Proteins are assembled from a standard set of 20 molecules known as canonical amino acids, encoded by an organism’s genetic material. A noncanonical amino acid (ncAA) is any amino acid that falls outside this standard set because it is not part of the universal genetic code. While the 20 canonical amino acids are defined by their incorporation during protein synthesis, ncAAs represent a much larger and more diverse group of molecules, with hundreds identified. Their classification is based on their relationship to protein production, not their abundance.
Natural and Synthetic Origins
Noncanonical amino acids originate from natural biological processes and laboratory synthesis. In nature, many organisms produce ncAAs that serve specific roles but are not inserted into proteins by the ribosome. For example, gamma-aminobutyric acid (GABA) is a derivative of glutamate and functions as a primary neurotransmitter, while ornithine is an intermediate in the urea cycle for metabolic waste disposal.
Another route to natural ncAAs is through post-translational modification, where canonical amino acids are chemically altered after being incorporated into a protein. The hydroxylation of proline to form hydroxyproline is an example that helps maintain the structural integrity of collagen. Similarly, the carboxylation of glutamate residues allows proteins to bind calcium, a process involved in blood clotting.
Chemists can also design and create a vast variety of synthetic ncAAs. These molecules are engineered with novel side chains that introduce chemical properties not found among the canonical amino acids. Scientists can create amino acids with fluorescent tags, heavy atoms for X-ray crystallography, or reactive groups, tailoring them for specific research or industrial purposes.
Genetic Code Expansion
Site-specific incorporation of ncAAs into proteins is achieved through genetic code expansion, a technique that reprograms the cell’s protein-synthesis machinery. This process involves transfer RNA (tRNA) molecules and aminoacyl-tRNA synthetase enzymes, which attach the correct amino acid to its corresponding tRNA.
To insert an ncAA, scientists introduce an “orthogonal” tRNA-synthetase pair into a cell. This pair functions independently of the host cell’s machinery to prevent cross-reactions. The engineered synthetase is designed to recognize a particular ncAA and attach it exclusively to its partner tRNA, ensuring only the desired ncAA is prepared for delivery to the ribosome.
This specialized tRNA is modified to recognize a repurposed codon, often a stop codon like UAG. When the ribosome encounters this codon in a gene, the orthogonal tRNA delivers the ncAA, incorporating it into the growing polypeptide chain. This method allows for creating proteins containing an ncAA at a predetermined site.
Applications in Scientific Research
Placing ncAAs at specific positions in proteins provides useful tools for research. One application is mapping protein interactions by incorporating ncAAs with photo-crosslinking groups. When exposed to UV light, these groups permanently link a protein to its binding partners. This technique captures transient interactions to help identify the components of complex biological pathways.
Fluorescent ncAAs offer another way to study protein behavior in living cells. These amino acids can be incorporated into a protein of interest, making it glow without a large, disruptive tag like GFP. This approach allows for precise tracking of its location, movement, and concentration in real-time.
Furthermore, ncAAs are used for detailed biophysical analyses of protein structure and function. Scientists can insert ncAAs containing unique spectroscopic probes into an enzyme’s active site. These probes act as reporters, providing information about the local chemical environment and how it changes as the enzyme carries out its catalytic function.
Therapeutic and Industrial Development
The properties of noncanonical amino acids are being harnessed to create advanced therapeutics and novel biomaterials. In medicine, incorporating ncAAs into protein-based drugs, or biologics, can enhance their performance. For example, adding specific ncAAs can increase a drug’s stability in the bloodstream, extending its effective half-life and reducing the required dosage frequency.
This technology also enables the precise construction of antibody-drug conjugates (ADCs), a class of targeted cancer therapies. An ncAA can be placed at a specific site on an antibody that targets tumor cells. A potent chemotherapy agent is then attached to the ncAA, ensuring the toxin is delivered specifically to cancer cells while minimizing damage to healthy tissue. This site-specific conjugation leads to more uniform ADCs compared to traditional methods.
In the industrial sector, ncAAs are used as building blocks for creating new biomaterials with tailored properties. By incorporating ncAAs into protein polymers, scientists can design materials that respond to specific stimuli, such as light or changes in temperature. This has led to the development of self-healing hydrogels, biocompatible adhesives, and new types of biosensors.