If you work in the rapidly expanding and exciting discipline of synthetic biology, chances are you’ll know all about unnatural amino acids.
If you’re not familiar with unnatural amino acids, we’ve got the lowdown on what are becoming powerful tools in synthetic biology and protein engineering.
Synthetic biology applications use an expanded genetic code that goes beyond the 20 naturally occurring amino acid codons and assigns novel codons to unnatural amino acids. 
Why would we want to do this, I hear you ask? Well, researchers are using synthetic biology to help fight disease, develop new drugs, improve the environment, and find new sources of food.
For example, the unnatural amino acid p-acetylphenylalanine was incorporated into an antibody to allow conjugation with a small molecule that binds to an antigen on prostate cells. The engineered antibody acts like a bridge that connects a cytotoxic T-cell with the tumor cell, allowing the T-cell to make contact and destroy the cancer cell. The unnatural amino acid in this case was key to efficiently creating this special antibody and would be very difficult, if not impossible to do, with the natural protein. 
Unnatural amino acids can also be incorporated into synthetic peptides and proteins made synthetically.
In this article, we explore what unnatural amino acids are and how they’re used so that you’ll be clued up in no time!
What Are Unnatural Amino Acids?
In a nutshell, unnatural amino acids are non-proteinogenic amino acids, meaning that they are not among the 20 amino acids attached to tRNAs in living cells used to polymerize proteins.
Some unnatural amino acids do occur naturally, but most are chemically synthesized.
Citrulline is a good example of a non-proteinogenic amino acid produced in vivo through the oxidation of arginine, and p-benzoyl-phenylalanine is an example of one not found in nature at all.
Some unnatural amino acids are derived from the 20 naturally occurring varieties but altered using chemical modifications. We’ve summarized the different types of unnatural amino acids in Table 1.
Table 1: Different types of unnatural amino acids
Type of UAA Modification
Non-proteinogenic (e.g., citrulline)
No natural codon exists; usually made
through the post-translational modification of a natural amino acid
Addition of a second carbon between the amino group and carboxy groups
Addition of a methylene group between the alpha carbon and the side group
Addition of a second carbon between the amino and carboxy groups and the addition of a methylene group between the alpha carbon and the side group
Modified side group (e.g., p-benzoyl-phenylalanine)
Modification of a naturally occurring side group to confer additional functionality
N-methyl amino acids
The attachment of a methyl group to the nitrogen in the amino group
Alpha-methyl amino acids
A methyl group replaces the hydrogen on the alpha carbon
Chirality at the alpha carbon is in the D rather than the L configuration found in naturally occurring amino acids
How Are Unnatural Amino Acids Used?
The applications of unnatural amino acids are as varied as the scientific problem being addressed. Still, most involve protein engineering, where unnatural amino acids are used to study or enhance a protein’s function or to create bioactive peptides that have therapeutic effects.
Let’s take a look at some examples of the use of unnatural amino acids.
p-benzoyl-phenylalanine is used to study protein–protein and protein–nucleic acid interactions through exposure to UV light. This causes a reaction with inactivated C–H bonds.  By creating this covalent linkage, you can catch these proteins or peptides in the act!
The use of light at specific wavelengths with photocaged amino acids can turn on a protein. A photocaged amino acid might contain a group such as 4,5-dimethoxy-2-nitrobenzyl (DMNB) attached, for example, to the side chain of serine. If you zap this with light, the DMNB group is cleaved, exposing the natural -OH group that can then be phosphorylated. 
You can engineer a protein to be “off” by introducing a photocaged amino acid at a critical residue within the active site of an enzyme; you can then remove the inhibition by exposing the protein to light and seeing what happens once you turn that protein on.
This includes FRET analysis. Tagging proteins and peptides with fluorescent dyes is well known. By tagging with multiple fluorescent groups, you can measure folding and interaction by FRET or simply visualize this in vivo. For instance, a fluorescing peptide ligand can be visualized binding to a cell surface receptor.
One great application of unnatural amino acids is replacing the natural amino acid within beta-lactamase with p-acrylamido-phenylalanine. This increases the activity of the protein by several fold.  Other unnatural amino acids confer enhanced stability. For instance, replacing a tyrosine at the T4 lysozyme with m-chlorotyrosine increases stability and enzymatic activity at higher temperatures. 
Unnatural amino acids can be used as the building blocks for more complex molecules. They can be incorporated into peptidomimetic (compounds that mimic peptides or proteins) drugs or be used directly as therapeutic agents. 
