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) and means "stranger, guest". So XB 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 canonial 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.
Difference between xeno-, exo-, and astro-
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 Goldilock 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 universial 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 selenomethionine 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. 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 outgoing motifs 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 extend.
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.
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 [universal genetic code]. The most promising approach to change the code is the re-assignment 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 re-assigned 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 314 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 Marliere 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 thymin (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. This approach thus sets two firewalls for any interaction with other bacteria, because the strain is auxotrophic for a non-natural chemical and it contains a form of DNA that cannot be deciphered by other organisms 
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. While a genetic firewall could implement semantic containment mechanisms into novel organisms, novel biochemical systems still have to be assessed for novel toxins and xenobiotics 
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.
- Expanded genetic code
- Directed evolution
- Nucleic acid analogue
- Xeno Nucleic Acid
- Glycol nucleic acid
- Threose nucleic acid
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