Artificially Expanded Genetic Information System

Artificially Expanded Genetic Information System (AEGIS) is a synthetic DNA analog experiment that uses some unnatural base pairs from the laboratories of the Foundation for Applied Molecular Evolution in Gainesville, Florida, especially the Steven A. Benner lab. AEGIS is a NASA-funded project to try to understand how extraterrestrial life may have developed.[1] In a 2024 article from the same laboratory, the concept has been broadened into anthropogenic evolvable genetic information systems, still with the same acronym.[2]

Hachimoji DNA is a strict subset of this system and comes from the same laboratory.[3]

Bases

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The system uses 12 to 14 different nucleobases in its genetic code, adding four types of base pairs on top of the two natural Watson-Crick base pairs.[1][4][5][6][7]

List of AEGIS base pairs (natural in highlight)
Purine Pyrimidine
Name Abbr. Structure Structure Abbr. Name
Adenine A
  • T
  • pyADA
 Thymine
  • AminoA
  • DAP
  • puDAD
Guanine
  • G
  • puADD
  • C
  • pyDAA
 Cytosine
Isoguanine
  • iG
  • B
  • puDDA
  • iC
  • rS
  • pyAAD
 Isocytosine
  • dS
  • pyAAD
 1-Methylcytosine
Xanthine
  • X
  • puADA
  • K
  • pyDAD
 2,4-Diaminopyrimidine
5-Aza-7-deazaguanine
  • P
  • puAAD
  • Z
  • pyDDA
 6-Amino-5-nitropyridin-2-one
4-Aminoimidazo[1,2-a][1,3,5]triazin-2(1H)-one
  • J
  • puDAA
  • V
  • PyADD
 6-Amino-3-nitropyridin-2-ol

Names such as "pyADA", "puDAD" belong to an AGEIS-specific system of denoting nucleobases. PyADA means that the base is a pyrimidine, and from top (5') to down (3') the hydrogen-bonding behavior is acceptor, donor, acceptor. PuDAD means the base is a purine with donor-acceptor-donor pattern. Under this system, all pairs form three hydrogen bonds.[8]

The three-bond system contains considerable flexibility for further modification of nucleosides. Functional groups can be added, removed, or replaced on the non-bonding side of the nucleobase without affecting bonding, much like how uridine (thymine without a methyl group) bonds like thymine in the natural genetic system. The original (1998) formulation only anticipated the possibility of replacing groups on pyAAD (dS), pyADA (T), pyADD (Z), pyDAA (V), and pyDAA (C),[8] but in Benner's 2014 paper all twelve types of bases have one site for group replacement.[9] Benner also indicates in a 2012 report for the DITC that all six purine bases have a second site for attaching another functional group.[10]

In addition, the purine and pyrimidine rings can be replaced with any ring with a similar size. This may prove to be advantageous when reduced tautomerization or some other form of additional stability is needed. For example, 6-amino-5-benzyl-3-methylpyrazin-2-one can replace isoguanine.[11]

Non-canonical bonding

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Like natural nucleobases, AEGIS bases can form non-canonical bonds. For example, B can pair with T by tautomerization, Z can pair with G at low pH (protonated hydrogen bond), and P can pair with C at low pH (also protonated). A DNA polymerase without access to the unnatural nucleobases would perform these pairings, causing bases to be replaced.[9]

An analogue of the G·U wobble base pair can be formed between aminoA and 5-methylisocytidine (m5iC).[12]

In 2021, it was found that isoguanine (B) can also base-pair with guanine (G) and 5-aza-7-deazaguanine (P) when put in DNA. The purine-purine base pair requires more space than the typical purine-pyrimidine base pair (the natural Watson-Crick A-T C-G pairs and the designed P-Z B-S pairs are all of these type), but the large groove of the DNA double helix provided enough space for this to happen. This "wider" base pair actually enhances the stability of DNA.[13]

Hoogsteen base pairing results in the formating of triplets in nucleic acid tertiary structure.[14]

