Manipulating the Genetic Alphabet – Molecular Biology

By Easha Vigneswaran

Biology is governed by one of the key laws that illustrate the idea that the genetic code consists of four nucleotide bases where adenine bonds with thymine and guanine with cytosine. For years, synthetic biologists have theorised and tested the possibility of increasing the number of DNA bases. In the last few years, numerous biotechnological companies have worked to synthesise these molecules and this article aims to discuss a few of the discoveries.

The four nucleotide bases are essential for the storage and transfer of DNA from one generation to the next across evolutionary history (Hoshika et al., 2019). DNA, as described by Watson and Crick, is composed of a double helix joined by hydrogen bonds between the 4 different bases in pairs (Watson & Crick, 1953). Studies into the hydrogen base-pairing have shown that it isn’t necessary for Darwinian molecular evolution, provided the general size of the molecules is the same (Hoshika et al., 2019).

Synthetic biologists at the Scripps Research Institute in California tested this theory and successfully engineered a part synthetic organism that can store genetic information in the form of DNA consisting of six different nucleotide bases (Zhang et al., 2017). The Romesberg Lab that undertook the research wanted to show the possibility of creating an organism that exhibits unnatural base-pairing inside living cells. They developed a base-pair termed X:Y formed between two lab synthesised nucleotides. The development of these nucleotides took over 14 years and required many trials and copies of the X and Y base pairs in order to find one form that was accurately replicated by the DNA polymerases (Weaver, 2018).

The bases were replicated in an Escherichia coli model which was also provided with a transporter protein to uptake the extracellular bases. Initially, the unnatural base pairs’ presence didn’t correlate with the growth of the E. coli model suggesting there was likely an issue with replication or base uptake. Upon amendments to their methods, the researchers were able to show the bacteria used these newly synthesised base pairs. These bacterial models also showed evidence of DNA that did not contain the new bases. The CRISPR-Cas9 technology was used to eliminate normal DNA sequences and retain the non-normal ones that incorporated the unnatural bases. This ultimately resulted in the formation of their new “semisynthetic organism” that is able to successfully grow and store a larger amount of genetic information. This method of increasing the genetic information stored by organisms was described as inexpensive and only requires a small number of biological molecules and systems to successfully achieve an outcome (Weaver, 2018).

Another group of scientists have also created an expanded genetic alphabet by synthesizing 4 artificial bases giving 8 bases in total thereby creating hachimoji DNA (“hachi” = eight and “moji” = letter). Adenine and Guanine are both purines and the lab created artificial equivalents are P and B; the pyrimidines (cytosine and thymine) artificial bases were Z and S. These bases formed the pairs as P:Z and B:S (Hoshika et al., 2019). These four artificial bases, in addition to the normal bases, formed molecular structures that resembled that of normal DNA. The main issue with the hachimoji DNA is that normal DNA enzymes were unable to transcribe and translate it into proteins. However, when the researchers created mutated versions of the normal DNA enzymes, the hachimoji bases were successfully transcribed (Agarwal, 2019).

Interestingly evidence of the expansion of the genetic alphabet can be found occurring naturally in the genome of viruses. An example is adenine being substituted by 2-aminoadenine (Z). The DNA formed from the ZTCG bases is dZ-DNA where Z:T bases pairs form three hydrogen bonds, which is one more than A:T base pairs. Due to the greater number of hydrogen bonds, dZ-DNA shows greater thermal stability. dZ-DNA also shows greater accuracy during binding and resistance to degradation from nuclease activity. For some viruses e.g., bacteriophages, this nuclease resistance proves beneficial. Bacteriophages replicate by injecting their genetic material into host cells which will then synthesise the viral proteins. Host cells protect themselves by using nucleases to break down viral DNA. However, dZ-DNA nuclease resistance may have been an advantageous evolutionary mechanism to allow persistence of these viruses in toxic environments (Grome & Isaacs, 2021).

At present, the existence of “semisynthetic organisms” has no realized applications but there is a possibility that in the future these functional unnatural bases could be transcribed and translated into new amino acids and proteins used in therapeutics. The existence of non ATCG bases could provide answers to the possibility of life outside of the one we know. If the building blocks of life don’t necessarily need to be comprised of the canonical bases, there is a possibility to create life from unnatural/non-normal bases that remain undiscovered.


Hoshika, S., Leal, N. A., Kim, M., Kim, M., Karalkar, N. B., Kim, H., Bates, A. M., Watkins, N. E., Santa Lucia, H. A., Meyer, A. J., Das Gupta, S., Piccirilli, J. A., Ellington, A. D., Santa Lucia, J., Georgiadis, M. M. & Benner, S. A. (2019) Hachimoji DNA and RNA: A genetic system with eight building blocks. Science (New York, N.Y.). 363 (6429), 884-887. Available from: doi: 10.1126/science.aat0971.

Watson, J. D. & Crick, F. H. C. (1953) Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid. Nature. 171 (4356), 737-738. Available from: Available from: doi: 10.1038/171737a0.

Zhang, Y., Lamb, B. M., Feldman, A. W., Zhou, A. X., Lavergne, T., Li, L. & Romesberg, F. E. (2017) A semisynthetic organism engineered for the stable expansion of the genetic alphabet. Proceedings of the National Academy of Sciences of the United States of America. 114 (6), 1317-1322. Available from: Available from: doi: 10.1073/pnas.1616443114.

Weaver, J. (2017) Expanding the genetic alphabet. BioTechniques. 62 (6), 252-253. Available from: Available from: doi: 10.2144/000114554.

Agarwal. R (2019) Scientists create an expanded 8-letter DNA genetic code. Science in the News. -02-27T18:00:16+00:00. Available from:

Grome, M. W. & Isaacs, F. J. (2021) ZTCG: Viruses expand the genetic alphabet. Science (New York, N.Y.). 372 (6541), 460-461. Available from: doi: 10.1126/science.abh3571.

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