A synthetic biology approach to fighting Francisella tularensis: Development of aptamer presenting DNA-nanorings

Pathogenic bacteria rely on molecules on their surfaces to bind to and infect people. These same molecules are also often triggers for our immune systems to develop antibodies, and are additionally used in assays to detect the presence of infectious bacteria. The studies proposed in this project aim to exploit the above principles by combining them with the emerging field of synthetic biology to develop new vaccine components and devices to rapidly detect potentially fatal pathogens. Synthetic biology uses the same principles from which life has evolved to design and develop novel life-reminiscent and biologically-active reagents.

In this study we aim to exploit our recent discovery that specified sequences of DNA can be used to assemble discrete 'nanoparticles' - chemicals with dimensions compatible with internal cellular sizes. We have combined a series of short lengths of DNA to form a ring-like structure we term a 'DNA nanoring'. These particles are all the same size, and their simple chemistry provides sites into which we can easily engineer other chemical groups. In this project we aim to incorporate a number of different groups at these sites, which are automatically multiplied by the symmetric construction of the nanoring. Through the addition of these inserted groups we will introduce both binding and sensing functionalities to the nanoring. These will: (1) enable nanorings to recognise and selectively bind to certain pathogenic bacteria; (2) change their chemical & physical properties in response to pathogen binding, enabling sensing and detection of these bacteria; and (3) safely present multiple copies of bacterial surface proteins to the immune system as components of vaccines delivering an enhanced response, hence providing a new strategy for effective vaccine development.

If these uses can be successfully incorporated and demonstrated using the bacterial test systems in this study, these principles could be deployed against a wide range of pathogens including those believed to be incorporated in bioweapons. We propose their main uses would be in portable sensor units to detect pathogens in circulation, and in novel and improved vaccines targeting pathogens for which no effective vaccines currently exist. The nanorings therefore represent a novel development of new designed functionalities arising directly from the new field of synthetic biology.

Recently, Zaccai & Brady (Biochemistry, Bristol) designed and characterized by atomic force microscopy and small angle neutron scattering a novel DNA architecture, in which intertwined ssDNA molecules assemble into a discrete and cylindrical particle. We call this assembly a DNA-nanoring. The complex is approximately 20 nm across and 4 nm long, and is formed from 3 copies each of 4 different primers, each comprising 75 nucleic acids. In conjunction with Burley (Chemistry, Strathclyde), the DNA-nanoring was subsequently rationally modified and simplified, but still retained the ability to assemble into a hollow cylinder of similar dimensions.

Here, in collaboration with Virji (Cellular and Molecular Microbiology, Bristol), we propose to functionalize these DNA-nanorings as presentation devices for bio-security applications. Specifically, we propose to explore the potential for DNA-nanorings to be used (1) as components for a molecular detector of pathogens, and (2) to engineer them to present multiple copiers of antigens, but also genetic information, to the immune system. These objectives will be achieved through the incorporation of (a) DNA aptamers to direct binding; (b) chemical groups for the linking of protein ligands; and (c) fluorescent labels capable of sensing binding and additions to other groups in the nanoring. All of these additions will be at sites distant from the ring-forming double-helical segments, and hence are not anticipated to disrupt the overall nanoring structure. In these proof-of-principle studies, the bacterial targets would be Francisella tularensis, Moraxella catarrhalis and Neisseria meningitidis.

This multidisciplinary synthetic biology approach brings together cutting edge expertise in nucleic acid chemistry and bioconjugation (GAB), structural biology (NZ, LB) and microbiology (MV)

If we succeed in making functionalised DNA-nanorings as described in the proposal, we envisage potential applications both for biosensing, and as vaccine components.

The multivalent use of aptamer DNA-nanorings specific for different proteins of a particular bacterial strain will provide a very sensitive and real-time system to detect and identify that strain. The relative simplicity of the required spectroscopic equipment also means that this molecular detector can be readily implemented for use outside a laboratory setting, for example on a hospital ward or even on a battle field. We therefore envisage that the biotech industry will express a keen interest in this molecular detector. This research will also strengthen our current partnerships with the NHS in both Bristol and Glasgow, as well as bring forward new collaborations with the biodefense research community in Porton Down. It is moreover important to note that if this biosensor design proves successful, this same approach could readily be deployed to further biosecurity and other medically important pathogens.

The data obtained from DNA-nanorings that display bacterial proteins will provide proof-of-principle that a designed and quantified multivalent display of antigen on DNA-nanorings, in comparison to an individual antigen, can react more strongly to sera from convalescing patients. The next step would be to undertake animal studies as part of a more general program to develop vaccines against Moraxella catarrhalis and Neisseria meningitidis, which is likely to involve the NHS and industrial collaborators. One of the applicants (MV) is already progressing studies on other vaccine components (not DNA nanorings) with large pharma, and we would use this experience to engage commercial partners. There are existing structures within both universities to establish and promote such links. We are particularly interested in ascertaining a quantitative relationship between multivalency and effectiveness of a vaccine: this would have extended impact for the general field of immunology and the preparation of commercial vaccines in general. These new data would subsequently guide us in testing the effectiveness of our DNA-nanoring design to elicit a protection against infection by Francisella tularensis.

More generally, DNA-nanorings that present multivalent aptamers and proteins will also be valuable tools with which to manipulate cellular adhesion and recognition events. These molecules would also be relatively straightforward to produce and adapt to displaying other ligands. For example, tools to interfere with cellular adhesion open up opportunities to target metastatic tumours and could be exploited using existing collaborations (e.g. GAB in collaboration with Prof. Hing Leung at the Beatson Institute). Hence, this research in synthetic biology could potentially have a far-reaching impact in many fields, including those of immunology and cell biology, with the attendant interest of the medical research community and of pharmaceutical companies. Translation of research at Bristol is developed and promoted by continuing liaison with our Research and Enterprise Department (RED), and at Strathclyde with the Research Knowledge Exchange Services (RKES) who have proven success in the biomedical arena (e.g. MGB Biopharma). Any outcomes of this work that are exploitable, notably in terms of materials, intellectual property, and knowledge transfer to the private sector, would be handled by the highly experienced team within RED, who engage closely with BBSRC.

Finally, Synthetic Biology is an emerging area of research that combines engineering and biology. Although public opinion is largely positive about the potential benefits of this work, it is clear that the research, its likely impacts and limitations, should be made clear. We shall therefore continue to publicise research papers and new grants awarded through our universities.