Unnatural Amino Acids for Genetic Codon Expansion (GCE)

Proteins are the basis of life activities and participate in various processes such as transport, catalysis, and regulation in life. These biological functions are closely related to the sequence of proteins. In order to explore the relationship between protein sequence and function, the site-directed mutation of specific amino acids into other amino acids and the study of the corresponding functional changes of proteins are the classic means to study this problem. However, amino acid mutations are usually limited to 20 common amino acids, and the functional groups carried by natural amino acids themselves are limited, which makes it difficult to meet the needs of changing or even giving proteins more abundant and diverse biological functions.

Genetically Encoded Unnatural Amino Acids

In recent years, important progress has been made in the study of introducing non-natural amino acids (UAAs) into proteins and giving target proteins new biological functions through gene codon expansion technology. There are many types of UAAs encoded by genes, and the functional groups they carry include alkenyl, halogenated alkanes, sulfonyl, alkynyl, azide, quinone methyl, quinolyl, phosphate, acetyl, etc. This type of chemical group can give proteins new properties through various reactions such as nucleophilic substitution, photoactivation, and click chemistry, helping to clarify the physicochemical properties and biological functions of proteins and their specific domains and sites. Existing UAAs have been encoded and inserted into different types of living cells (such as Escherichia coli, yeast, mammalian cells, etc.), which can be used to increase the light and heat stability of target proteins, locate the distribution of proteins in the body, reveal unknown protein-protein interactions, and study the regulation of protein function by post-translational modifications.

Fig. 1. Mechanism of genetic code expansion by site-specific incorporation of unnatural amino acids (Essays Biochem. 2019, 63(2): 237-266).

It is worth mentioning that the site-specific introduction of UAAs containing chemically reactive or photoactivated cross-linking groups into the target protein can enable the target protein to form a covalent bond with the target protein with which it interacts in the interaction region, thereby obtaining analogs with stronger affinity for the target protein, which has great transformation value in the field of biopharmaceutical research and development. This is because compared with traditional small molecules, highly reactive covalent biopharmaceuticals have higher selectivity for target proteins and can significantly reduce off-target side effects, bringing new opportunities for the design of biopharmaceuticals with clear binding targets. Currently, more than 200 UAAs can be introduced into proteins through gene codon expansion technology.

* List of unnatural amino acids:

Genetic Code

The genetic code is a set of rules in biology by which the sequence of nucleotides stored in genetic material (DNA or RNA) is translated into the sequence of amino acids in proteins. Every three nucleotides constitute a codon, and the way they are combined determines the order of amino acid arrangement in each protein molecule in the organism. The genetic code has some special properties. First, it is a triplet code, and the amino acids of each protein are determined by a codon consisting of 3 nucleotides. Second, it is degenerate, that is, multiple codons can correspond to the same amino acid, which increases the fault tolerance of biological systems. Third, it is universal, that is, almost all organisms use the same genetic code, showing the unity of life at the molecular level. During transcription, DNA is transcribed into mRNA, and then during translation, the ribosome reads the codons in the mRNA and uses tRNA to add the corresponding amino acids to the polypeptide chain being formed through anticodon matching. The deciphering of the genetic code was one of the major breakthroughs in molecular biology in the 20th century, laying the foundation for modern biotechnology and genetic engineering.

Genetic Code Expansion

Gene codon expansion technology refers to the technology of inserting UAAs into proteins at specific sites by using bio-orthogonal aminoacyl-tRNA synthetase and tRNA molecule pairs. This technology first synthesizes aminoacyl tRNAs that bind to UAAs by evolving aminoacyl-tRNA synthetases and orthogonal tRNAs that specifically recognize UAAs. Subsequently, the aminoacyl tRNA recognizes nonsense codons on mRNA and inserts UAAs at specific sites in the target protein.

Fig. 2. Genetic code expansion (Biochemistry. 2021, 60(46): 3455-3469).

Amino Acids in Genetic Code

UAAs introduce a variety of functional groups not present in the natural repertoire, such as photoreactive groups, bioorthogonal handles, and post-translational modification mimics, enabling sophisticated biochemical manipulations and analyses. For example, photocrosslinked UAAs can be used to capture transient protein-protein interactions within living cells, providing insights into dynamic cellular processes. Similarly, UAAs equipped with bioorthogonal groups enable site-specific labeling of proteins with fluorescent dyes or therapeutic drugs, enhancing imaging techniques and targeted drug delivery. The introduction of UAAs can also improve stability and enhance catalytic properties, making engineered proteins more suitable for industrial and medical applications. Proteins with customized functionality can be created by combining a variety of UAAs to map the folding and stability landscape of proteins.

