The central dogma of microbiology is an underappreciated system that governs every living organism; The dogma is the process of converting DNA into RNA and then into proteins. This system can be broken down into three completely different processes, DNA replication, transcription, and translation. The first main component that separates these systems apart is for the fact that DNA replication involves making double-stranded (ds)DNA. More specifically, the process of replication involves the production of two identical double stranded nucleic acids (Aryal, 2018). On the other hand, the two processes of transcription and translation do not involve dsDNA. Transcription and translation involve single-stranded (ss)DNA. Transcription is the process of converting the information derived from DNA and turning it into messenger (m)RNA (Aryal, 2018). Finally, translation involves the creation of proteins from using the information that is taken from the mRNA.

Understanding the central dogma of molecular biology aids in the ability to comprehend DNA nucleotides, which make up DNA and thus the flow of information. The normal polar nucleotides that are found in nature are adenine (A), thymine (T), cytosine (C), and guanine (G). The four naturally found nucleotides, which are the universal alphabet for every living organism here on Earth, have strict rules that govern their molecular laws. Only A can bind to T, and G can only bind to C. The reasoning for this strict rule is through the hydrogen bonds that are formed join the two strands and stabilize the molecule, which allows it to form the ladder-like double helix (Walsh, 2019). However, only until recently, there are six known nucleotides that have been documented. These new non-polar nucleotides—deoxy 5SICS and deoxy NaM—have been scientifically developed and show great promise in the world of technology, specifically biotechnology (Hirao and Kimoto, 2012). What is known about these unnatural nucleotides is that they are hydrophobic nucleotides that chemically bond with one another using hydrogen binding, the same as the natural nucleotides, as well as only bonding with each other and not with A, T, C, or G. These unnatural base pairs are not stable and have three phosphates attached to them. These three phosphates attached causes them to move slower compared to one with two phosphates. Furthermore, these molecules degrade a lot faster. The promise of utilizing these two new nucleotides is offering the ability of scientists to be able to work with DNA with more control, for this system allows DNA to reproduce as a trinary instead of a binary grouping. These two nucleotides are also considered a lot more safe, when playing with genetic code, for organisms that are introduced with these two new unnatural nucleotides are not able to replicate without a constant supply of the chemicals that make up all three pairs; without a constant supply of these chemicals needed, the organism would revert right back to its natural state (Diwo and Budisa, 2018). 

Unnatural base pairs (UBP) are chemically synthesized in the lab, these deoxy 5SICS (Y) and deoxy NaM (X) nucleotides have never been seen before by living organisms on this planet. These unnatural nucleotides therefore need to be introduced alongside varying mechanisms to be able to have the end result of these UBP within the cell to be expressed. The exploration of X and Y nucleotides in the bacteria E. coli. is researched and the data obtained is compiled together in the article, A semi-synthetic organism with an expanded genetic alphabet, by the group of scientists: Malyshev, Dhami, Lavergne, Chen, Dai, Foster, Corrêa, and Romesberg. The actual result that allowed for these UBP to enter a cell was through the inclusion of two main mechanisms: including a transport protein (PtNTT2) and through using electroporation. The inclusion of a transport protein is needed as shown by figure 2D—With a transporter protein there is tenfold increase of intracellular levels of X and Y nucleotides as compared to one without the transporter protein (Malyshev, Dhami, Lavergne, Chen, Dai, Foster, Corrêa, and Romesberg, 2014). The inclusion of using electroporation is shown through figure 2C; electroporation is a mechanism that simply shocks bacteria with electricity at a low current causing the bacteria to be permeable, especially for the when both the triphosphates of X and Y were present with the chemical IPTG. IPTG is simply a chemical that stimulates the pACS to express the transporter protein (Malyshev, Dhami, Lavergne, Chen, Dai, Foster, Corrêa, and Romesberg, 2014). Meanwhile, there are problems that arise with attempting to get the UBP into the cell and thus scientists had to figure out a way of combating this. The problem with including UBP is having the bacteria to take up synthetic DNA and the actual placement of X and Y into the cell. The solution for this problem of including PtNTT2, is the scientists needed to include two plasmids: pACS which codes for the required transporter protein and pINF which is the template DNA strand that has both the X and Y nucleotides (Malyshev, Dhami, Lavergne, Chen, Dai, Foster, Corrêa, and Romesberg, 2014). Once the UBP was in the cell, how scientists were able to determine that the X and Y nucleotides were actually present in the cell, was through keeping the bacteria in an antibiotic media with streptomycin (SM) and amoxicillin (AmpR). The bacteria that survived the medium proved the UBP had entered the cell. The survival of antibiotics only arises through the successful addition of the SM antibiotic resistant gene proving this mechanism actually worked for the bacteria were able to survive the antibiotics.

