Xenobiology (XB) is a subfield of synthetic biology, the study of synthesizing and manipulating biological devices and systems. Xenobiology derives from the term xenos (Greek), which means "stranger, guest". Xenobiology describes a form of biology that is not (yet) familiar to science and is not found in nature. In practice it describes novel biological systems and biochemistries that differ from the canonical DNA-RNA-20 amino acid system (see the classical central dogma in molecular biology). For example, instead of DNA or RNA, XB explores nucleic acid analogues, termed Xeno Nucleic Acid (XNA) as information carriers. It also focuses on an expanded genetic code  and the incorporation of non-proteinogenic amino acids into proteins.
- 1 Difference between xeno-, exo-, and astro-biology
- 2 Aims of xenobiology
- 3 Scientific approach
- 4 Biosafety
- 5 Governance and Regulatory issues
- 6 See also
- 7 External links
- 8 References
Difference between xeno-, exo-, and astro-biology
Astro means star and exo means outside. Both exo- and astrobiology deal with the search for naturally evolved life in the Universe, mostly on other planets in Goldilocks zones. Whereas astrobiologists are concerned with the detection and analysis of (hypothetically) existing life elsewhere in the Universe, xenobiology attempts to design forms of life with a different biochemistry or different genetic code on planet Earth.
Aims of xenobiology
- Xenobiology has the potential to reveal fundamental knowledge about biology and the origin of life. In order to better understand the origin of life, it is necessary to know why life evolved seemingly via an early RNA world to the DNA-RNA-protein system and its nearly universal genetic code. Was it an evolutionary "accident" or were there constraints that ruled out other types of chemistries? By testing alternative biochemical "primordial soups", it is expected to better understand the principles that gave rise to life as we know it.
- Xenobiology is an approach to develop industrial production system with novel capabilities by means of enhanced biopolymer engineering and pathogen resistance. The genetic code encodes in all organisms 20 canonical amino acids that are used for protein biosynthesis. In rare cases, special amino acids such as selenocysteine, pyrrolysine or formylmethionine, can be incorporated by the translational apparatus in to proteins of some organisms. By using additional amino acids from among the over 700 known to biochemistry, the capabilities of proteins may be altered to give rise to more efficient catalytical or material functions. The EC-funded project METACODE, for examples, aims to incorporate metathesis (a useful catalytical function so far not known in living organisms) into bacterial cells. Another reason why XB could improve production processes lies in the possibility to reduce the risk of virus or bacteriophage contamination in cultivations since XB cells would no longer provide suitable host cells, rendering them more resistant (an approach called semantic containment)
- Xenobiology offers the option to design a ‘genetic firewall’, a novel biocontainment system, which may help to strengthen and diversify current bio-containment approaches. One concern with traditional genetic engineering and biotechnology is horizontal gene transfer to the environment and possible risks to human health. One major idea in XB is to design alternative genetic codes and biochemistries so that horizontal gene transfer is no longer possible. Additionally alternative biochemistry also allows for new synthetic auxotrophies. The idea is to create an orthogonal biological system that would be incompatible with natural genetic systems.
In xenobiology, the aim is to design and construct biological systems that differ from their natural counterparts on one or more fundamental levels. Ideally these new-to-nature organisms would be different in every possible biochemical aspect exhibiting a very different genetic code. The long-term goal is to construct a cell that would store its genetic information not in DNA but in an alternative informational polymer consisting of xeno nucleic acids (XNA), different base pairs, using non-canonical amino acids and an altered genetic code. So far cells have been constructed that incorporate only one or two of these features.
Xeno nucleic acids (XNA)
Originally this research on alternative forms of DNA was driven by the question of how life evolved on earth and why RNA and DNA were selected by (chemical) evolution over other possible nucleic acid structures. Two hypotheses for the selection of RNA and DNA as life's backbone are either they are favored under life on Earth's conditions, or they were coincidentally present in pre-life chemistry and continue to be used now. Systematic experimental studies aiming at the diversification of the chemical structure of nucleic acids have resulted in completely novel informational biopolymers. So far a number of XNAs with new chemical backbones or leaving group of the DNA have been synthesized, e.g.: hexose nucleic acid (HNA); threose nucleic acid (TNA), glycol nucleic acid (GNA) cyclohexenyl nucleic acid (CeNA). The incorporation of XNA in a plasmid, involving 3 HNA codons, has been accomplished already in 2003. This XNA is used in vivo (E coli) as template for DNA synthesis. This study, using a binary (G/T) genetic cassette and two non-DNA bases (Hx/U), was extended to CeNA, while GNA seems to be too alien at this moment for the natural biological system to be used as template for DNA synthesis. Extended bases using a natural DNA backbone could, likewise, be transliterated into natural DNA, although to a more limited extent.