• Chemically Reactive UAAs

Introducing chemically reactive UAAs into target proteins can regulate the affinity of target proteins with their interacting proteins, affect interaction-mediated signal transduction, have the potential to be transformed into biological drugs for treating diseases, and can be used to reveal unknown protein interactions. Conventional chemical cross-linkers mostly rely on reacting with active groups such as amino groups and sulfhydryl groups of amino acids in target proteins. This non-specific cross-linking reaction can lead to the risk of target proteins being off-target and binding to other proteins. The use of proximity-activated UAAs can ensure that the covalent cross-linking reaction is triggered only when a specific region of the target protein is close enough to the target protein into which the UAA is inserted. This technology of genetically encoded UAAs that endow target proteins with the ability to covalently bind to target proteins has shown great translational potential in protein drug development.

• Photo-Crosslinked UAAs

By introducing photoactivated groups into genetically encoded UAAs to react with adjacent amino acid residues, light-controlled crosslinking reactions can be achieved, which can be used to capture transient and dynamically changing protein interactions, and contribute to the study of biological processes and controllable drug design. Aryl azides are the most widely used photocrosslinkers. For example, Chin et al. mutated the tyrosyl tRNA synthetase of Methanococcus jannaschii and screened a new orthogonal aminoacyl tRNA synthetase/tRNA pair, which can selectively introduce the UAA AziF (p-azido-L-phenylalanine) into proteins to obtain recombinant proteins with higher yields. It has been successfully used in the study of the dimer topology of glutathione S-transferase (GST).

• Fluorescently Labeled UAAs

Fluorescent labeling of proteins helps to explore the dynamic processes of biomacromolecules in the cellular environment and visualize the physiological processes in cells. Chatterjee et al. reported a UAA Anap (3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid) with a fluorescent group. The fluorescence intensity of the protein encoded by the gene Anap can be observed with the help of a conventional fluorescence microscope. It has been successfully used to locate histone H3 expressed in mammalian cells. Charbon et al. designed the UAA CouAA (coumarin-derived amino acid) based on coumarin as a prototype, and used FtsZ protein as a model. It was found that the fluorescent CouAA can determine the subcellular localization of FtsZ protein without affecting the function of FtsZ protein.

In addition to introducing UAAs with fluorescent groups, gene codon expansion technology can also site-specifically insert UAAs carrying click chemistry groups, and then introduce fluorescent groups through click chemistry reactions. For example, Jagadish et al. introduced the non-natural amino acid AziF carrying an azide group into the cyclic peptide MCoTI-I, and used azide and a derivative of dibenzylcyclooctyne (DBCO) with a fluorescent group to carry out a cycloaddition reaction, so that the cyclic peptide carried fluorescence, which was used for subsequent research on the interaction between the cyclic peptide and protein in living cells.

• Post-translationally Modified UAAs

Post-translational modification is one of the key elements in regulating protein function, affecting life processes such as gene transcription, proliferation, signal transduction, and immune regulation in cells. Therefore, the level and disorder of post-translational modification are closely related to a variety of diseases. Gene codon expansion technology helps to clarify the structural and functional changes of substrate proteins induced by post-translational modifications by introducing post-translational modifications and their mimics to the target protein. For example, for the most important post-translational modification, protein phosphorylation, Hoppmann et al. introduced pTyr (phosphotyrosine) analogs through genetic coding, removed the protecting group through acid treatment, and efficiently obtained the target protein with tyrosine phosphorylation at a specific site. Polyacrylamide gel electrophoresis and mass spectrometry data proved that this method successfully introduced pTyr specifically into calmodulin and green fluorescent protein sites. At present, modifications such as lysine ubiquitination, fatty acylation, and serine phosphorylation have been designed and introduced into target proteins through gene codon expansion technology, becoming a powerful tool to reveal the regulation of proteins by post-translational modifications.

* List of unnatural amino acids:

Unnatural amino acids have endowed proteins with new properties by introducing reactive groups, fluorescent groups, and post-translational modification groups. Among them, chemically reactive and photo-crosslinked unnatural amino acids can covalently bind to adjacent amino acid residues with the help of reactive functional groups, and can be used to target target proteins bound by target proteins. They not only have the potential to discover new interaction partners of target proteins and elucidate biological processes, but can also be used to construct derivatives of target proteins with the ability to covalently bind to target proteins, thereby transforming and developing new biopharmaceuticals. Fluorescently labeled and post-translationally modified unnatural amino acids provide new means for clarifying the functions of proteins. According to the scientific problems studied, a variety of unnatural amino acids have been continuously optimized, improved, and innovated. We expect that the fusion of genetic codon expansion technology and unnatural amino acids of different properties can be used to design a rich variety of biomacromolecules, bringing important breakthroughs in many fields such as biotherapy, biological research, and protein engineering.

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