02/08/2013
UNNATURAL AMINO ACIDS
Louie Iselin
Unnatural Amino Acids
There are 20 naturally occurring amino acids which have a limited number of functions. If we could
create more amino acids, either through the use of nonsense stop codons or quadruplet codons, we
could increase the productivity of cells, as well as adding entirely new functionalities to amino acids.
By using the 4 base codons alone, we could incorporate another 200 unnatural amino acids into the
genetic code.
Amino acids
Amino acids are often considered the most important building block of life despite not being the only
aspect essential to it. They make up proteins which make up everything from our hair to our heart and
are what really make us living beings. Amino acids are present in every cell and make up nearly every
component of the cell. Altering one aspect of an amino acid’s genetic make-up could be catastrophic
but, with improving tools and knowledge, we can harness the structure and efficient mechanisms that
life has evolved over billions of years to create entirely new amino acids with new and exciting
functions. This can help us to better understand the functions of cells and possibly even direct us
towards more efficient therapies.
Amino acids are made up of 3 main parts: The amino group, the carboxyl group and a side chain
which is what causes variation which are all based around a central carbon. The 20 major amino acids
found in living organisms are usually split up into 4 groups based on the properties of the variable ‘R’
group. Amino acids can be acidic (polar), basic (polar), positively charged or negatively charged but
they can be classified in many other ways as well. The 20 most commonly studied amino acids are not
the only ones. There are also many ‘minor’ amino acids that are simply slight alterations of major
ones. There are also around 5 unnatural amino acids found in the human body. To alter the structure
of amino acids, all that is needed is to alter the structure of the R group. Amino acids are synthesised
through a process called Strecker synthesis. This creates an alpha-amino acid from ammonia (the
amine precursor), cyanide (the carboxyl precursor), and an aldehyde. Acid or base catalysis can then
take place to hydrolyze the then amino nitrile and form an amino acid.
02/08/2013
UNNATURAL AMINO ACIDS
Louie Iselin
Stop codon use
Stop codons are the codons that come at the end of every gene and are what stops mRNA synthesis.
There are 3 of them in total and none of them code for an amino acid so they offer exciting new
prospects in synthetic biology.
Harnessing the uselessness of stop codons has been a new and popular area of research because they
can be manipulated so easily without affecting the test subject. One of the most well-known areas of
work has been in replacing one stop codon with another throughout an organism through the CAGE
or MAGE methods.
This technique can make an organism resistant to viral infection because the stop codons that the
virus is used to when replicating in a host cell would be changed and so the proteins of the virus
would be made ineffective. Another useful application for stop codons, and the one that I am focused
on is the attachment of an amino acid to the stop codon which could then be placed into genes to alter
protein structure or serve functions that I will address.
The amber codon was first successfully used to code for an artificial amino acid in E coli in 1998 by
Furter. It took many modifications and positive and negative selection to reach the desired outcome,
making sure that the AARS is specific to the amino acid (using site-directed mutagenesis and other
selective processes) but there are now over 100 UAAs across a wide range of organisms. One
example was the use of the amber stop suppressor from E coli being placed into yeast. This prevented
the crossing of the orthogonal pair with the native tRNA/ tRNA aminoacylase pair. The amber
suppressor is the tRNA that binds to the amber stop codon and so is its anticodon. It usually carries
tyrosine so a mutation library of aminoacylases can be built
from tyrosine aminoayclase. The most efficient aminoacylase
can then be paired with the tRNA and the AARS, which
attaches the artificial amino acid onto the tRNA. Many
different AARS and tRNA/ tRNA aminoacylase pairs can be
created to form new UAAs.
This diagram shows the orthogonal nature
of the AARS and the attachment of an
artificial amino acid (x) to a stop codon
tRNA.
02/08/2013
UNNATURAL AMINO ACIDS
Louie Iselin
Quadruplet codons
Another method that scientists have experimented with is creating codons which are 4 letters long
instead of the natural 3. If mastered, this would dramatically expand the genetic code, adding a
potential of 200 new amino acids that could serve a variety of purposes. Using quadruplet codons
allows us to start with a completely blank canvas, rather than trying to work with what we have when
it comes to the stop codons. The process could also be quicker and more efficient because you could
potentially have more than one artificial amino acid being produced at once. There are downsides
however. Unlike using the stop codon, orthogonal ribosomes are required to prevent the reading of 3
letter combinations within the 4 letter codons which is what could happen if we were to try carrying
out the process in a natural ribosome. The amino acids cannot easily be incorporated into a regular
protein because orthogonal ribosomes do not recognise natural, 3 letter codons other than the amber
stop codon which is what the ribosome codes the amino acid in response to. There are still many
functions for these amino acids because artificial peptides can be formed to have specific functions
from strings of 4 letter codons and, in future, the technology could be improved to incorporate them
into the genetic code itself.