Aside being used as extensions to template DNA strands, XNA activity has been tested for use as genetic catalysts. Although proteins are the most common components of cellular enzymatic activity, nucleic acids are also used in the cell to catalyze reactions. One study found several different kinds of XNA, most notably FANA (2'-fluoroarabino nucleic acids), as well as HNA, CeNA and ANA (arabino nucleic acids) could be used to cleave RNA during post-transcriptional RNA processing acting as XNA enzymes, hence the name XNAzymes. FANA XNAzymes also showed the ability to ligate DNA, RNA and XNA substrates. Although XNAzyme studies are still preliminary, this study was a step in the direction of searching for synthetic circuit components that are more efficient than those containing DNA and RNA counterparts that can regulate DNA, RNA, and their own, XNA, substrates.
Expanding the genetic alphabet
While XNAs have modified backbones, other experiments target the replacement or enlargement of the genetic alphabet of DNA with unnatural base pairs. For example, DNA has been designed that has - instead of the four standard bases A,T,G, and C - six bases A, T, G, C, and the two new ones P and Z (where Z stands for 6-Amino-5-nitro3-(l'-p-D-2'-deoxyribofuranosyl)-2(1H)-pyridone, and P stands for 2-Amino-8-(1-beta-D-2'-deoxyribofuranosyl)imidazo[1,2-a]-1,3,5-triazin-4 (8H)). In a systematic study, Leconte et al. tested the viability of 60 candidate bases (yielding potentially 3600 base pairs) for possible incorporation in the DNA.
In 2002, Hirao et al. developed an unnatural base pair between 2-amino-8-(2-thienyl)purine (s) and pyridine-2-one (y) that functions in vitro in transcription and translation toward a genetic code for protein synthesis containing a non-standard amino acid. In 2006, they created 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) as a third base pair for replication and transcription, and afterward, Ds and 4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole (Px) was discovered as a high fidelity pair in PCR amplification. In 2013, they applied the Ds-Px pair to DNA aptamer generation by in vitro selection (SELEX) and demonstrated the genetic alphabet expansion significantly augment DNA aptamer affinities to target proteins.
In May 2014, researchers announced that they had successfully introduced two new artificial nucleotides into bacterial DNA, alongside the four naturally occurring nucleotides, and by including individual artificial nucleotides in the culture media, were able to passage the bacteria 24 times; they did not create mRNA or proteins able to use the artificial nucleotides.
Neither the XNA nor the unnatural bases are recognized by natural polymerases. One of the major challenges is to find or create novel types of polymerases that will be able to replicate these new-to-nature constructs. In one case a modified variant of the HIV-reverse transcriptase was found to be able to PCR-amplify an oligonucleotide containing a third type base pair. Pinheiro et al. (2012) demonstrated that the method of polymerase evolution and design successfully led to the storage and recovery of genetic information (of less than 100bp length) from six alternative genetic polymers based on simple nucleic acid architectures not found in nature Xeno nucleic acids.
Genetic code engineering
One of the goals of xenobiology is to rewrite the genetic code. The most promising approach to change the code is the reassignment of seldomly used or even unused codons. In an ideal scenario, the genetic code is expanded by one codon, thus having been liberated from its old function and fully reassigned to a non-canonical amino acid (ncAA) (“code expansion”). As these methods are laborious to implement, and some short cuts can be applied (“code engineering”), for example in bacteria that are auxotrophic for specific amino acids and at some point in the experiment are fed isostructural analogues instead of the canonical amino acids for which they are auxotrophic. In that situation, the canonical amino acid residues in native proteins are substituted with the ncAAs. Even the insertion of multiple different ncAAs into the same protein is possible. Finally, the repertoire of 20 canonical amino acids can not only be expanded, but also reduced to 19. By reassigning transfer RNA (tRNA)/aminoacyl-tRNA synthetase pairs the codon specificity can be changed. Cells endowed with such aminoacyl-[tRNA synthetases] are thus able to read [mRNA] sequences that make no sense to the existing gene expression machinery. Altering the codon: tRNA synthetases pairs may lead to the in vivo incorporation of the non-canonical amino acids into proteins. In the past reassigning codons was mainly done on a limited scale. In 2013, however, Farren Isaacs and George Church at Harvard University reported the replacement of all 321 TAG stop codons present in the genome of E. coli with synonymous TAA codons, thereby demonstrating that massive substitutions can be combined into higher-order strains without lethal effects. Following the success of this genome wide codon replacement, the authors continued and achieved the reprogramming of 13 codons throughout the genome, directly affecting 42 essential genes.
