ERASynBio2 - Orthogonal biosystems based on phosphonate XNAs (invivoXNA)

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 establish a novel XNA, based on oxymethylphosphonates (PMTs), as a genetic material with a view to developing a PMT 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.

The chemical modifications in PMT are expected to make natural enzymes that normally interact with DNA or RNA, unable to interact or degrade PMT molecules, minimising PMT toxicity and creating a natural separation between natural and synthetic genes. That separation will be maximised by adapting a stable genetic element from yeast, which we believe will replicate in bacteria independently from the bacterial genetic material.
Using rational design, high-throughput assays and directed evolution, we will first establish PMT as a genetic material and then obtain a specialist enzyme, based on natural enzymes that replicate DNA, capable of replicating PMTs.

PMT maintenance in vivo, in addition to the replicase and a stable genetic element, also requires efficient precursor uptake and a link between PMT and cell survival. Phosphonates, like PMT precursors, can be readily taken up by bacterial cells using dedicated transporters but we will also explore other approaches to ensure maximum uptake. In addition, we will identify PMT molecules capable of carrying out chemical reactions essential for cell viability. Synthesis of these PMT molecules in vivo will enable cell survival, ensuring stability for the PMT information and genetic elements.

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 PMTs and a safer transgenic organism - a contained organism dependent on the continuous supply of synthetic precursors and encoding information inaccessible to natural organisms.

Although RNA and DNA are the only genetic polymers in nature, synthetic nucleic acids (XNAs) can be suitable genetic materials. If not toxic and if unable to interact with the cellular machinery, such XNAs can be further developed into orthogonal genetic materials in vivo - rewriting the topology of information transfer in biology, redesigning the Central Dogma.
An XNA episome, maintained independent from and unable to interact with the cell's genetic information storage, would give us insights into how information is stored and propagated in biology as well as establishing a minimal system from which complex functions based on XNA could be systematically developed. If XNA precursors cannot be made in the cell, then an XNA episomecan be immediately applied to develop safer, genetically-contained, engineered organisms.
Redesign of the Central Dogma pose a number of challenges that can be systematically overcome. It requires an XNA that is bio-orthogonal, a replication system for maintaining the information, and a route of communication with the cell. Recent advances highlight that this is a feasible goal.
Using oxymethylphosphonates as a bio-orthogonal XNA and an orthogonal DNA replication system, we propose to develop all key components required to establish and maintain an XNA episome in vivo. This will include engineered polymerases to transfer information to XNA in vitro (DNA>XNA and XNA>DNA) as well as an XNA replicase (XNA>XNA) dedicated to replicating the linear XNA plasmid being developed. Functional XNAzymes, will link genetic information stored in XNA to cell survival, ensuring episome maintenance in the semi-synthetic cell and establishing the episome as a platform for further creation and evolution of XNA function and circuits.
In addition to forging the first orthogonal genetic element, the methodologies and molecules generated in the project will in themselves be of great scientific interest and enable new avenues of research.

The proposed research will deliver multiple landmark results in synthetic biology culminating on the development of a stable orthogonal XNA episome in vivo: novel XNA backbones compatible with in vivo applications, the tools to generate, maintain and link XNA information to cellular function, and orthogonal genetic enclaves that will remain isolated and allowed to evolve at different rates from the bacterial host.
The resulting episome will greatly extend the current efforts on engineering semi-synthetic organisms and importantly, it will create the minimal Darwinian genetic element from which every component of a wholly synthetic organism can be developed. These are ambitious goals at the forefront of synthetic biology.
Our results will provide a number of significant scientific advances in multiple disciplines and generate platforms of considerable potential economic and societal impact. Selection methodologies being developed are general, enabling their application to other protein and nucleic acid enzymes of direct commercial relevance. An orthogonal episome in bacteria in DNA would provide a powerful platform for in vivo directed evolution of novel enzymes and pathways of industrial relevance. Notably, a bacterial platform based on K. lactis pGKL1 may also generate a shuttle system, functional in both prokaryotic and eukaryotic hosts. Functional XNA molecules are proof of principle that the proposed XNA can be used in the development of novel materials, sensors and other biomedical applications.
Development of a stable XNA episome can also deliver significantly safer genetically engineered microorganisms. Genetic information maintained in XNA and dependent on exogenously added precursors, creates a genetically contained organism that can be engineered to pose negligible ecological and 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.
Project landmarks will be of sufficient general interest and significant advances on the current state-of-the-art to merit publication in world-leading journals. Open access will be secured to maximise public access to the research output. Where the option is available, supplementary data will be included with journal submissions 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), on a consortium website or on individual partners' websites.
Scientific meetings will provide opportunities to all members of the consortium to present their work and network with the field at large. 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. A website will be set up for the consortium, allowing a coherent and manageable interface for science communication and public engagement, linking the online presence of individual partners (organisations and funding agencies) and providing a platform to increase the profile of the post-doctoral researchers associated with the consortium.
Engagement with stakeholders can be carried through established initiatives (e.g. UK Royal Society MP pairing scheme) and through more targeted dialogue with groups that have reservations against genetic modification and synthetic biology (e.g. Friends of the Earth), to better understand their concerns, while fostering discussion of our proposed research and how it can contribute towards safer genetically modified organisms.