ERASynBio2 - Orthogonal biosystems based on phosphonate XNAs (invivoXNA)

Lead Research Organisation: University College London
Department Name: Structural Molecular Biology

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Cozens C (2018) Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis. in Nucleic acids research

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Cozens C (2018) XNA Synthesis and Reverse Transcription by Engineered Thermophilic Polymerases. in Current protocols in chemical biology

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Jabgunde AM (2018) Methylated Nucleobases: Synthesis and Evaluation for Base Pairing In Vitro and In Vivo. in Chemistry (Weinheim an der Bergstrasse, Germany)

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Kestemont D (2018) XNA ligation using T4 DNA ligase in crowding conditions. in Chemical communications (Cambridge, England)

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Liu C (2018) Phosphonomethyl Oligonucleotides as Backbone-Modified Artificial Genetic Polymers. in Journal of the American Chemical Society

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Pinheiro VB (2018) Engineering-driven biological insights into DNA polymerase mechanism. in Current opinion in biotechnology

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Tizei PA (2016) Selection platforms for directed evolution in synthetic biology. in Biochemical Society transactions

 
Title Darwin Assembly 
Description The Darwin assembly is a novel DNA library assembly methodology that allows the fast and accurate synthesis of bespoke libraries containing targeted mutations targeting distal sites in a single construct. It is also cost-effective delivering libraries for little more than £20 per library (c.f. £5,000 of currently available commercial libraries of equivalent complexity). 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact The method has been patented by UCLB and a local facility is being set up to further improve the technology and set up the structure for a spin out. 
 
Description Marliere 
Organisation University Paris Sud
Department University of Évry Val-d'Essonne
Country France 
Sector Academic/University 
PI Contribution None
Collaborator Contribution Partner lab has long experience in gene knock-outs in E. coli, including essential genes. They have generated to date two E. coli knock-outs: LeuRS and OmpT
Impact None yet.
Start Year 2016
 
Description invivoXNA 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Public 
PI Contribution We are working as a consortia to develop an episome in vivo based on synthetic genetic materials (XNAs). I am the coordinator of this consortia, ensuring data are shared and that the teams are collaborating effectively. My group is tasked with establishing a novel genetic system based on phosphonate nucleic acids, developing novel selection methodologies and isolating an XNA replicase based on the K. lactis system.
Collaborator Contribution Each partner is bringing the know-how required to tackle at least one of the steps in the research. 1. Prof. Herdewijn (Leuven and Evry) is contributing with nucleic acid chemistry, 2. Marliere (Evry) with in vivo evolution platforms and microbiology, 3. Delarue (Pasteur) with polymerase bioinformatics and modelling, 4. Liu (UCI) with the K. lactis episome upon which our research is based. 5. Holliger (MRC-LMB) contributing with the development of functional XNAs that will create a selectable marker for the XNA episome
Impact None yet.
Start Year 2015
 
Description invivoXNA 
Organisation Pasteur Institute, Paris
Country France 
Sector Academic/University 
PI Contribution We are working as a consortia to develop an episome in vivo based on synthetic genetic materials (XNAs). I am the coordinator of this consortia, ensuring data are shared and that the teams are collaborating effectively. My group is tasked with establishing a novel genetic system based on phosphonate nucleic acids, developing novel selection methodologies and isolating an XNA replicase based on the K. lactis system.
Collaborator Contribution Each partner is bringing the know-how required to tackle at least one of the steps in the research. 1. Prof. Herdewijn (Leuven and Evry) is contributing with nucleic acid chemistry, 2. Marliere (Evry) with in vivo evolution platforms and microbiology, 3. Delarue (Pasteur) with polymerase bioinformatics and modelling, 4. Liu (UCI) with the K. lactis episome upon which our research is based. 5. Holliger (MRC-LMB) contributing with the development of functional XNAs that will create a selectable marker for the XNA episome
Impact None yet.
Start Year 2015
 