Once researchers were able to get the UBP into the cell, they had used methods to test for determining if the bacteria were able to hold onto this newly introduced plasmid and actually replicate it. The group of scientists found that the bacteria were able to replicate these new nucleotides in their cells through two different methods: using PCR and through sequencing technologies. In PCR, one just simply looks at the bands that arise and then compares the bands. When the PCR reaction works, then there will be seen to have more copies of isolated plasmids after than compared to before the reaction. If the bacteria actually grew and divided, there will be more plasmids than that which was originally present. More base pairs means DNA is replicated which is shown by figure 2E, for the figure shows a band with more base pairs in the one case there was both the transporter chemical and the X and Y base pairs with streptavidin present—there were a lot more base pairs present after the reaction. Furthermore, sequencing showed that UBP were present after replication because with the presence of UBP sequencing stopped (Malyshev, Dhami, Lavergne, Chen, Dai, Foster, Corrêa, and Romesberg, 2014).

The inclusion of these nucleotides are therefore proven to cause no harm for the bacteria were able to continue replication and survive a medium that otherwise would have killed them. Having the bacteria accept the plasmid, researchers were able to determine if the plasmid caused any problems and how long the bacteria were able to actually hold onto the plasmid. These UBP actually have no damaging effects on the bacteria, for Figure 2C shows bacterial growth. Having bacterial growth proves these UBPs do not harm the bacteria because there is bacterial growth. In figure 2C, the OD600 value shows there is bacterial growth (Malyshev, Dhami, Lavergne, Chen, Dai, Foster, Corrêa, and Romesberg, 2014). A higher OD600 value means there is more bacterial growth which is caused from it being more cloudier. The bacteria in the experiment were able to deal with the X and Y base pairs as long as they were present. The bacteria continued to divide for 24 generations as shown by figure 3. After the 24 generations, the UBP simply just becomes lost and are replaced with A and T (Malyshev, Dhami, Lavergne, Chen, Dai, Foster, Corrêa, and Romesberg, 2014).

The newly scientifically developed nucleotides, X and Y, show huge potential in medically related fields. The deoxy 5SICS and deoxy NaM nucleotides are unstable by nature and have strict guidelines for the conditions they are able to be present under, making them a safe tool to be used in combating the on-going war between humans versus the universal genetic code. The article, A semi-synthetic organism with an expanded genetic alphabet, relates Bio 293 course material such as the transport system of cells and gene expression to the real world application of fighting antibiotic resistance.

References:

Aryal, S. (2018). Central Dogma- Replication, Transcription, Translation. Microbe Notes, https://microbenotes.com/central-dogma-replication-transcription-translation/.

Hirao, I. and Kimoto, M. (2012). Unnatural base pair systems toward the expansion of the genetic alphabet in the central dogma. Proceedings of the Japan Academy Series B Physical and biological sciences, 88, 345–367.

Walsh, E. (2019). What Is the Complementary Base Pairing Rule? Sciencing, https://sciencing.com/complementary-base-pairing-rule-8728565.html.

Diwo, C. and Budisa, N. (2018). Alternative Biochemistries for Alien Life: Basic Concepts and Requirements for the Design of a Robust Biocontainment System in Genetic Isolation. Genes, 10, 1-16.

Malyshev, D.A., Dhami, K., Lavergne, T., Chen, T., Dai, N., Foster, J.M., Corrêa, I.R., and Romesberg, F.E. (2014). A semi-synthetic organism with an expanded genetic alphabet. Nature 509, 385-388.