Nature has a set recipe for making proteins.

Triplets of DNA letters translate into 20 molecules called amino acids. These basic building blocks are then variously strung together into the dizzying array of proteins that makes up all living things. Proteins form body tissues, revitalize them when damaged, and direct the intricate processes keeping our bodies’ inner workings running like well-oiled machines.

Studying the structure and activity of proteins can shed light on disease, propel drug development, and help us understand complex biological processes, such as those at work in the brain or aging. Proteins are becoming essential in non-biological contexts too, like for example, in the manufacturing of climate-friendly biofuels.

Yet with only 20 molecular building blocks, evolution essentially put a limit on what proteins can do. So, what if we could expand nature’s vocabulary?

By engineering new amino acids not seen in nature and incorporating them into living cells, exotic proteins could do more. For example, adding synthetic amino acids to protein-based drugs—such as those for immunotherapy—could slightly tweak their structure so they last longer in the body and are more effective. Novel proteins also open the door to new chemical reactions that chew up plastics or more easily degradable materials with different properties.

But there’s a problem. Exotic amino acids aren’t always compatible with a cell’s machinery.

A new study in Nature, led by synthetic biology expert Dr. Jason Chin at the Medical Research Council Laboratory of Molecular Biology in Cambridge, UK, brought the dream a bit closer. Using a newly developed molecular screen, they found and inserted four exotic amino acids into a protein inside bacteria cells. An industrial favorite for churning out insulin and other protein-based medications, the bacteria readily accepted the exotic building blocks as their own.

All the newly added components are different from the cell’s natural ones, meaning the additions didn’t interfere with the cell’s normal functions.

“It’s a big accomplishment to get these new categories of amino acids into proteins,” Dr. Chang Liu at the University of California, Irvine who was not part of the study, told Science.

A Synthetic Deadlock

Adding exotic amino acids into a living thing is a nightmare.

Picture the cell as a city, with multiple “districts” performing their own functions. The nucleus, shaped like the pit of an apricot, houses our genetic blueprint recorded in DNA. Outside the nucleus, protein-making factories called ribosomes churn away. Meanwhile, RNA messengers buzz between the two like high-speed trains shuttling genetic information to be made into proteins.

Like DNA, RNA has four molecular letters. Each three-letter combination forms a “word” encoding an amino acid. The ribosome reads each word and summons the associated amino acid to the factory using transfer RNA (tRNA) molecules to grab onto them.

The tRNA molecules are tailormade to pick up particular amino acids with a kind of highly specific protein “glue.” Once shuttled into the ribosome, the amino acid is plucked off its carrier molecule and stitched into an amino acid string that curls into intricate protein shapes.

Clearly, evolution has established a sophisticated system for the manufacture of proteins. Not surprisingly, adding synthetic components isn’t straightforward.

Back in the 1980s, scientists found a way to attach synthetic amino acids to a carrier inside a test tube. More recently, they’ve incorporated unnatural amino acids into proteins inside bacteria cells by hijacking their own inner factories without affecting normal cell function.

Beyond bacteria, Chin and colleagues previously hacked tRNA and its corresponding “glue”—called tRNA synthetase—to add an exotic protein into mouse brain cells.

Rewiring the cell’s protein building machinery, without breaking it, takes a delicate balance. The cell needs modified tRNA carriers to grab new amino acids and drag them to the ribosome. The ribosome then must recognize the synthetic amino acid as its own and stitch it into a functional protein. If either step stumbles, the engineered biological system fails.

Expanding the Genetic Code

The new study focused on the first step—engineering better carriers for exotic amino acids.

The team first mutated genes for the “glue” protein and generated millions of potential alternative versions. Each of these variants could potentially grab onto exotic buildings blocks.

To narrow the field, they turned to tRNA molecules, the carriers of amino acids. Each tRNA carrier was tagged with a bit of genetic code that attached to mutated “glue” proteins like a fishing hook. The effort found eight promising pairs out of millions of potential structures. Another screen zeroed in on a group of “glue” proteins that could grab onto multiple types of artificial protein building blocks—including those highly different from natural ones.

The team then inserted genes encoding these proteins into Escherichia coli bacteria cells, a favorite for testing synthetic biology recipes.

Overall, eight “glue” proteins successfully loaded exotic amino acids into the bacteria’s natural protein-making machinery. Many of the synthetic building blocks had strange backbone structures not generally compatible with natural ribosomes. But with the help of engineered tRNA and “glue” proteins, the ribosomes incorporated four exotic amino acids into new proteins.

The results “expand the chemical scope of the genetic code” for making new types of materials, the team explained in their paper.

A Whole New World

Scientists have already found hundreds of exotic amino acids. AI models such as AlphaFold or RoseTTAFold, and their variations, are likely to spawn even more. Finding carriers and “glue” proteins that match has always been a roadblock.

The new study establishes a method to speed up the search for new designer proteins with unusual properties. For now, the method can only incorporate four synthetic amino acids. But scientists are already envisioning uses for them.

Protein drugs made from these exotic amino acids are shaped differently than their natural counterparts, protecting them from decay inside the body. This means they last longer, and it lessens the need for multiple doses. A similar system could churn out new materials such as biodegradable plastic which, similar to proteins, also relies on stitching individual components together.

For now, the technology relies on the ribosome’s tolerance of exotic amino acids—which can be unpredictable. Next, the team wants to modify the ribosome itself to better tolerate strange amino acids and their carriers. They’re also looking to create protein-like materials made completely of synthetic amino acids, which could augment the function of living tissues.

“If you could encode the expanded set of building blocks in the same way that we can proteins, then we could turn cells into living factories for the encoded synthesis of polymers for everything from new drugs to materials,” said Chin in an earlier interview. “It’s a super-exciting field.”

Image Credit: National Institute of Allergy and Infectious Diseases, National Institutes of Health

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• Source: https://singularityhub.com/2024/01/23/scientists-coax-bacteria-into-making-exotic-proteins-not-found-in-nature/