Xenobiology (XB) is a subfield of synthetic biology, the study of synthesizing and manipulating biological devices and systems. Xenobiology derives from the Greek word xenos, 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 central dogma of 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-biology
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.[5] 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.[6] 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,[7] for example, 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.[4] 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.[8] 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.[9]
Scientific approach
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.[10] 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.[11] 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.[12] 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,[1][13][14][15] e.g.: hexose nucleic acid (HNA); threose nucleic acid (TNA),[16] glycol nucleic acid (GNA) cyclohexenyl nucleic acid (CeNA).[17] The incorporation of XNA in a plasmid, involving 3 HNA codons, has been accomplished already in 2003.[18] 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.[19] Extended bases using a natural DNA backbone could, likewise, be transliterated into natural DNA, although to a more limited extent.[20]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. A 2015 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.[12] 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.[24]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.[25] 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,[26] and afterward, Ds and 4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole (Px) was discovered as a high fidelity pair in PCR amplification.[27][28] 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.[29]
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.[30][31][32]
Novel polymerases
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.[33][34] 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.[35]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.[36] 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.[37] Finally, the repertoire of 20 canonical amino acids can not only be expanded, but also reduced to 19.[38] 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.[39] Altering the codon: tRNA synthetases pairs may lead to the in vivo incorporation of the non-canonical amino acids into proteins.[40][41] 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.[42] 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.[43]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 [44] and by Schultz in bacteria.[45] Finally, non-natural base pairs can be used to introduce novel amino acid in proteins.[46]
