Evolution, Editing, and Clicking: Nobel Laureates Unpack the Biochemistry Revolution

“I wanted to be an engineer in the biological world and I fell in love with enzymes,” said Frances Arnold, on the #LINO23 main stage, “because they can do chemistry better than human chemists”. Arnold, an engineer by training, was awarded the 2018 Nobel Prize in Chemistry for her work on directed evolution to engineer enzymes. Her lecture kickstarted an intriguing series about biochemistry and how we can harvest the power of biochemistry for the benefit of mankind.

Evolving Enzymes

Proteins and enzymes are remarkable in their ability to adapt and optimize various conditions, Arnold explained in a thoroughly engaging lecture. This adaptability can be seen in the rapid development of antibiotic resistance, for instance. This poses not only a problem – but also an opportunity.

Arnold wanted to create enzymes that did not exist in human chemistry. Like many scientists, she was experiencing frustration because her work wasn’t progressing the way she was hoping. Other researchers were designing enzymes that barely functioned, but she wasn’t producing anything. Then she realised there was one excellent chemist who could help her: nature.

Nature has already done everything in chemistry, Arnold explained, and it does it through evolution.

“How do enzymes create new chemistry? It comes out of diversity. Novelty is already there because there’s massive diversity in the natural world. You may not know where it is, but nature will find it and it will appear in natural selection.”

So Arnold got started with creating new proteins through the process of directed evolution. Directed evolution is a method used in biochemical engineering that mimics natural evolution to create new and improved enzymes proteins. The process involves iterative rounds of genetic mutation, selection or screening, and amplification.

Directed evolution is analogous to climbing a hill on a ‘fitness landscape‘ where elevation represents the desired property. Each round of selection samples mutants on all sides of the starting template and selects the mutant with the highest elevation, thereby climbing the hill. This is repeated until a local summit is reached.

The biochemist acts as a ‘breeder’, mutating a gene in a careful way. Then, the chemist screens the results and looks for improvements in a desired function. The beneficial mutations are selected and fed back into the process, pushing it more and more towards the desired functional peak.

When you’re creating new enzymes through evolution, you’re not exactly understanding all the genetic mechanisms at play. But then again, this isn’t new. Just look at the many different and specialised breeds of dogs, Arnold mentions. They were selected through breeding, without the breeders truly understanding the process. More recently, the process has been used in various products, including hair gels and detergents.

“Directed evolution worked incredibly well for many years and you might be surprised to know that laundry detergents were made by natural evolution. After all, what natural enzyme wants to work in your laundry machine?,” Arnold quipped.

But it gets even more interesting. Directed evolution can be used not only to create things, but also to destroy them. This is particularly important when you consider that the world generates 50 million metric tons of e-waste every year, and we only recycle a fraction of that. These materials, like many cleaning and household products, stay in the environment for a long time and we don’t have a good way to disintegrate them.

Arnold says she can’t say too much about that because there’s an upcoming paper in the works — proof that even when you’re a Nobel Laureate, there’s still plenty of work to be done. But she’s not the only one pushing the boundaries of biochemistry.

‘Thanks, Emmanuelle!’

At one point in her talk, Arnold mentioned our ability to read and edit DNA, after which she emphatically said “Thanks, Emmanuelle!” She was of course referring to Emmanuelle Charpentier, who was awarded the Nobel Prize in Chemistry in 2020 for the development of CRISPR/Cas9, along with Jennifer Doudna.

The story of CRISPR/Cas9 as a genome editing tool starts with an unlikely character: Streptococcus pyogenes. S. pyogenes is a human pathogen that infects up to 5% of all humans and up to 17% of all children. Around 700 million infections occur every year, and symptoms can range from mild to life-threatening. But S. pyogenes is itself vulnerable to infections — viral infections, that is.

Several researchers have looked at how bacteria react to viral infections, explained Charpentier. This is how the groundwork for understanding CRISPR was laid out. CRISPR (an acronym for clustered regularly interspaced short palindromic repeats) is an accessory system in some bacteria and archaea that provides immunity against viruses and harmful plasmids.

First, explains Charpentier, you have a recognition of the invasion by the phage. The CRISPR system will recognise the invasion DNA, will take a portion of it, and will insert this portion into an array that allows the memorisation of the infection. Basically, CRISPR-Cas immunity integrates sequences of invading DNA to recognise, to remember and to destroy the invasive element.