Description invivoXNA 
Organisation University Paris Sud
Department University of Évry Val-d'Essonne
Country France 
Sector Academic/University 
PI Contribution We are working as a consortia to develop an episome in vivo based on synthetic genetic materials (XNAs). I am the coordinator of this consortia, ensuring data are shared and that the teams are collaborating effectively. My group is tasked with establishing a novel genetic system based on phosphonate nucleic acids, developing novel selection methodologies and isolating an XNA replicase based on the K. lactis system.
Collaborator Contribution Each partner is bringing the know-how required to tackle at least one of the steps in the research. 1. Prof. Herdewijn (Leuven and Evry) is contributing with nucleic acid chemistry, 2. Marliere (Evry) with in vivo evolution platforms and microbiology, 3. Delarue (Pasteur) with polymerase bioinformatics and modelling, 4. Liu (UCI) with the K. lactis episome upon which our research is based. 5. Holliger (MRC-LMB) contributing with the development of functional XNAs that will create a selectable marker for the XNA episome
Impact None yet.
Start Year 2015
 
Description invivoXNA 
Organisation University of California, Irvine
Country United States 
Sector Academic/University 
PI Contribution We are working as a consortia to develop an episome in vivo based on synthetic genetic materials (XNAs). I am the coordinator of this consortia, ensuring data are shared and that the teams are collaborating effectively. My group is tasked with establishing a novel genetic system based on phosphonate nucleic acids, developing novel selection methodologies and isolating an XNA replicase based on the K. lactis system.
Collaborator Contribution Each partner is bringing the know-how required to tackle at least one of the steps in the research. 1. Prof. Herdewijn (Leuven and Evry) is contributing with nucleic acid chemistry, 2. Marliere (Evry) with in vivo evolution platforms and microbiology, 3. Delarue (Pasteur) with polymerase bioinformatics and modelling, 4. Liu (UCI) with the K. lactis episome upon which our research is based. 5. Holliger (MRC-LMB) contributing with the development of functional XNAs that will create a selectable marker for the XNA episome
Impact None yet.
Start Year 2015
 
Description invivoXNA 
Organisation University of Leuven
Country Belgium 
Sector Academic/University 
PI Contribution We are working as a consortia to develop an episome in vivo based on synthetic genetic materials (XNAs). I am the coordinator of this consortia, ensuring data are shared and that the teams are collaborating effectively. My group is tasked with establishing a novel genetic system based on phosphonate nucleic acids, developing novel selection methodologies and isolating an XNA replicase based on the K. lactis system.
Collaborator Contribution Each partner is bringing the know-how required to tackle at least one of the steps in the research. 1. Prof. Herdewijn (Leuven and Evry) is contributing with nucleic acid chemistry, 2. Marliere (Evry) with in vivo evolution platforms and microbiology, 3. Delarue (Pasteur) with polymerase bioinformatics and modelling, 4. Liu (UCI) with the K. lactis episome upon which our research is based. 5. Holliger (MRC-LMB) contributing with the development of functional XNAs that will create a selectable marker for the XNA episome
Impact None yet.
Start Year 2015
 
Description Naked Scientists 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Radio interview with the BBC Naked Scientists (29.11.16)
It covered synthetic genetic materials and biocontainment. This is broadcast live to BBC Cambridgeshire, played in BBC Australia and also released as a podcast.
Year(s) Of Engagement Activity 2016
URL https://goo.gl/UUQpKR
 
Description SBUK2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I chaired the Second Synthetic Biology UK conference, which took palce in Edinburgh, on 14-16 November.
The conference with more than 180 participants, included researchers (PIs and PDRAs) and funding organization represenatives (BBSRC, KTN).
Year(s) Of Engagement Activity 2016
URL https://www.biochemistry.org/Events/tabid/379/MeetingNo/SA186/view/Conference/Default.aspx
 
Description Xenobiology: biosafety, biosecurity and biocontainment 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact We organised a small workshop (approximately 40 participants) including researchers, students, social scientists, funders and government representatives to discuss some of the challenges of releasing genetically engineered microorganisms into the environment. The workshop also explored biological orthogonality and how that can be adapted for biological containment. Some of the issues relating to the development of Xenobiology were also covered. The workshop consisted of a mixture of talks and plenary discussions with participation from the audience.

In addition to it being a consultative exercise, the workshop was also used to establish a network of researchers and social scientists involved in ethics, risk assessment and governance. Our goal was to further develop the engagement between lab-based and social scientists and to improve the dialogue on Xenobiology, Synthetic Biology and biotechnology. Based on feedback from participants, the workshop was a useful introduction ot the area and created a depth of discussion beyond what is currently present in the field.

We are now in the process of generating a podcast based on the workshop for public distribution and creating a webpage to host the talks presented on the day.
Year(s) Of Engagement Activity 2017