Hachimoji DNA: The Eight-letter Genetic Code

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DNA is the biopolymer used by all living organisms on Earth to store and transmit genetic information. DNA is a mixed-sequence polymer of four nucleotides, each of which contains a deoxyribose sugar, a phosphate group, and a nitrogenous base. There are four types of nitrogenous bases, including adenine (A), thymine (T), cytosine (C), and guanine (G). In 1953, Watson and Crick proposed that DNA assembles into a double helix, with the two strands bound together by highly specific patterns of hydrogen bonding called Watson-Crick base pairs [1]. A forms a base pair with T and G forms a base pair with C. Hence, the ordering of nitrogenous bases encodes genetic information, and the complementary base pairing allows for reliable DNA replication from one generation to another.

In 1962, Alexander Rich suggested the possibility of a third unnatural base-pair (UBP), composed of isoguanine (isoG) and isocytosine (isoC), that interact with each other using a hydrogen bond pattern that differs from that of from natural base pairs [2]. However, it was not until the late 1980’s to early 1990’s that the Rappaport and Benner groups demonstrated the incorporation of UBPs by DNA polymerase [3,4]. The development of UBPs to expand the genetic code is still of great interest in many research groups today. For instance, transcription and translation in a semi-synthetic organism that uses UBPs to store genetic information have been developed and optimized by the Romesberg group in 2019 [5].

Recently, Hoshika and coworkers reported a genetic system that uses an 8-letter code called hachimoji DNA to store genetic information and can be transcribed into hachimoji RNA (from Japanese 八文字 hachimoji, “eight letters”) in a form of a fluorescent hachimoji RNA aptamer [6]. Hachimoji DNA contains four natural bases (A, T, C, G) and four extra unnatural bases (P, Z, S, B) where P forms a base pair with Z, and S forms a base pair with B. To assess whether hachimoji DNA can be used as an informational system, first the researchers need to confirm that hachimoji DNA can bind or unbind with another complementary strand as predicted. 94 hachimoji duplexes with varied sequences were experimentally tested to compare with theoretical predictions of how temperature would affect the structure of double-stranded hachimoji DNA. It was shown that this man-made DNA meets the requirement of predictable thermodynamic stability and can act as an informational system. In addition, the crystal structures of three different hachimoji duplexes were the same regardless of the composition.

Next, the researchers showed that the information encoded in hachimoji DNA can be transmitted, as it can be transcribed into hachimoji RNA. Using a specific type of RNA polymerase, all UBPs in hachimoji DNA can be recognized and incorporated ribonucleotides to reliably generate hachimoji RNA. The researchers showed that they could use this hachimoji DNA/RNA system to generate a hachimoji version of a fluorescent RNA aptamer called ‘Spinach’ that turns green when bound to a specific small molecule. Hachimoji spinach was shown to be as functional as spinach that was transcribed with natural DNA.

This study has shown that Hachimoji DNA/RNA system can act as an informational system, which can be used to expand the genetic code, increase data storage, and form new nanostructures. It also suggests that a genetic system does not have to be limited to 4 building blocks. The research in making unnatural base pairs is still developing, and the number of genetic letters keeps growing more and more.

  1. Watson, J.D., and Crick, F.H.C. (1953). Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid. Nature 171, 737–738.
  2. Rich, A. (1962). On the problems of evolution and biochemical information transfer. in: Kasha M. Puhlman B. Horizons in Biochemistry. Academic Press, 103-126.
  3. Rappaport, H.P. (1993). Replication of the base pair 6-thioguanine/5-methyl-2-pyrimidinone with the large Klenow fragment of Escherichia coli DNA polymerase I. Biochemistry 32, 3047–3057.
  4. Piccirilli, J.A., Benner, S.A., Krauch, T., Moroney, S.E., and Benner, S.A. (1990). Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet. Nature 343, 33–37.
  5. Feldman, A. W., Dien, V. T., Karadeema, R. J., Fischer, E. C., You, Y., Anderson, B. A., … Romesberg, F. E. (2019). Optimization of Replication, Transcription, and Translation in a Semi-Synthetic Organism. Journal of the American Chemical Society, 141(27), 10644–10653.
  6. Hoshika, S., Leal, N.A., Kim, M.-J., Kim, M.-S., Karalkar, N.B., Kim, H.-J., Bates, A.M., Watkins, N.E., Jr., SantaLucia, H.A., Meyer, A.J., et al. (2019). Hachimoji DNA and RNA: A genetic system with eight building blocks. Science 363, 884–887.

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