In order to do this, the system has to be very good at cutting and pasting DNA, and this is what Charpentier and Doudna were most interested in. “The beauty of this mechanism is that it’s simple enough and yet versatile enough to make it a powerful genome editing technology,” Charpentier told the audience in Lindau.

It wasn’t straightforward or simple. In fact, the journey of CRISPR/CAS9 started with Darwin and the understanding of the laws of inheritance, and progressed with the understanding that DNA was a carrier of genetic information, the isolation of DNA, and the understanding that there was a genetic code involved – and this only paved the groundwork for CRISPR/CAS9.

“This was followed by a lot of effort in the scientific community to really use the mechanisms existing in bacteria, specifically to understand how bacteria defend themselves against viral infections, which led to the development of molecular biology, and a lot of genetic tools that have been developed over the past 50-60 years, with the ability to recombine DNA,” Charpentier adds.

There were plenty of challenges along the way, she adds. In particular, the difficulty stemmed from understanding that there are actually two types of RNA involved in the CRISPR process. Notably, trans-activating crispr RNA (tracrRNA), a small trans-encoded RNA, was found to also play a role in the process. This came as a surprise for Charpentier and was very frustrating, but after the role of tracrRNA was uncovered, things progressed “quite fast”.

“Sometimes it’s good to be a bit frustrated and have your first hypothesis contradicted, says Charpentier. This enables you to take a second, closer look at what is going on.”

Nowadays, applications for CRISPR/CAS9 are aplenty, which is striking for such a new technology. In fact, Charpentier says she’s almost left the field because there are already so many talented people working on it. But she draws great satisfaction from knowing her work makes a difference in the world.

“I have to say that what makes me most happy is the application for treating human genetic disorders. This is something I wanted to see quite early on.”

It’s definitely rewarding when your research clicks together, but sometimes, it’s the molecules themselves that do the clicking.

The ‘Click’ Moment

Up next was Morten Meldal, whose lecture focused on click chemistry.

Meldal, the 2022 Nobel Laureate in Chemistry, is also a Lindau alumni, having attended the meeting as a young researcher in 1986, so it was quite a triumphant return. But Meldal wasted no time. After a brief introduction on the imperfection of funding systems in science, he started discussing the advantages of click chemistry, a field he pioneered.

Generally speaking, chemists have two toolboxes for creation. The first is the large, organic toolbox. “You can make all sorts of molecules with that, but there’s a lot of waste, it’s not green chemistry, and that of course is a problem,” says Meldal.

Then, there is the click toolbox. The term “click chemistry” was inspired by the metaphor of clicking together two pieces of a puzzle. In the metaphor, these pieces are two chemical components coming together to form a new compound. But in order for something to be a click reaction, it has to fulfill specific characteristics.

Click reactions occur in one pot, don’t react to water, and generate minimal and inoffensive byproducts. Essentially, the molecular building blocks ‘click’ together seamlessly and efficiently.

The classic click reaction is the copper-catalysed reaction of an azide with an alkyne (CuAAC), which was demonstrated independently by Barry Sharpless at the Scripps Research Institute in California and Morten Meldal in the Carlsberg Laboratory, Denmark. Now, although the click chemistry toolbox is still small, there are several important reactions in the work.

An intriguing example Meldal mentioned focuses on the horseshoe crab. This species has remained largely unchanged for over 400 million years, and a good part of that is probably to do with its blood and its immune system. “They are like a pharmaceutical factory,” Meldal says. To this day, the pharmaceutical industry relies on horseshoe crab blood to test new medicines, vaccines, and medical supplies. But click chemistry could be used to synthesise a similar substance, Meldal mentions – or maybe an even better one.

Science for the People

Meldal ended his talk with a call to action, saying we should teach more science to children, and this can help the betterment of our society.

In fact, this shaped up to be a major the theme of #LINO23: science for the benefit of people.

All three laureates mentioned their hope and passion to do science not just for the sake of science, but to make a positive impact in society. From creating new, useful molecules to curing genetic disease, modern biochemistry is doing just that.

“Make science good for people,” Arnold concluded. A noble goal indeed!