life is how droplets of RNA floating around the primordial soup turned into the membrane-protected packets of life we call cells.

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

In the paper, published today in Science Advances, UChicago 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 could have helped create a meshy wall around protocells 3.8 billion years ago, a critical step in the transition from tiny beads of RNA to every bacterium, plant, animal, and human that ever lived.

"This is a distinctive and novel observation," Tirrell said.

The research looks at "coacervate droplets" -- naturally occurring compartments of complex molecules like proteins, lipids, and RNA. The droplets, which behave like drops of cooking oil in water, have long been eyed as a candidate for the first protocells. But there was a problem. It wasn't that these droplets couldn't exchange molecules between each other, a key step in evolution, the problem was that they did it too well, and too fast.

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

And that means no life.

"If molecules continually exchange between droplets or between cells, then all the cells after a short while will look alike, and there will be no evolution because you are ending up with identical clones," Agrawal said.

Engineering a solution

Life is by nature interdisciplinary, so Szostak, the director of UChicago's Chicago Center for the Origins of Life, said it was natural to collaborate with both UChicago PME, UChicago's interdisciplinary school of molecular engineering, and the chemical engineering department at the University of Houston.

"Engineers have been studying the physical chemistry of these types of complexes -- and polymer chemistry more generally -- for a long time. It makes sense that there's expertise in the engineering school," Szostak said. "When we're looking at something like the origin of life, it's so complicated and there are so many parts that we need people to get involved who have any kind of relevant experience."

In the early 2000s, Szostak started looking at RNA as the first biological material to develop. It solved a problem that had long stymied researchers looking at DNA or proteins as the earliest molecules of life.

"It's like a chicken-egg problem. What came first?" Agrawal said. "DNA is the molecule which encodes information, but it cannot do any function. Proteins are the molecules which perform functions, but they don't encode any heritable information."

Researchers like Szostak 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 which, like DNA, can encode information, but it also folds like proteins so that it can perform functions such as catalysis as well," 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 a natural next step.

That is until Szostak poured cold water on this theory, publishing a paper in 2014 showing that RNA in coacervate droplets exchanged too rapidly.

"You can make all kinds of droplets of different types of coacervates, but they don't maintain their separate identity. They tend to exchange their RNA content too rapidly. That's been a long-standing problem," Szostak said. "What we showed in this new paper is that you can overcome at least part of that problem by transferring these coacervate droplets into distilled water -- for example, rainwater or freshwater of any type -- and they get a sort of tough skin around the droplets that restricts them from exchanging RNA content."

'A spontaneous combustion of ideas'

Agrawal started transferring coacervate droplets into distilled water during his PhD research at the University of Houston, studying their behavior under an electric field. At this point, the research had nothing to do with the origin of life, just studying the fascinating material from an engineering perspective.

"Engineers, particularly Chemical and Materials, have good knowledge of how to manipulate material properties such as interfacial tension, role of charged polymers, salt, pH control, etc.," said University of Houston Prof. Alamgir Karim, Agrawal's former thesis advisor and a senior co-author of the new paper. "These are all key aspects of the world popularly known as 'complex fluids' -- think shampoo and liquid soap."

Agrawal wanted to study other fundamental properties of coacervates during his PhD. It wasn't Karim's area of study, but Karim had worked decades earlier at the University of Minnesota under one of the world's top experts -- Tirrell, who later became founding dean of the UChicago Pritzker School of Molecular Engineering.

During a lunch with Agrawal and Karim, Tirrell brought up how the research into the effects of distilled water on coacervate droplets might relate to the origin of life on Earth. Tirrell asked where distilled water would have existed 3.8 billion years ago.

"I spontaneously said 'rainwater!' His eyes lit up and he was very excited at the suggestion," Karim said. "So, you can say it was a spontaneous combustion of ideas or ideation!"

Tirrell brought Agrawal's distilled water research to Szostak, who had recently joined the University of Chicago to lead what was then called the Origins of Life Initiative. He posed the same question he had asked Karim.

"I said to him, 'Where do you think distilled water could come from in a prebiotic world?'" Tirrell recalled. "And Jack said exactly what I hoped he would say, which was rain."

Working with RNA samples from Szostak, Agrawal found that transferring coacervate droplets into distilled water increased the time scale of RNA exchange -- from mere minutes to several days. This was long enough for mutation, competition, and evolution.