An even more radical change in the genetic code is the change of a triplet codon to a quadruplet and even pentaplet codon pioneered by Sisido in cell-free systems  and by Schultz in bacteria. Finally, non-natural base pairs can be used to introduce novel amino acid in proteins.
The goal of substituting DNA by XNA may also be reached by another route, namely by engineering the environment instead of the genetic modules. This approach has been successfully demonstrated by Marlière and Mutzel with the production of an E. coli strain whose DNA is composed of standard A, C and G nucleotides but has the synthetic thymine analogue 5-chlorouracil instead of thymine (T) in the corresponding positions of the sequence. These cells are then dependent on externally supplied 5-chlorouracil for growth, but otherwise they look and behave as normal E. coli. These cells, however, are currently not yet fully auxotrophic for the Xeno-base since they are still growing on thymine when this is supplied to the medium.
Xenobiological systems are designed to convey orthogonality to natural biological systems. A (still hypothetical) organisms that uses XNA, different base pairs and polymerases and has an altered genetic code will hardly be able to interact with natural forms of life on the genetic level. Thus, these xenobiological organisms represent a genetic enclave that cannot exchange information with natural cells. Altering the genetic machinery of the cell leads to semantic containment. In analogy to information processing in IT, this safety concept is termed a “genetic firewall”. The concept of the genetic firewall seems to overcome a number of limitations of previous safety systems. A first experimental evidence of the theoretical concept of the genetic firewall was achieved in 2013 with the construction of a genomically recoded organism (GRO). In this GRO all known UAG stop codons in E.coli were replaced by UAA codons, which allowed for the deletion of release factor 1 and reassignment of UAG translation function. The GRO exhibited increased resistance to T7 bacteriophage, thus showing that alternative genetic codes do reduce genetic compatibility. This GRO, however, is still very similar to its natural “parent” and cannot be regarded as a genetic firewall. The possibility of reassigning the function of large number of triplets opens the perspective to have strains that combine XNA, novel base pairs, new genetic codes etc. that cannot exchange any information with the natural biological world. Regardless of changes leading to a semantic containment mechanism in new organisms, any novel biochemical systems still has to undergo a toxicological screening. XNA, novel proteins etc. might represent novel toxins, or have an allergic potential that needs to be assessed.
Governance and Regulatory issues
Xenobiology might challenge the regulatory framework, as currently laws and directives deal with genetically modified organisms and do not directly mention chemically or genomically modified organisms. Taking into account that real xenobiology organisms are not expected in the next few years, policy makers do have some time at hand to prepare themselves for an upcoming governance challenge. Since 2012 policy advisers in the US, four National Biosafety Boards in Europe, and the European Molecular Biology Organisation  have picked up the topic as a developing governance issue.
- Directed evolution
- Expanded genetic code
- Glycol nucleic acid
- Nucleic acid analogue
- Threose nucleic acid
- Xeno Nucleic Acid
- XB1: The First Conference On Xenobiology May 6–8, 2014 – Genoa, Italy.
- Pinheiro, V.B. and Holliger, P., 2012. The XNA world: Progress towards replication and evolution of synthetic genetic polymers. Current Opinion in Chemical Biology, 16, 245
- Bain, J. D., Switzer, C., Chamberlin, R., & Steven A. Bennert, S.A. (1992). Ribosome-mediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code, Nature 356, 537 – 539
- Noren, C.J., Anthony-Cahill, S.J., Griffith, M.C., Schultz, P.G.(1989). A general method for site-specific incorporation of unnatural amino acids into proteins. Science 44, 82-88
- Schmidt M. Xenobiology: a new form of life as the ultimate biosafety tool Bioessays Vol 32(4):322-331
- Pace NR. 2001. The universal nature of biochemistry. Proc Natl Acad Sci USA 98: 805–8.
- Wiltschi, B. and N. Budisa, Natural history and experimental evolution of the genetic code. Applied Microbiology and Biotechnology, 2007. 74: p. 739-753
- Herdewijn P, Marlière P. Toward safe genetically modified organisms through the chemical diversification of nucleic acids.Chem Biodivers. 2009 Jun;6(6):791–808.
- Eschenmoser, A. (1999) Chemical etiology of nucleic acid structure. Science. 284, 2118–2124.