History

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The Benner Lab reported in 1987 that it had expanded the number of bases available for making RNA in an article about the RNA world hypothesis. It is unclear whether the whole AEGIS six-pair arrangement had been planned out by then because the text was not accessible to the author of this article.[15]

The Benner Lab reported in 1989 that the T7 RNA polymerase and the Klenow fragment, in their unmodified forms, can insert an isoguanine (iG) residue when guided by a DNA template containing isocytidine (iC). The study was funded by the Swiss government and Sandoz AG, not by NASA.[16]

A 1990 Benner Lab article reports that T7 RNA polymerase and the Klenow fragment are able to insert 2,4-diaminopyrimidine (kappa, κ) opposite xanthosine (X). The T7 RNA polymerase could insert X opposite of κ but the Klenow fragment could not. This research was also funded by Swiss organizations. It also includes the six-base-pair arrangement of AEGIS, unlike the earlier article.[17]

An 1992 cooperation with Jim Bain and Dick Chamberlin (UC Irvine) produced an example of pyAAD in mRNA and puDDA in tRNA. The result can be translated using a standard ribosome to incorporate the non-standard amino acid iodotyrosine. When the tRNA is absent, the ribosome exhibits a frame shift and continues translating. This is the first study with any funding provided by the US government as one of the authors (Christopher Switzer of UC Riverside) was funded by a NSF fellowship.[18]

A 1998 article from the Benner Lab was funded by the Swiss National Science Foundation, the Danish Natural Science Research Council, the National Institutes of Health, and the Office of Naval Research. It provides an overview of Benner's research so far.[8]

The term "AEGIS" appeared in a short 2003 paper from the Benner Lab which explicitly mentioned a possible connection with extraterrestrial life and NASA. This paper mainly reports on the modification of the HIV reverse transcriptase to incorporate pyDAD (K)-puADA (X) with higher fidelity than earlier solutions. It also claims that AEGIS has been used in FDA-approved commercial tests for HIV and hepatitis C as well as a detection tool for SARS virus but does not mention them by name.[5] The paper was expanded upon by a full length 2004 article.[19] The 2004 article also cites the 1997 research paper for branched DNA assay supported by the Aaron Diamond AIDS Research Center.[20]

Besides interaction with biological enzymes and other systems, the Benner Lab also works to refine the production route and the structure of the bases themselves. For example, the synthesis of Z and P was only reported in 2006[4] and dS was only discovered in 2009.[21]

Benner Lab's research made the headlines in 2019, when it was published under a catchy name "Hachimoji DNA". The paper did not mention AEGIS. The system used a "FAL" variant of T7 RNA polymerase and produced an RNA with secondary structure. It also showed that AEGIS bases do not distort DNA structure outside natural ranges.[22]

Despite the notoriety, the Benner Lab soon stopped using the "Hachimoji" name, preferring to instead keep their options open among all bases designed for AEGIS. In a 2023 article, the group reports on a way to synthesize DNA with all 12 bases of AEGIS using commercially available enzymes, which would decrease the cost of producing them compared to the chemical method. It also describes a way to apply nanopore sequencing to 12-base DNA.[23]

In a 2024 article from the same laboratory, the concept has been broadened into "anthropogenic evolvable genetic information systems", still with the same acronym.[2]