"If you have protocell populations that 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 against exchange so that they store their genetic information well enough, at least for several days so that the mutations can happen in their genetic sequences, then a population can evolve."

Rain, checked

Initially, Agrawal experimented with deionized water, which is purified under lab conditions. "This prompted the reviewers of the journal who then asked what would happen if the prebiotic rainwater was very acidic," he said.

Commercial lab water is free from all contaminants, has no salt, and lives with a neutral pH perfectly balanced between base and acid. In short, it's about as far from real-world conditions as a material can get. They needed to work with a material more like actual rain.

What's more like rain than rain?

"We simply collected water from rain in Houston and tested the stability of our droplets in it, just to make sure what we are reporting is accurate," Agrawal said.

In tests with the actual rainwater and with lab water modified to mimic the acidity of rainwater, they found the same results. The meshy walls formed, creating the conditions that could have led to life.

The chemical composition of the rain falling over Houston in the 2020s is not the rain that would have fallen 750 million years after the Earth formed, and the same can be said for the model protocell system Agrawal tested. The new paper proves that this approach of building a meshy wall around protocells is possible and can work together to compartmentalize the molecules of life, putting researchers closer than ever to finding the right set of chemical and environmental conditions that allow protocells to evolve.

Read more at Science Daily

What gave the first molecules their stability?

The origins of life remain a major mystery. How were complex molecules able to form and remain intact for prolonged periods without disintegrating? A team at ORIGINS, a Munich-based Cluster of Excellence, has demonstrated a mechanism that could have enabled the first RNA molecules to stabilize in the primordial soup. When two RNA strands combine, their stability and lifespan increase significantly.

In all likelihood, life on Earth began in water, perhaps in a tide pool that was cut off from seawater at low tide but flooded by waves at high tide. Over billions of years, complex molecules like DNA, RNA and proteins formed in this setting before, ultimately, the first cells emerged. To date, however, nobody has been able to explain exactly how this happened.

"We know which molecules existed on the early earth," says Job Boekhoven, Professor of Supramolecular Chemistry at the Technical University of Munich (TUM). "The question is: Can we use this to replicate the origins of life in the lab?" The team led by Boekhoven at the ORIGINS Cluster of Excellence is primarily interested in RNA. "RNA is a fascinating molecule," says Boekhoven. "It can store information and also catalyze biochemical reactions." Scientists therefore believe that RNA must have been the first of all complex molecules to form.

The problem, however, is that active RNA molecules are composed of hundreds or even thousands of bases and are very unstable. When immersed in water, RNA strands quickly break down into their constituent parts -- a process known as hydrolysis. So, how could RNA have survived in the primordial soup?

How did double strands form in the primordial soup?

In laboratory testing, the researchers from TUM and LMU used a model system of RNA bases that join together more easily than naturally occurring bases in our cells today. "We didn't have millions of years available and wanted an answer quickly," explains Boekhoven. The team added these fast-joining RNA bases into a watery solution, provided an energy source and examined the length of the RNA molecules that formed. Their findings were sobering, as the resulting strands of up to five base pairs only survived for a matter of minutes.

The results were different, however, when the researchers started by adding short strands of pre-formed RNA. The free complementary bases quickly joined with this RNA in a process called hybridization. Double strands of three to five base pairs in length formed and remained stable for several hours. "The exciting part is that double strands lead to RNA folding, which can make the RNA catalytically active," explains Boekhoven. Double-stranded RNA therefore has two advantages: it has an extended lifespan in the primordial soup and serves as the basis for catalytically active RNA.

But how could a double strand have formed in the primordial soup? "We're currently exploring whether it's possible for RNAs to form their own complementary strand," says Boekhoven. It is conceivable for a molecule comprising three bases to join with a molecule comprising three complementary bases -- the product of which would be a stable double-strand. Thanks to its prolonged lifespan, further bases could join with it and the strand would grow.

Evolutionary advantage for protocells

Another characteristic of double-stranded RNA could have helped bring about the origin of life. It is firstly important to note that RNA molecules can also form protocells. These are tiny droplets with an interior fully separated from the outside world. Yet, these protocells do not have a stable cell membrane and so easily merge with other protocells, which causes their contents to mix. This is not conducive to evolution because it prevents individual protocells from developing a unique identity. However, if the borders of these protocells are composed of double-stranded DNA, the cells become more stable and merging is inhibited.

Read more at Science Daily