Xenobiology

Xenobiology (XB ) is a sub-discipline of synthetic biology that deals with the synthesis and manipulation of complex biological circuits and systems. From the Greek Xenos (Greek for "guest, stranger " ) derived xenobiology describes biological forms that are previously unknown to science or not of natural origin. In experimental practice, the XB means novel biological and biochemical systems that differ from the canonical DNA-RNA system 20 amino acids (see central dogma of molecular biology ). In this sense, the natural DNA and RNA molecules can be replaced by synthetic nucleic acid analogs in xenobiology and under the designation Xenonukleinsäuren (XNA ) is used as an information carrier. Also located xenobiology focused on the expansion of the genetic code and the installation nonproteinogenic amino acids ( non-canonical amino acids) into proteins.

Demarcation between Xeno -, exo-, and astrobiology

The prefix astro- (Greek astron = star ( picture) ) than determining word the meaning of star, star, universe, with Exo (Greek exo to: ex = (her ) off) for the determination of word meaning outside, outside assigned will. Exobiology and astrobiology deal with the possible existence and emergence of extraterrestrial life and the general search for life in space, with this interest usually focuses on planets in the habitable zone. In contrast to astrobiologists who are trying to detect a possible extraterrestrial life in the universe and analyze Exobiologist to deal with the attempt to develop life forms with fundamentally different biochemistry or deviant genetic code on Earth.

Objectives of xenobiology

The xenobiology has the potential to uncover fundamental principles of biology and knowledge about the origin of life. To better understand this, it is important to find out why his own life ( most likely) has developed over the early RNA world to a DNA - RNA-protein system with a universal genetic code. In this context, on the questions whether life is evolutionary " accident" was, or whether specific selective constraints existed that precluded a different biochemistry of life from the beginning. By creating alternative biochemical " primeval soup " is expected to explore fundamental principles that led to the development of life as we know it today, contributed.

Away from the basic research provides the xenobiology many new approaches for the development of industrial production systems with which new production possibilities in the field of biopolymer engineering and pathogen resistances are created. The genetic code encodes a canonical 20 amino acids in all organisms, which are used for protein synthesis. In rare cases, the specific amino acids selenomethionine, selenocysteine ​​and pyrrolysine, are incorporated by additional translational components into proteins. However, there are another 700 amino acids, which are known in biochemistry and its properties could be useful to improve the potential of the proteins in view of more efficient catalytic function or material properties. The EU-funded project METACODE example aims metathesis - a useful catalytic process that is unknown so far in living organisms - to establish in bacterial cells. Another potential for the improvement of production processes by the XB is the ability to minimize the risk of viruses or Bakteriophagenbefall during cultivation. Xenobiologische cells no longer were suitable as hosts for viruses and phages, as they have through a so-called " semantic containment" a higher resistance.

Xenobiology enables the development of new systems of containment of genetically modified organisms ( biocontainment ). Here, the aim is to enhance current containment approaches with a " genetic firewall ' and diversify. A many services mentioned above criticism of the tranditionellen genetic engineering and biotechnology is the possibility of horizontal gene transfer from genetically modified organisms into the environment and resulting potential risks to nature and human health. One of the main ideas of the XB it is now to develop alternative genetic codes and biochemical building blocks, so that horizontal gene transfer is no longer possible. An altered biochemistry would allow new synthetic auxotrophy and use this to generate orthogonal biological systems that are no longer compatible with the natural genetic systems.

Scientific Approach

The xenobiology aims to design and manufacture products that differ from their natural templates on one or more fundamental levels of biological systems. Ideally, these new creatures would be different in every possible biochemical aspect and contained a highly modified genetic code. The long-term goal is to develop a cell that no longer stores its genetic information in DNA and translated with 20 amino acids, but in alternative information-carrier polymers consisting of XNA, alternative base pairing and noncanonical amino acids (that is, is an altered genetic code ) exist. So far succeeded only to produce cells that had implements one or two of the aforementioned properties.

