A small turn for DNA; a large twist for biology
If, like me, you thought that in cells, X and Y were reserved for chromosomal nomenclature, then think again, because there are some new bases on the block! Synthetic DNA bases dNaM and dTPT3 now claim the status of X and Y, respectively. This represents a major milestone in biology, adding two new ‘letters’ to the natural four-letter DNA code. What I’ll do in this article is to summarise the impressive feat that has been accomplished, and supply a bit of context regarding practical and ethical implications. For more information, you can read a good review (Callaway 2017) of the recent primary article (Zhang et al. 2017).
So, what is all the fuss about? Basically, the authors engineered in vitro a new DNA base pair, X and Y. But this isn’t big news. They went further and introduced the freshly made base pair into the wild-type gene for green fluorescent protein (GFP). Their aim, however, was to make the mutation as silent as possible. That is, they hoped that upon transferring the mutant GFP gene into a bacterial cell, the host cell wouldn’t pick up on the mutation, and would, therefore, use the mutant GFP gene as a template to produce messenger RNA (mRNA) (in a process termed transcription), and subsequently protein (by a process known as translation). Indeed, when they provided the host cell with the mutant GFP gene and extra copies of X and Y bases (to make mRNA), the cell successfully made mutant GFP mRNA. So far, so good.
But then the question comes, how can the host cell translate this mutant GFP mRNA into protein? The way translation works in a cell is amazing. Let me give a quick run through. Firstly, mRNA is ‘read’ in sequences of triplet base pairs (known as codons). For example, let’s say in the sequence of DNA we see ‘CAT’; not literally of course. As well as CAT, there are 63 other possible permutations of any codons containing the bases G, C, A or T (as shown in Figure 1).
Figure 1 – mRNA codons and their corresponding amino acids
When transcribed into mRNA, the DNA codon sequence CAT becomes the complementary sequence, which would be GTA. This ‘three-lettered code’ is recognised by a specific RNA molecule called transfer RNA (tRNA), as depicted in Figure 2. Now, here’s a fascinating piece of trivia for you; given the 64 possible 3-lettered codons within DNA/mRNA, how many corresponding amino acids would you expect? Well, to give you a clue, the answer is not 64. That would make sense, however, only 20 amino acids exist. As such, somehow nature has engineered redundancy into the system. Let’s take our example, GTA. If you check the bottom right corner of Figure 1, you will see that the mRNA codon GTA corresponds to the amino acid Valine. What is fascinating, and is illustrated in Figure 1, is that Valine is also coded by three other codons: GTT, GTC and GTG. As shown in Figure 2, the way translation of mRNA into protein works is that the cell contains specific tRNA molecules that on one of their sides recognise certain codons. And on their other end, they are bound to an amino acid. In this way, you can imagine that there are four types of ‘Valine tRNAs’ in our cells.
Figure 2 – tRNAs shuttling amino acids for translation of mRNA into protein
To recap, I’ve mentioned that the authors of the recent study managed to design two new DNA/mRNA base pairs, X and Y. They successfully made a mutant GFP gene that contains this base pair, replacing a codon TAC with AXC, and this was transcribed into mRNA once inserted into the host cell. Looking at Figure 1, you’ll notice that TAC codon is translated into the amino acid Tyrosine, however, there is no amino acid encoded by the codon AXC…obviously! How could there be? After all, X is a synthetic base not recognised by the endogenous cell. As such, it was necessary for the authors to create a special tRNA molecule that was able to recognise the AXC codon. They could then attach a ‘non-canonical amino acid’ (ncAA) to the other side of this alien tRNA; in this case, they attached a molecule called ‘PrK.’ Upon feeding the host cell with the alien tRNA and ncAA PrK, the incredible result was that the bacterial ribosome used these exogenous components to add PrK to the growing chain of amino acids, finally creating mutant GFP protein. This mutant protein was not functionally affected by the replacement of Tyrosine with PrK.
Overall, this study represents a remarkable feat within the fields of synthetic and molecular biology. Not only were the authors able to create new DNA/mRNA base pairs (X and Y) that could be read by RNA polymerase in bacteria in vivo, they were also able to introduce new information into the resultant protein. By linking an unnatural amino acid, PrK, to a tRNA that recognised the mutant codon AXC, the translated mutant GFP protein became partially synthetic. This study will no doubt pave the way for future efforts in therapeutic protein design.
So now that it has been possible to modify a pre-existing organism to include semi-synthetic components, what implications does this have?
For example, the statement below is taken from a review of a prior study on semi-synthetic life (Callaway 2014):
Hailed as a breakthrough by other scientists, the work is a step towards the synthesis of cells able to churn out drugs and other useful molecules. It also raises the possibility that cells could one day be engineered without any of the four DNA bases used by all organisms on Earth.
I agree with the first sentence of the paragraph, which essentially purports that we can, in a parasitic fashion, hijack life (the cell and its machinery) to produce proteins that we are interested in. In fact, this isn’t really abnormal at all given that viruses do exactly this.
However, I think we should be more careful before making the leap of faith that the author then proceeds to take in the second paragraph (italicised). The unspoken assumption here is that one day we will be able to engineer life from synthetic ingredients. The major roadblock, however, is that we don’t actually understand the cause of life. We’ve been very adept at breaking Humpty Dumpty (‘the cell’) into its component parts, but this hasn’t yielded a definition of life. That DNA is the ‘blueprint of life’ is an analogy that was adopted far too prematurely, and it is becoming apparent that organisms operate as a whole, and are not reducible to their parts. DNA is indeed a template, but its function is to serve the cell and the organism. No one puts it more elegantly than the Nobel Prize winner in physiology Barbara McClintock, in her 1983 award ceremony speech:
…DNA is a highly sensitive organ of the cell.
The key point here is to acknowledge that the organism is in control, and their molecular biological outfit follows suit. The ethical and practical considerations of this point are extraordinary, and indeed there are numerous discussions due to be had on this topic. It also begs the question, what is an organism?
I would very much like to open it up in future articles, especially in relation to Professor Denis Noble’s new book, Dance to the Tune of Life. It is becoming more and more evident that we are entering a new phase in understanding the theory of evolution, and as esteemed scientists start to come together, it is clear that this time it is not DNA, but the organism who comes first.
Callaway, Ewen. 2014. “First Life with ‘Alien’ DNA.” Nature News, May. https://doi.org/10.1038/nature.2014.15179.
Callaway, Ewen. 2017. “‘Alien’ DNA Makes Proteins in Living Cells for the First Time.” Nature 551 (7682):550–51.
Zhang, Yorke, Jerod L. Ptacin, Emil C. Fischer, Hans R. Aerni, Carolina E. Caffaro, Kristine San Jose, Aaron W. Feldman, Court R. Turner, and Floyd E. Romesberg. 2017. “A Semi-Synthetic Organism That Stores and Retrieves Increased Genetic Information.” Nature 551 (7682):644–47.