All life (that we know of) is built from the 4 nucleotides of DNA (Adenine, Guanine, Cytosine, Thymine and in RNA Uracil instead of Thymine), which provide the code for creating 22 amino acids, which are then combined into proteins. An important part of the process is the reading of the DNA code by RNA (to be precise transcription RNA, tRNA) before creating amino acids and proteins. All life is created by reading the nucleotides in groups of 3 ‘letters’ (ACG etc.), called codons. With 4 nucleotides in combinations of 3-letters per codon – that’s 64 possible combinations, more than enough to code the 22 amino acids. What if, instead of reading codons with groups of 3, they had groups of 4 letters (ACGT etc.)? This is no longer idle speculation.

A team of researchers from Jason Chin’s group at the Medical Research Council Laboratory of Molecular Biology, Cambridge (UK) have succeeded with a proof of concept demonstration for codons containing four nucleotides. Before going into the details of how this was done, a question: Why is it important? The question is (or should be) asked of every experiment, but here is a case where it begs another question – If Nature only needs codons with 3-letter nucleotides, why bother with 4-letter?

For one thing, it increases the number of nucleotide combinations to 256. From the point of view of synthetic biology, that’s 192 more blocks in the kit for building proteins – creating proteins that have never been seen before…and all that might entail. More immediately, the techniques used to create a codon of four nucleotides will have great impact on proteomics (the systemic study of proteins) because it presents a new and better way to conduct the experiments.

To achieve the new approach, the research team had to solve a number of difficult points: Proteins are made in small enclosures called ribosomes within a cell. Ribosomes receive instructions for manufacturing proteins from messenger RNA (mRNA) coming from DNA in the cell nucleus. The mRNA brings the instructions, normally, in 3-letter nucleotide codons. Since the ‘new’ codons can’t be used in the normal ribosomes, the researchers had to create a new type of ribosome and a new type of mRNA. They accomplished this by inserting genes into our old friend the bacteria E. coli that caused the creation of ‘extra’ ribosomes, so called orthogonal (independent) ribosomes or o-ribosomes. These o-ribosomes exist alongside the normal ribosomes and very importantly do not interfere with the creation of normal proteins needed by the cell. The extra ribosomes were also modified genetically so that they could accept the 4-letter nucleotide codons. Likewise altered tRNA was introduced into the o-ribosome, which would accept 4-letter codons. Finally, to introduce altered codons and their amino acid instructions, the E. coli mRNA was modified.

Once the machinery was in place, the team put it to the test. A normal 3-letter anti-codon, CTT in mRNA, would specify the amino acid phenylalanine. In turn this would be the tRNA codon AAG inside the ribosome. Shifting to a 4-letter codon, they decided to make an unnatural amino acid p-azido-l-phenylalanine. This was assigned – made up, since there are no naturally occurring 4-letter codes – mRNA AGGA, tRNA UCCU. This coding they ran through the o-ribosomes to produce a mutant form of the protein calmodulin, which uses p-azido-l-phenylalanine in its construction.

In essence, the 4-letter codon and the machinery to process it is a technology. At least for now there is no direct use within human cells, but that’s not the point. This is an enabling technology, one that will make it possible for an incredible number of new experiments in protein development, which will hopefully lead to better understanding of the microbiology of proteomics and epigenetics. It will also enable ‘a new laboratory space’ for creation of synthetic proteins – the building blocks of cells and living tissue. Dr. Frankenstein never had it so fundamentally good.