See also

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References

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  1. ^ a b Lloyd, Robin (February 14, 2009). "New Artificial DNA Points to Alien Life". LiveScience. Retrieved 5 July 2016.
  2. ^ a b Wang, B; Kim, HJ; Bradley, KM; Chen, C; McLendon, C; Yang, Z; Benner, SA (25 December 2024). "Joining Natural and Synthetic DNA Using Biversal Nucleotides: Efficient Sequencing of Six-Nucleotide DNA". Journal of the American Chemical Society. 146 (51): 35129–35138. doi:10.1021/jacs.4c11043. PMID 39625448.
  3. ^ Hoshika S, Leal NA, Kim MJ, Kim MS, Karalkar NB, Kim HJ, Bates AM, Watkins NE, SantaLucia HA, Meyer AJ, DasGupta S, Ellington AD, SantaLucia J, Georgiadis MM, Benner SA (February 2019). "Hachimoji DNA and RNA: A genetic system with eight building blocks". Science. 363 (6429): 884–887. Bibcode:2019Sci...363..884H. doi:10.1126/science.aat0971. PMC 6413494. PMID 30792304.
  4. ^ a b Yang, Z.; Hutter, D.; Sheng, P.; Sismour, A. M.; Benner, S. A. (29 October 2006). "Artificially expanded genetic information system: a new base pair with an alternative hydrogen bonding pattern". Nucleic Acids Research. 34 (21): 6095–6101. doi:10.1093/nar/gkl633. PMC 1635279. PMID 17074747.
  5. ^ a b Benner, SA; Hutter, D; Sismour, AM (1 September 2003). "Synthetic biology with artificially expanded genetic information systems. From personalized medicine to extraterrestrial life". Nucleic Acids Research. Supplement. 3 (3): 125–6. doi:10.1093/nass/3.1.125. PMID 14510412.
  6. ^ Benner, Steven A. (December 2010). "Defining Life". Astrobiology. 10 (10): 1021–1030. Bibcode:2010AsBio..10.1021B. doi:10.1089/ast.2010.0524. PMC 3005285. PMID 21162682.
  7. ^ Klotz, Irene (February 27, 2009). "Synthetic life form grows in Florida lab". Science. Archived from the original on January 13, 2016. Retrieved 5 July 2016.
  8. ^ a b c Benner, SA; Battersby, TR; Eschgfaller, B; Hutter, D; Kodra, JT; Lutz, S; Arslan, T; Baschlin, DK; Blattler, M; Egli, M; Hammer, C; Held, HA; Horlacher, J; Huang, Z; Hyrup, B; Jenny, TF; Jurczyk, SC; Konig, M; von Krosigk, U; Lutz, MJ; MacPherson, LJ; Moroney, SE; Muller, E; Nambiar, KP; Piccirilli, JA; Switzer, CY; Vogel, JJ; Richert, C; Roughton, AL; Schmidt, J; Schneider, KC; Stackhouse, J (February 1998). "Redesigning nucleic acids". Pure and applied chemistry. Chimie pure et appliquee. 70 (2): 263–6. doi:10.1351/pac199870020263. PMID 11542721.
  9. ^ a b Bradley, K. M.; Benner, S. A. (2014). "OligArch: A software tool to allow artificially expanded genetic information systems (AEGIS) to guide the autonomous self-assembly of long DNA constructs from multiple DNA single strands". Beilstein Journal of Organic Chemistry. 10: 1826–1833. doi:10.3762/bjoc.10.192. PMC 4142867. PMID 25161743.
  10. ^ Benner, Steve A (August 5, 2012). "Design Automation Software for DNA-based Nano-Sensor Architecture" (PDF). apps.dtic.mil.
  11. ^ Voegel, Johannes J.; von Krosigk, Ulrike; Benner, Steven A. (December 1993). "Synthesis and tautomeric equilibrium of 6-amino-5-benzyl-3-methylpyrazin-2-one. An acceptor-donor-donor nucleoside base analog". The Journal of Organic Chemistry. 58 (26): 7542–7547. doi:10.1021/jo00078a038.
  12. ^ Strobel, SA; Cech, TR; Usman, N; Beigelman, L (22 November 1994). "The 2,6-diaminopurine riboside.5-methylisocytidine wobble base pair: an isoenergetic substitution for the study of G.U pairs in RNA". Biochemistry. 33 (46): 13824–35. doi:10.1021/bi00250a037. PMID 7524665.