Xenonukleinsäuren ( XNA)

Originally the research was for alternative DNA forms from the question of the origin of life and why RNA and DNA were given by the ( chemical ) evolution in preference to other possible nucleic acid structures. A systematic study, which aimed at the diversification of chemical Nukleinssäurenstruktur, resulted in completely new information-carrying biopolymers. To date, several XNAs were synthesized with a new chemical backbone or nucleobases novel, for example hexose nucleic acid (HNA ) nucleic acid threose (TNA ), glycol nucleic acid ( GNA ) and cyclohexenyl nucleic acid ( CeNA ). The incorporation of XNA in a plasmid in the form of three HNA codon was successfully completed in 2003. This Xenonukleinsäuren are already being used in vivo in E. coli as a template for DNA synthesis. Here, a binary genetic cassette ( G / T ) and two non-DNA - bases were (Hx / U ) is used. During the installation of CeNA could also be performed successfully, failed so far any attempt to use GNA as the backbone, as in this case are too large differences of the natural system to serve as a template for the biosynthesis of DNA by the natural machinery. These expanded base pairs that exist on the chemistry of natural DNA backbone, but could probably be converted back into natural DNA to a limited extent.

Expansion of the genetic alphabet

While XNA only based on modification in the polymer backbone or on the nucleobases, aim other attempts from it to replace the natural alphabet of DNA or expand with unnatural base pairs or to be replaced. For example, DNA was prepared, which contained, instead of the four Standardnukleobasen (A, T, G and C), an extended alphabet 6 nucleobases ( A, T, G, C, P, and Z). This is in these two new centers P of 2-amino- 8-( 1 -beta-D -2'- deoxyribofuranosyl ) imidazo [ 1,2-a] -1,3,5-triazine -4 (8H ), and Z for 6 -amino -5- nitro3 - ( l' - pD -2'- deoxyribofuranosyl ) -2 (1H) -pyridones. In a systematic study, Leconte et al. the possible mounting designs of 60 base candidates ( this would correspond to 3600 possible base pairs ) in the DNA.

Novel polymerases

Neither XNA nor the unnatural bases are recognized by natural polymerases. Accordingly, one of the biggest challenges the development and production of new types of polymerases, which are able to replicate these novel structures. As already a modified variant of the HIV reverse transcriptase was detected, which was capable of producing in a PCR amplification, a Oligonukleotidamplifikat containing an additional third base pair. Pinheiro et al (2012 ) demonstrated that by means of the evolution and structure of polymerases genetic information can be saved and restored successfully ( less than 100 bp in length ). This was done on the basis of six alternative information storage polymers ( Xenonukleinsäuren ).

Expanding the Genetic Code

One of the goals of xenobiology is the redesign of the universal genetic code. The currently most promising approach to achieving this goal is the replacement of rare or even unused codons. Ideally, this would produce " gaps " in the current code, the ( NCAA) can be filled with new, non-canonical amino acids ( "Expansion of the genetic code ," engl. Code expansion).

Since such strategies are very difficult to implement and require a lot of time, short also abbreviations can be taken. Thus, the " genetic code engineering " (English code engineering), for example, bacteria, certain amino acids can not be produced by, offered under certain culture conditions isostructural analogues of the natural amino acids, which they then install instead of the natural amino acids into proteins. However, only a canonical amino acid is replaced by a non-canonical With this method, and it does not, strictly speaking, an "extension " of the genetic code. In this manner, it is easily possible to incorporate several nichkanonische amino acids into proteins simultaneously. The Aminosäurereportoire may, however, are not just expanding but also reduced. The Codonspezifität can be changed by new tRNA / aminoacyl-tRNA synthetase pairs are modified so that they recognize different codons. Cells with such new configuration will then be able to decipher the mRNA sequences were unsuitable for the natural Proteinbiosynthesemaschinerie. Novel tRNA / aminoacyl-tRNA synthetase pairs can on this basis for the site-specific in vivo incorporation of non-canonical amino acids are used. In the past, the reorganization of codons mainly happened only in a very limited context. In 2013, however, a complete codon was removed from a genome, which is now free for the occupancy of new amino acids for the first time. Specifically, could replace all 314 TAG stop codons in the genome of E. coli by TAA stop codon groups to Farren Isaac and George Church at Harvard University, where they demonstrated that a massive exchange of individual codons by other lethal effects for the particular organism is possible. Building on this success of genome-wide Codonaustausches, the working groups were able to replace 13 codons in 42 essential genes by their synonyms and reduce the genetic code of 64 to 51 codons used as in these genes.

