Pushing the boundaries of biological design, researchers at Scripps Research have just developed a novel method for slipping non-canonical amino acids into proteins. Their revolutionary approach, which was revealed in a September 11, 2024, article in Nature Biotechnology, is predicated on a bit of a twist: instead of coding for each new amino acid with the standard three RNA nucleotides, they use four. For protein engineering, this tiny but significant change creates a plethora of new possibilities.
Ahmed Badran, PhD, senior author and assistant professor of chemistry at Scripps Research, states, “We want to create proteins with unique functions that could transform industries like bioengineering and drug discovery.”
To understand the scope of this, let’s go back to the fundamentals: cells must first convert RNA sequences into chains of amino acids in order to produce proteins. Typically, a codon is a group of three RNA nucleotides that codes for a particular amino acid. However, certain amino acids have several codons and are a little greedy, such as tyrosine, which is encoded by both UAU and UAC. The hard work is done by tiny molecules known as transfer RNAs (tRNAs), which connect each amino acid to its corresponding codon.
The problematic part is that researchers have been attempting to alter the codons in proteins for a very long time in an attempt to add whole new amino acids. Consider altering the tRNA associated with UAU to substitute a non-canonical amino acid for tyrosine. Doesn’t it sound simple? However, to prevent that new amino acid from appearing in the wrong proteins, every instance of UAU in the genome would need to be replaced with UAC—a major pain, to be sure.
Badran’s crew changed course at that point. Instead of laboriously altering entire genomes, they sought a more efficient and focused method that would enable scientists to introduce non-canonical amino acids to particular locations inside a single protein without affecting the organism’s biology as a whole. The four-nucleotide codon is now present.
Ironically, they discovered a hint in the natural world. Four-nucleotide codons actually evolve on their own in some severe circumstances, such as when bacteria are frantically trying to evade antibiotics. Intriguingly, Badran’s group found that the surrounding sequences of these four-base codons were crucial. Nearby codons made it easier for the organism to decipher and employ the four-nucleotide codon to secrete a new amino acid.
What comes next? To see if they could modify a single gene to incorporate a functional four-nucleotide codon. You know what? It was successful! The cell joyfully generated new amino acids to match the four-nucleotide tRNA by cramming the target site with frequently used three-letter codons and making sure there was enough four-nucleotide tRNA in the mixture.
That’s not all, though. By using 12 distinct four-nucleotide codons, the team was able to create over 100 unique macrocycles, or cyclic peptides, each of which could contain up to three non-canonical amino acid groups.
Even more astounding is how easy the procedure is. Adding non-canonical amino acids by traditional methods typically necessitates rearranging the entire genome. Badran’s strategy? It’s more simpler and quicker to change just one gene. Additionally, this approach creates even more possibilities for combining non-canonical amino acids into a single protein because four-nucleotide codons are more likely to exist than three-nucleotide ones.
This approach has a wide range of possible applications. This technique could revolutionize manufacturing, chemical detection, and possibly medicine by re-engineering or even synthesizing new proteins. The design of proteins has never been more fascinating.