  13. ^ Zhang, A; Kondhare, D; Leonard, P; Seela, F (6 May 2021). "5-Aza-7-deazaguanine-Isoguanine and Guanine-Isoguanine Base Pairs in Watson-Crick DNA: The Impact of Purine Tracts, Clickable Dendritic Side Chains, and Pyrene Adducts". Chemistry (Weinheim an der Bergstrasse, Germany). 27 (26): 7453–7466. doi:10.1002/chem.202005199. PMC 8251886. PMID 33443814.
  14. ^ Jena, N. R.; Shukla, P. K. (2023). "Structure and stability of different triplets involving artificial nucleobases: clues for the formation of semisynthetic triple helical DNA". Scientific Reports. 13: 19246. Bibcode:2023NatSR..1319246J. doi:10.1038/s41598-023-46572-4. PMC 10630353.
  15. ^ Benner, SA; Allemann, RK; Ellington, AD; Ge, L; Glasfeld, A; Leanz, GF; Krauch, T; MacPherson, LJ; Moroney, S; Piccirilli, JA (1987). "Natural selection, protein engineering, and the last riboorganism: rational model building in biochemistry". Cold Spring Harbor symposia on quantitative biology. 52: 53–63. doi:10.1101/sqb.1987.052.01.009. PMID 2456885.
  16. ^ Switzer, Christopher; Moroney, Simon E.; Benner, Steven A. (October 1989). "Enzymatic incorporation of a new base pair into DNA and RNA". Journal of the American Chemical Society. 111 (21): 8322–8323. doi:10.1021/ja00203a067.
  17. ^ Piccirilli, JA; Krauch, T; Moroney, SE; Benner, SA (4 January 1990). "Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet". Nature. 343 (6253): 33–7. doi:10.1038/343033a0. PMID 1688644.
  18. ^ Bain, J. D.; Switzer, Christopher; Chamberlin, Richard; Benner, Steven A. (April 1992). "Ribosome-mediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code". Nature. 356 (6369): 537–539. doi:10.1038/356537a0.
  19. ^ Sismour, AM; Lutz, S; Park, JH; Lutz, MJ; Boyer, PL; Hughes, SH; Benner, SA (2004). "PCR amplification of DNA containing non-standard base pairs by variants of reverse transcriptase from Human Immunodeficiency Virus-1". Nucleic acids research. 32 (2): 728–35. doi:10.1093/nar/gkh241. PMID 14757837.
  20. ^ Collins, ML; Irvine, B; Tyner, D; Fine, E; Zayati, C; Chang, C; Horn, T; Ahle, D; Detmer, J; Shen, LP; Kolberg, J; Bushnell, S; Urdea, MS; Ho, DD (1 August 1997). "A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml". Nucleic acids research. 25 (15): 2979–84. doi:10.1093/nar/25.15.2979. PMID 9224596.
  21. ^ Kim, HJ; Leal, NA; Benner, SA (15 May 2009). "2'-deoxy-1-methylpseudocytidine, a stable analog of 2'-deoxy-5-methylisocytidine". Bioorganic & medicinal chemistry. 17 (10): 3728–32. doi:10.1016/j.bmc.2009.03.047. PMID 19394831.
  22. ^ Hoshika, S; Leal, NA; Kim, MJ; Kim, MS; Karalkar, NB; Kim, HJ; Bates, AM; Watkins NE, Jr; SantaLucia, HA; Meyer, AJ; DasGupta, S; Piccirilli, JA; Ellington, AD; SantaLucia J, Jr; Georgiadis, MM; Benner, SA (22 February 2019). "Hachimoji DNA and RNA: A genetic system with eight building blocks". Science (New York, N.Y.). 363 (6429): 884–887. doi:10.1126/science.aat0971. PMID 30792304.
  23. ^ Kawabe, Hinako; Thomas, Christopher A.; Hoshika, Shuichi; Kim, Myong-Jung; Kim, Myong-Sang; Miessner, Logan; Kaplan, Nicholas; Craig, Jonathan M.; Gundlach, Jens H.; Laszlo, Andrew H.; Benner, Steven A.; Marchand, Jorge A. (26 October 2023). "Enzymatic synthesis and nanopore sequencing of 12-letter supernumerary DNA". Nature Communications. 14 (1). doi:10.1038/s41467-023-42406-z.
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