- Taylor, Alexander I.; Pinheiro, Vitor B.; Smola, Matthew J.; Morgunov, Alexey S.; Peak-Chew, Sew; Cozens, Christopher; Weeks, Kevin M.; Herdewijn, Piet; Holliger, Philipp. "Catalysts from synthetic genetic polymers". Nature 518 (7539): 427–430. doi:10.1038/nature13982. PMC 4336857. PMID 25470036.
- Vastmans K, Froeyen M, Kerremans L, et al. (2001). Reverse transcriptase incorporation of 1,5-anhydrohexitol nucleotides. Nucleic Acids Res 29: 3154–63. 42
- Jang, M et al. (2013). A synthetic substrate of DNA polymerase deviating from the bases, sugar, and leaving group of canonical deoxynucleoside triphosphates. Chemistry & Biology, 20 (3), art.nr. 10.1016/j.chembiol.2013.02.010, 416-23
- Pinheiro, V.B. and Holliger, P., (2012) The XNA world: Progress towards replication and evolution of synthetic genetic polymers. Current Opinion in Chemical Biology, 16, 245
- Pinheiro, V.B., Loakes, D. and Holliger, P. (2013) Synthetic polymers and their potential as genetic materials. Bioessays, 35, 113
- Ichida JK, Horhota A, Zou K, et al. (2005). High fidelity TNA synthesis by Therminator polymerase. Nucleic Acids Res 33: 5219–25
- Kempeneers V, Renders M, Froeyen M, et al. (2005). Investigation of the DNA-dependent cyclohexenyl nucleic acid polymerization and the cyclohexenyl nucleic acid-dependent DNA polymerization. Nucleic Acids Res. 33: 3828–36
- Pochet S. et al. (2003). Replication of hexitol oligonucleotides as a prelude to the propagation of a third type of nucleic acid in vivo. Comptes Rendus Biologies. 326:1175–1184
- Pezo V. et al. (2012). Binary Genetic Cassettes for Selecting XNA-Templated DNA Synthesis In Vivo. Angew Chem. 52: 8139–8143
- Krueger AT. et al. (2011). Encoding Phenotype in Bacteria with an Alternative Genetic Set. J. Am. Chem. Soc. 133 (45):18447–18451
- Sismour, A.M., et al. (2004) PCR amplification of DNA containing non-standard base pairs by variants of reverse transcriptase from Human Immunodeficiency Virus-1. Nucleic Acids Res. 32, 728–735
- Yang, Z., Hutter, D., Sheng, P., Sismour, A.M. and Benner, S.A. (2006) Artificially expanded genetic information system: a new base pair with an alternative hydrogen bonding pattern. Nucleic Acids Res. 34, 6095–6101
- Yang, Z., Sismour, A.M., Sheng, P., Puskar, N.L. and Benner, S.A. (2007) Enzymatic incorporation of a third nucleobase pair. Nucleic Acids Res. 35, 4238–4249
- Leconte, A.M., Hwang, G.T., Matsuda, S., Capek, P., Hari, Y. and Romesberg, F.E. (2008) Discovery, characterization, and optimization of an unnatural base pair for expansion of the genetic alphabet. J. Am. Chem. Soc. 130, 2336–2343
- Hirao, I. et al. (2002) An unnatural base pair for incorporating amino acid analogs into proteins. Nat. Biotechnol. 20, 177-182
- Hirao, I. et al. (2006) An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA. Nat. Methods 6, 729-735
- Kimoto, M. et al. (2009) An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules. Nucleic acids Res. 37, e14
- Yamashige, R. et al. Highly specific unnatural base pair systems as a third base pair for PCR amplification. Nucleic Acids Res. 40, 2793-2806
- Kimoto, M. et al. (2013) Generation of high-affinity DNA aptamers using an expanded genetic alphabet. Nat. Biotechnol. 31, 453-457
- Pollack, Andrew (May 7, 2014). "Researchers Report Breakthrough in Creating Artificial Genetic Code". New York Times. Retrieved May 7, 2014.
- Callaway, Ewen (May 7, 2014). "First life with 'alien' DNA". Nature (journal). doi:10.1038/nature.2014.15179. Retrieved May 7, 2014.
- Malyshev, Denis A.; Dhami, Kirandeep; Lavergne, Thomas; Chen, Tingjian; Dai, Nan; Foster, Jeremy M.; Corrêa, Ivan R.; Romesberg, Floyd E. (May 7, 2014). "A semi-synthetic organism with an expanded genetic alphabet". Nature (journal). doi:10.1038/nature13314. Retrieved May 7, 2014.
