ERASynBio2 - Design and Synthesis of a Bio-orthogonal Genetic System Based on Threose Nucleic acids In Vivo (TNAepisome)

Every living organism on Earth relies solely on two molecules to store its genetic information: DNA and RNA (deoxy- and ribonucleic acids). These molecules have unique properties (such as base-pairing) that make them ideally suited to their role. It is now clear that some synthetic nucleic acids (or XNAs) can also store genetic information, enabling a number of potential applications, based on the chemical properties of the individual XNAs.

Our goal is to further develop TNA (threose nucleic acids) as a genetic material with a view to developing a TNA genetic system that can be introduced and maintained in vivo, in bacterial cells.

There are many challenges to introduce a stable genetic element into a cell that is not based on DNA; a number of conditions must be fulfilled for that to be achieved:
1. The genetic element and its precursors cannot be toxic to the cells and should be maintained isolated from the cell machinery.
2. Precursors must be readily assimilated by the cells but not interfere with the their metabolism.
3. The genetic element has to be replicated in vivo, so as to be maintained as cells divide.
4. The genetic information in the XNA system must be of use to the cell, otherwise it will be lost.

TNA is poorly incorporated by natural enzymes specialised in DNA synthesis and it is not expected to interact with other cellular enzymes. As such, TNA is not expected to be toxic or interfere with cellular metabolism. Further separation between TNA and natural systems will be achieved by adapting a genetic element from a bacterial virus for controlled replication in cellular conditions. Viral replication in bacteria occurs independently from the main bacterial genome and relies on few cellular machinery components, further isolating the TNA element from the cell.

Although TNA synthesis has been demonstrated, currently available polymerases are too error prone. Using rational design, high-throughput assays and directed evolution, we will optimise polymerases for improved TNA synthesis and to obtain TNA replication.

TNA maintenance in vivo, in addition to the replicase and a stable genetic element, also requires efficient precursor uptake and a link between TNA information and cell survival. TNA nucleosides can be efficiently taken up by bacterial cells but require further activation, converting them into the appropriate polymerase substrates. We will engineer enzymes capable of activating the TNA nucleosides in vivo, minimising the cost of synthesis while delivering an additional layer of regulation.

We will also engineer viral enzymes capable of converting TNA information into RNA, thus making use of the cellular machinery and natural information processing pathways to link TNA information to proteins. By introducing an essential gene in TNA, only cells capable of accessing that information, that is, converting it to RNA using the engineered enzymes, will survive.

Establishing an XNA element in vivo should give us great insight into the origin of life and into how to define life itself. It will establish a platform for the development of therapeutic agents based on TNA and a safer transgenic organism - a contained organism dependent on the continuous supply of synthetic precursors and encoding information inaccessible to natural organisms.

The development of synthetic biopolymers as an effective engineering tool for in vivo applications has been limited by our ability to demonstrate an intra-cellular system that replicates and transfers encoded chemical information. The maintenance and interpretation of cellular chemical information is central to biology and best known as the Central Dogma. Through the re-design of information storage systems (genetic material) and the enzymes involved in accessing and maintaining biological information (polymerases, ribosomes), synthetic biology has the potential to re-write the Central Dogma and extend our understanding of life's most basic processes.
Our proposed synthesis of an all-TNA episome based on the bacteriophage Phi29 would revolutionize synthetic biology by providing an in vivo, stable replicon for storing sequence-defined genetic information completely isolated from the cellular genome and able to replicate independently and therefore function as a safe and effective tool for synthetic biology.

Our proposal reaches out to engage a broader community, provides education and training opportunities for young scholars, and advances fundamental underlying concepts that we believe will contribute towards making SB safer for future generations.
The proposed research activities constitute a convergent and focused approach aimed at the generation, manipulation and application of an in vivo TNA genetic element, called a TNA episome. The combined interdisciplinary capabilities of the partners span all of the key aspects of the project and we provide proof-of-principle demonstrations whenever possible to establish convincing evidence of our work in the fields of chemistry, molecular biology, and genetics. These interdisciplinary activities solidify our synergy between the various partners and promote our willingness to advance XNA research in SB.
This proposal will provide a number of significant scientific advances in various disciplines and it holds considerable potential for economic and societal impact. Selection methodologies being developed are general, enabling their application to other protein and nucleic acid enzymes. We recognize the serious role that safety plays in SB, and we realize that concerns about the release of genetically modified microorganisms into the environment has delayed the production of new SB technologies that could benefit society. In addition to its scientific interest, our proposal would also deliver a new route towards the development of substantially safer genetically engineered microorganisms. Our integrated end goal is equivalent to a genetically contained organism that can be engineered to perform defined functions but at the same time posing negligible ecological or informational risk in the environment. It can be developed into a safety standard in the industrial synthesis of biological products - one that can be legislated and monitored.
Once IP rights are secured, results will be publicly disseminated. This will be achieved primarily through publication in international peer-reviewed journals and through presentations at local and international scientific meetings, and our own annual workshop events.
We anticipate that significant project landmarks will be of sufficient general interest to merit publication in world-leading journals with accompanying press releases. Open access will be secured to maximize public access to the research output. Whenever available, supplementary data will be included to allow detailed information on protocols and datasets to be disclosed. Alternatively, additional material, such as engineered enzyme sequences, can be deposited in public databases, other open access repositories (e.g. figshare, DNASU), or on a consortium website or on individual partners' websites.
Scientific meetings such as IS3NA roundtables, FEBS congresses and Synthetic Biology conferences such as SB 7.0 will provide opportunities to all members of the consortium to present their work and network with the field. This will allow the profile of the consortium partners and funding agencies to be raised, new collaborations forged and potential industrial partners contacted. Potential commercial partners can also be contacted through the consortium's extensive contacts.
The proposal includes three workshops in three different countries (FR, UK, and US), one each year of the project. The workshops will assemble participants from all the partners' groups plus an invited keynote speaker prominent in SB. The topics will be focused on the concepts and progress of all the Work Plans of the partners, and at the end there will be discussion tables to coordinate future plans.