It's a dogma taught in every introductory biology class: Proteins are composed of combinations of 20 different amino acids, arranged into diverse sequences like words. But researchers trying to engineer biologic molecules with new functions have long felt limited by those 20 basic building blocks and strived to develop ways of putting new building blocks -- called non-canonical amino acids -- into their proteins. Now, scientists at Scripps Research have designed a new paradigm for easily adding non-canonical amino acids to proteins. "Our goal is to develop proteins with tailored functions for applications in fields spanning bioengineering to drug discovery," says senior author Ahmed Badran, PhD, an assistant professor of chemistry at Scripps Research. "Being able to incorporate non-canonical amino acids into proteins with this new method gets us closer to that goal."

 

For a cell to produce any given protein, it must translate a strand of RNA into a string of amino acids. Every three nucleotides of RNA, called a codon, correspond to one amino acid. But many amino acids have more than one possible codon. Recently, researchers aiming to add completely new amino acids to a protein have created strategies to reassign a codon. For instance, the UAU codon could be linked to a new amino acid by changing the tRNA for UAU. Badran and his colleagues wanted to create an efficient plug-and-play strategy that would only incorporate the chosen non-canonical amino acid(s) into specific sites in a target protein, without disrupting the cell's normal biology or requiring the entire genome to be edited. That meant using tRNA that wasn't already assigned to an amino acid. Their solution: a four-nucleotide codon. They discovered that the identities of the sequences nearby to the four-base codon were critical -- frequently used codons enhanced how the cell could read a four-nucleotide codon to incorporate a non-canonical amino acid.

 

Badran's group then tested whether they could alter the sequence of a single gene so that it had a new four-nucleotide codon that would be correctly used by the cell. "These cyclic peptides are reminiscent of bioactive small molecules that one might find in nature," says Badran. "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." Compared with previous approaches to non-canonical amino acid incorporation, this new method is easy to use since it involves altering only one gene rather than a cell's entire genome. He notes that the technique could be used to re-engineer existing proteins -- or create entirely new ones -- that have utility in a range of sectors, including medicine, manufacturing and chemical sensing.