There are many uses for unnatural amino acids and quite a few ways to put them into peptides and proteins. It’s worth checking out SwissSidechain, a curated database from the Swiss Institute of Bioinformatics that can be used to model the insertion of hundreds of unnatural amino acid side chains into peptides in silico. 
How to Incorporate Unnatural Amino Acids into Proteins and Peptides
So, now you know how you can use unnatural amino acids, how do you get them into a protein or peptide?
There are a few approaches: one is to synthesize a peptide using the unnatural amino acid. Other approaches rely on engineering the translation system in vivo or in vitro using cell-free conditions.
Let’s take a look at the first approach, chemical synthesis. unnatural amino acids can be built into synthetic peptides in much the same way as their natural counterparts, through blocking reactive groups and joining the carboxy group to the amino group of the adjoining amino acid.
When it comes to using translational machinery, there are two methods – selective pressure incorporation (SPI)  and stop codon suppression (SCS).  Let’s look at these in a bit more detail.
Selective Pressure Incorporation
SPI requires an auxotrophic strain of bacteria grown on medium supplemented with an unnatural amino acid that is structurally similar to the natural amino acid the organism cannot produce.
While not as efficient, the auxotrophic strain can be coaxed into using the unnatural amino acid in lieu of its natural counterpart. For instance, say the auxotroph must be cultured on medium supplemented with tyrosine.
If you supplemented the medium with an unnatural amino acid structurally similar to tyrosine, the organism would charge the tyrosine tRNA with the structurally similar unnatural amino acid and incorporate the unnatural amino acid into the protein.
Stop Codon Suppression
Stop codon suppression (SCS) engineers the translational system of a host cell to recognize one of the three stop codons (usually the amber codon, UAG) as the codon corresponding to the unnatural amino acid. An orthogonal translation system that will not “crosstalk” with the endogenous translation system is needed for SCS to work.
For example, a methanogenic archea known as Methanosarcina barkeri produces a very unique tRNA (tRNAPyl) with an anticodon complementary to the amber (UAG) codon. M. barkeri also produces a tRNA synthetase (PylRS) that will charge tRNAPyl with an unnatural amino acid known as pyrolysine.
If you were to introduce the M. barkeri genes encoding these into a naïve strain like E. coli, the translation machinery within this now-modified E. coli strain would read through the UAG codon and incorporate pyrolysine at that position, instead of terminating translation.  PylRS will not charge any of the native E. coli tRNAs with pyrolysine or other amino acids nor will the native E. coli tRNA synthetases charge tRNAPyl with any other amino acids. In other words, there is no crosstalk.
The M. barkeri pyrolysine tRNA synthetase can also charge tRNAPyl with other lysine-derivative unnatural amino acids besides pyrolysine, and researchers have used PylRS mutants to charge the tRNAPyl with unnatural amino acids phenylalanine derivatives. This synthetic biology system has been used to produce native proteins engineered with dozens of unnatural amino acids in vivo. 
If you’ve ever used unnatural amino acids in your research and have any tips or tricks of the trade, please leave a comment below!
- Neumann-Staubitz P, Neumann H (2016). The use of unnatural amino acids to study and engineer protein function. Curr Opin Struct Biol 38:119–28.
- Kim C H et al. (2013) Bispecific antibody targeting prostate cancer. PNAS 110(44): 17796-17801.
- Chin, JW et al. (2002) Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. PNAS 99(17): 11020–4.
- Nguyen, DP et al. (2014) Genetic encoding of photocaged cysteine allows photoactivation of TEV protease in live mammalian cells. J Am Chem Soc 136(6): 2240–3.
- Xiao, H et al. (2015) Exploring the potential impact of an expanded genetic code on protein function. PNAS 112(22): 6961–6.
- Carlsson, A-CC et al. (2018) Increasing enzyme stability and activity through hydrogen bond-enhanced halogen bonds. Biochemistry 57(28): 4135–47.
- Blaskovich, MAT (2016) Unusual amino acids in medicinal chemistry. J Med Chem 59(24): 10807–36.
- Gfeller, D et al. (2013) SwissSidechain: a molecular and structural database of non-natural sidechains. Nucleic Acids Res 41: D327–32.
- Johnson, JA et al. (2010) Residue-specific incorporation of non-canonical amino acids into proteins: recent developments and applications. Current Opin Chem Biol 14(6):774–80.
- Dumas, A et al. (2015) Designing logical codon reassignment – Expanding the chemistry in biology. Chem Sci 6(1): 50–69.
- Srinivasan, G et al. (2002) Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA. Science 296(5572): 1459–62.
- Wan, W et al. (2014) Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool. Biochim Biophys Acta 1844(6): 1059–70.