A Novel Method for Incorporating Non-Canonical Amino Acids into Proteins

Every basic biology lesson teaches that proteins are made up of combinations of 20 distinct amino acids, which are ordered in various patterns similar to words. However, researchers attempting to construct biological molecules with novel functionalities have long felt constrained by those 20 fundamental building blocks, and have worked to discover methods of incorporating other building blocks—known as non-canonical amino acids—into their proteins.

Scripps researchers have developed a new paradigm for simply introducing non-canonical amino acids to proteins. Their strategy, disclosed in Nature Biotechnology on September 11^th, 2024, involves encoding each new amino acid with four RNA nucleotides rather than the standard three.

Our goal is to develop proteins with tailored functions for applications in fields spanning bioengineering to drug discovery. Being able to incorporate non-canonical amino acids into proteins with this new method gets us closer to that goal.”

Ahmed Badran, PhD, Study Senior Author and Assistant Professor, Department of Chemistry, Scripps Research

To generate any particular protein, a cell must first convert an RNA strand into a string of amino acids. Every three nucleotides of RNA, known as a codon, corresponds to one amino acid. However, many amino acids have many potential codons; for example, RNA reading the sequences UAU and UAC both correspond to the amino acid tyrosine. Small molecules known as transfer RNAs (tRNAs) are responsible for linking each amino acid to its matching codons.

Recently, researchers that want to add completely new amino acids to a protein devised techniques to reassign a codon. For example, the UAU codon can be connected to a different amino acid by modifying the tRNA for UAU, causing the cell to read UAU as a building block other than tyrosine.

At the same time, every instance of UAU in the cell's genome would have to be converted into UAC to prevent the new amino acid from being incorporated into thousands of other proteins where it does not belong.

Badran added, “Creating free codons by whole genome recoding can be a powerful strategy, but it can also be a challenging undertaking since it requires considerable resources to build new genomes. For the organism itself, it can be difficult to predict how such codon changes influence genome stability and host protein production.”

Badran and his colleagues aimed to develop an efficient plug-and-play technique that would only introduce the selected non-canonical amino acid(s) into specified places in a target protein, without compromising the cell's normal biology or needing the entire genome be altered. That meant employing tRNA that hadn't already been allocated to an amino acid. Their answer was a four-nucleotide codon.

The team was aware that four-nucleotide codons have spontaneously developed in a few cases, such as bacteria rapidly evolving to drug resistance. So, in their latest study, the researchers investigated why cells used a codon with four nucleotides rather than three. They observed that the identities of the sequences adjacent to the four-base codon were critical—frequently used codons improved the cell's ability to detect a four-nucleotide codon and integrate a non-canonical amino acid.

Badran's group then investigated if they could change the sequence of a single gene to create a new four-nucleotide codon that would be appropriately employed by cells. The approach worked.

When the researchers surrounded a target location with three-letter, commonly used codons while maintaining adequate amounts of the four-nucleotide tRNA, the cell absorbed any new amino acid connected to the associated four-letter tRNA.

The researchers repeated the experiment with 12 alternative four-nucleotide codons before applying the approach to create more than 100 unique cyclic peptides known as macrocycles, each containing up to three non-canonical amino acids.

Badran noted, “These cyclic peptides are reminiscent of bioactive small molecules that one might find in nature. By capitalizing on the programmability of protein synthesis and the diversity of building blocks accessible by this approach, we can create new-to-nature small molecules that will have exciting applications in drug discovery.”

He adds that, unlike earlier techniques to non-canonical amino acid incorporation, this new method is simple to utilize because it entails changing only one gene rather than a cell's complete genome. Furthermore, more non-canonical amino acids might be employed in a single protein since four-nucleotide codons are more common than three-nucleotide codons.

Badran concluded, “Our results suggest that one can now easily and effectively incorporate non-canonical amino acids at diverse sites in a wide array of proteins. We are excited about these possibilities for our ongoing work and to provide this capability to the broader community.”

He noted that the process might be used to re-engineer existing proteins or build wholly new ones with applications in a variety of fields such as medicine, manufacturing, and chemical sensing.