To create an amino acid for a codon made up of 4 letters a certain type of ribosome is needed. The
ribosomes used are called orthogonal ribosomes and detect only certain types of heterologous
mRNAs, not the ones coming from the cell itself. The ribosome found to be the most productive in
this process is the Ribo-Q orthogonal ribosome, which was created by a process of artificial evolution
by saturation mutagenesis. This means that researchers created all possible mutations within a small
stretch of DNA. In this case the 16s rRNA of Ribo-X was used as the starting point because it had
already been evolved to efficiently decode the amber stop codon which is what is necessary in
quadruplet decoding and they created the most efficient ribosome possible through the use of the
reporter construct AAGA. The resulting orthogonal ribosome was of a similar efficiency to the natural
triplet-decoding ribosomes. It included two main mutations: A1196G and A1197G. These enabled the
ribosome to more efficiently accept and translate tRNA with a 4-letter anticodon.
For amino acid synthesis to occur in these orthogonal ribosomes the orthogonal set is also required.
This is composed of the tRNA, the aminoacyl-tRNA synthetase and the codon itself. In this case, the
tRNA/ tRNA aminoacyl pair for lysine from the archaebacterium P. horikoshii can be (and has been)
02/08/2013
UNNATURAL AMINO ACIDS
Louie Iselin
used. The pair is only in contact with lysine at two points: Glu 41 and Tyr 268 so these could simply
be mutated to stop incorporating lysine and instead take up an artificial amino acid and transport it to
the orthogonal ribosome to be added in response to orthogonal mRNAs or a codon inserted into the
organism on a plasmid. This has been done with the codon AGGA, which was placed in position 24
on the gene for myoglobin in yeast and coded successfully for the required amino acid:
homoglutamine.
Functions and applications for unnatural amino acids
There are many ways that scientists hope to harness unnatural amino acids in the future. These vary
from posttranslational modifications to spectroscopic probes and, although the technology is still very
new, is certainly a viable option when considering certain questions faced by the scientific
community.
Posttranslational modifications
Artificial amino acids can be incorporated into proteins following translation. This has been
successfully tried in E coli where they inserted the unnatural amino acid 3-axidotyrosine using the
amber stop codon and the tRNA/ mutated tyrosyl-tRNA synthetase pair. With the unnatural amino
acid placed at certain points, modifications could take place.
Photo reactive probes
Amino acids can now be used to regulate cellular activity and the activity of individual proteins and
pathways within that cell. This has been done in various different ways. Photodynamic regulation of
proteins such as the proapoptotic cysteine protease, caspase 3, has been achieved through the sitespecific incorporation of photocaged amino acids (o-nitrobenzyl cysteine in this case). This has also
been done with the Pho4 transcription factor in yeast, which had its phosphorylation blocked by a
photocaged serine. Irradiation with blue light uncaged the serine and allowed the phosphorylated
Pho4 to be followed by way of its nuclear export. Proteins can now also be cross-linked so they can
be linked to their interacting pair within the cell /and can be cleaved in the presence of light. Onitrophenylaline can site-specifically cleave a protein through a light-induced mechanism.
Protein evolution
Unnatural amino acids have been found to actually increase the productivity of proteins in some
situations, in particular in the example of CDR3H (complementary determining region 3 heavy chain).
This is an antibody and, in in vitro selection, the strains containing boronate or sulfotyrosine actually
performed better in their respective tasks. These amino acids could incorporate whole new sites and
aspects into cells that could increase both efficiency and functionality.
Metal Chelation
Amino acids can be altered to chelate metals. These could be used in a new generation of
metallaproteins (proteins that can bind to metal ions) which could be used in chelation therapy and in
transportation and storage of metals such as copper. One process that has been successfully attempted
was to introduce a Cu2+ -chelating amino acid that could site specifically cleave DNA.
Where next?
There is an ongoing effort among the scientific community to continue the genetic code expansion.
This requires both new amino acids and new orthogonal sets to allow synthesis to occur. Work is also
being done to try and extend this expansion beyond E coli and other simple organisms into C Elegans
and eventually even mice.