An even more radical step of changing the genetic code is the move away from the natural triplet codons and towards quadruplet or even Penta Plett codons. Masahiko Sisido and Schultz contributed to this field, pioneering work, which Sisido managed in a cell-free system to establish a pentablen code and Schultz even brought bacteria to work with quadruplet codons. Finally, it is possible to use even the above-mentioned non-natural nucleobases ( XNA) for introducing non-canonical amino acids into proteins.

Directed Evolution

Another possibility of replacing the DNA by XNA, it, rather than specifically to alter the genetic molecules the cell environment would be. This approach has already been successfully demonstrated by Marliere and Mutzel, by establishing a new E. coli strain, which has a DNA structure, which is composed of the Standardnukleotiden A, C and G and of a synthetic Thyminanalogon. Here, the Thyminanalogon was installed 5 - Chlorouracil sequence-specific manner at all positions of the natural thymine into the genome. In order to grow these cells are dependent on the external addition of the base 5 - Chlorouracil, but otherwise behave like normal E. coli bacteria. Using this approach results in two layers of protection in order to prevent any interaction between the non- natural and natural bacteria, since the master has a auxotrophy for a non-natural chemical substance and the body also has a DNA form which can be decrypted by any other organisms.

Biological Safety

Xenobiologische systems have been developed so as to be orthogonal to the natural biological systems of our planet. A (but so far purely hypothetical ) XNA organism that has XNA, other base pairs and new polymerases and uses a modified genetic code, will be very hard to be able to interact with the natural forms of life at the genetic level. In this sense xenobiologische organisms represented a genetic enclave, the genetic information with natural cells can not replace. Therefore, the change in the genetic replication machine of a cell leads to a so-called " semantic containment". As a security concept, this - in analogy to information processing in the IT sector - are referred to as a genetic firewall. This concept of a genetic firewall seems to address several limitations of existing biosafety systems. Initial experimental evidence that identify the theoretical concept of genetic firewall as an effective future instrument in 2013 with the creation of a genomrekodierten organism ( GRO) were delivered. In this organism, all TAG stop codons in E. coli were replaced by TAA codons. This allowed the deletion of the release factor 1 and, based on the replacement of the TAG codon for amino acid instead of stop signal. This GRO showed a concomitant increase in resistance to T7 Bakteriophageninfektionen. This emphasizes that alternative genetic codes can reduce the genetic compatibility. Nevertheless, this GRO is its natural predecessors still very similar and has accordingly not yet have a " genetic firewall '. The example shows, however, that the replacement of a large number of triplet codons opens the door not to further generate in the future bacterial strains XNA, use new base pairs, new genetic codes, etc.. With these semantic changes, these tribes would no longer be able to exchange genetic information with the natural environment. While such a genetic firewall would implement semantic containment mechanisms into new organisms, also new biochemical systems for toxins and xenobiotics have yet to be developed.

Legal and regulatory framework, regulatory

Xenobiology could blow up the currently applicable regulatory framework and lead to new legal challenges. Currently, laws and policies to deal with, although genetically modified organisms ( GMOs), but worth mentioning in any way chemically modified or genomrekodierte organisms. If you consider that right xenobiologische organisms are not expected in the coming years, policy makers still have time to prepare for the future regulatory challenges. Since 2012 there are in the U.S., under political consultant, four national committees for Biosafety in Europe, and the European Molecular Biology Organisation in order to work this issue as a future to be controlled field.