By Elaine To
In biology classes, everybody is taught that deoxyribonucleic acid (DNA, AKA the genetic information of a cell) has four and only four nucleotide bases. Adenine (A) and thymine (T) base pair together and cytosine (C) and guanine (G) base pair together. For the first time ever, researchers have expanded the genetic alphabet to include two additional bases: dNaM (X) and d5SICS (Y).
The researchers have previously shown that DNA polymerases, the enzymes responsible for replicating DNA, successfully replicate DNA containing the dNaM-d5SICS base pair. However these reactions were not carried out within living cells. The researchers decided to try this in the bacterium Escherichia coli due to the simplicity of the cells. Multiple factors had to be optimized in preparation for carrying out these reactions inside cells.
Firstly, the unnatural bases must be present inside the bacteria for DNA polymerase to use them as raw materials. Cells normally obtain A, C, T, and G from breaking down food or recycling previously used nucleotides. Both these pathways were not options for X and Y, so the researchers first tried passive diffusion across the cell membrane. Once X and Y diffused into the cell, they could then be phosphorylated by naturally occurring enzymes to their triphosphate form, which is the form that DNA polymerases recognize and use. The phosphorylation was unsuccessful.
The researchers then explored the idea of transporting the triphosphate forms (XTP and YTP) directly into the cells. Uptake of XTP and YTP by nucleotide triphosphate transporters from multiple other species was screened. The PtNTT2 transporter from the diatom Phaeodactylum tricornutum was most efficient at bringing XTP and YTP into the cells.
The next issue was the instability of XTP and YTP in the culture medium, especially when the E. coli were actively growing. Tests were first carried out on the natural triphosphate ATP. It was determined that addition of KPi to the culture medium increased ATP stability significantly and that KPi had the same effect on XTP and YTP.
And with that, the researchers were ready to generate their E. coli organism containing X and Y. They prepared two circular pieces of DNA, known as plasmids, which are easy to transport into bacteria. One plasmid contained the gene for the PtNTT2 transporter and the other contained a gene with an A-T base pair replaced by X and an analog of Y. Since YTP is the provided substrate, any newly produced plasmid will contain X and Y. This distinguishes it from the original template plasmid containing X and the Y analog.
After inserting both plasmids into the bacteria and growing them in KPi, XTP, and YTP containing medium, the plasmids were extracted from inside the cells. Analyzing the total nucleotide content with mass spectrometry showed that Y was clearly present. X was not detected, but it is known to fragment poorly and thus be difficult to detect with mass spectrometry.
To check the incorporation of XTP and YTP into the extracted plasmid, it was replicated in a PCR reaction using the natural nucleotides, YTP, and biotinylated XTP as substrates. The new product should contain biotin and thus react with streptavidin, which binds very strongly to biotin. As expected, streptavidin bound to the PCR product, confirming that the X-Y base pair is in the plasmid.
Sequencing of the plasmid shows that the nucleotide sequence is correct up until the expected location of the X-Y base pair. The sequencing reaction terminates at this location because there is no X nor Y provided in the sequencing reagents. This proves that X-Y is present in the right location in the plasmid.
In a series of landmark experiments, the researchers have shown replication of DNA containing an unnatural base pair inside living cells. The next step to be undertaken is the transcription of this DNA to mRNA and then hopefully translation into a functional protein. It is conceivable that the incorporation of X-Y into mRNA will soon transpire due to the similarity of DNA and RNA. Subsequently, work that has already been done in incorporating unnatural amino acids could be leveraged to facilitate the use of X-Y in codons that result in proteins.