- Sismour, A.M. and Benner, S.A. (2005) The use of thymidine analogs to improve the replication of an extra DNA base pair: a synthetic biological system. Nucleic Acids Res. 33, 5640–5646
- Havemann, S.A., Hoshika, S., Hutter, D. and Benner, S.A. (2008) Incorporation of multiple sequential pseudothymidines by DNA polymerases and their impact on DNA duplex structure. Nucleosides Nucleotides Nucleic Acids 27, 261–278
- Pinheiro VB et al. (2012) Synthetic genetic polymers capable of heredity and evolution. Science 336: 341-344
- Budisa, N. (2005). Engineering the Genetic Code - Expanding the Amino Acid Repertoire for the Design of Novel Proteins, WILEY-VHC Weinheim, New York, Brisbane, Singapore, Toronto
- Hoesl, M. G., Budisa, N., (2012). Recent advances in genetic code engineering in Escherichia coli. Curr. Opin. Biotechnol. 23, 751–757
- Pezo, V., Guérineau, V., Le Caer, J.-P., Faillon, L., Mutzel, R. & Marlière, P. (2013). A metabolic prototype for eliminating tryptophan from the genetic copde. Scientific Reports 3: 1359
- Rackham, O. and Chin, J.W. (2005) A network of orthogonal ribosome mRNA pairs. Nat. Chem. Biol. 1, 159–166
- Wang, L., Brock, A., Herberich, B. and Schultz, P.G. (2001) Expanding the genetic code of Escherichia coli. Science 292, 498–500
- Hartman, M.C., Josephson, K., Lin, C.W. and Szostak, J.W. (2007) An expanded set of amino acid analogs for the ribosomal translation of unnatural peptides. PLoS ONE 2, e972
- Lajoie MJ, et al. (2013) Genomically Recoded Organisms Expand Biological Functions. Science. 342, 357
- Lajoie MJ, Kosuri S, Mosberg JA, Gregg CJ, Zhang D, Church GM (2013) Probing the Limits of Genetic Recoding in Essential Genes. Science. 342(6156):361-3
- Hohsaka T, Sisido M. (2002) Incorporation of non-natural amino acids into proteins. Curr Opin Chem Biol. 6, 809-815
- Anderson, J.C., Wu, N., Santoro, S.W., Lakshman, V., King, D.S. and Schultz, P.G. (2004) An expanded genetic code with a functional quadruplet codon. Proc. Natl. Acad. Sci. USA 101, 7566–7571
- Hirao I, Ohtsuki T, Fujiwara T, Mitsui T, Yokogawa T, Okuni T, Nakayama H, Takio K, Yabuki T, Kigawa T, Kodama K, Yokogawa T, Nishikawa K, Yokoyama S. (2002). An unnatural base pair for incorporating amino acid analogs into proteins. Nat Biotechnol, 20, 177–182
- Marlière, P. et al. (2011) Chemical Evolution of a Bacterium’s Genome. Angewandte Chemie Int. Ed. 50(31): 7109–7114
- Herdewijn, P. and Marlière, P. (2009) Toward safe genetically modified organisms through the chemical diversification of nucleic acids. Chem. Biodivers. 6, 791–808
- Marlière, P. (2009) The farther, the safer: a manifesto for securely navigating synthetic species away from the old living world. Syst. Synth. Biol. 3, 77–84
- Acevedo-Rocha CG, Budisa N (2011). On the Road towards Chemically Modified Organisms Endowed with a Genetic Firewall. Angewandte Chemie International Edition. 50(31):6960–6962
- Moe-Behrens GH, Davis R, Haynes KA. (2013) Preparing synthetic biology for the world. Front Microbiol. 2013;4:5
- Wright O, Stan GB, Ellis T. (2013) Building-in biosafety for synthetic biology. Microbiology. 159 (7):1221-35
- Lajoie MJ, et al. Genomically Recoded Organisms Expand Biological Functions. Science, 2013, 342(6156):357-60
- Schmidt M, Pei L. 2011. Synthetic Toxicology: Where engineering meets biology and toxicology Toxicological Sciences. 120(S1), S204–S224
- Schmidt M. 2013. Safeguarding the Genetic Firewall with Xenobiology. In: ISGP. 2013. 21st Century Borders/Synthetic Biology: Focus on Responsibility and Governance.
- ISGP. 2013. 21st Century Borders/Synthetic Biology: Focus on Responsibility and Governance p.55-65
- Pauwels K. et al. (2013) Event report: SynBio Workshop (Paris 2012) – Risk assessment challenges of Synthetic Biology. Journal für Verbraucherschutz und Lebensmittelsicherheit. DOI 10.1007/s00003-013-0829-9
- Garfinkel M. (2013) Biological containment of synthetic microorganisms: science and policy. Report on a ESF/LESC Strategic Workshop