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Protein nano-patterning using DNA nanotechnology; control of surface-based immune system activation

Periodic Reporting for period 3 - BioNanoPattern (Protein nano-patterning using DNA nanotechnology; control of surface-based immune system activation)

Reporting period: 2021-01-01 to 2022-06-30

Protein nanopatterning concerns the geometric arrangement of individual proteins with nanometre accuracy. Protein nanopatterns are essential for cellular function, and our research has revealed that our immune system is activated by nanopatterned antibody platforms, which initiate the classical Complement pathway by binding to the first component of Complement, the C1 complex.

We currently have no control over these nanopatterns, and therefore do not understand the molecular details of how geometry leads to activation. DNA nanotechnology can be used to form self-assembled nanoscale structures, which are ideal for use as templates to pattern proteins with specific geometries and nanometre accuracy. This project uses DNA to nanopattern antigens and other biomolecular moieties with defined geometry to study and control Complement pathway activation by the C1 complex.

The first aim of this proposal is to understand the role of nanopatterns in complement activation. We will develop and demonstrate the potential use of DNA to nanopattern proteins by designing DNA nanotemplates suitable for patterning C1 binding sites. C1 will bind with specific geometry, and the relationship between geometry and Complement activation will be assessed using novel liposome assays. Using DNA to mimic antigenic surfaces will enable high-resolution structure determination of C1 complexes, both in solution and on lipid bilayer surfaces, using cryo-electron microscopy to elucidate the structure-activation relationship of complement.

The second aim of this proposal is exploit our understanding of nanopatterns to develop new and highly efficient ways to activate our immune system in vivo, with potential for targeted anti-tumour immunotherapies. This will be achieved by determining both the geometric and molecular requirements of activation, and distilling these down into discrete constituents to form rationally-designed small-molecule activators of complement. These will be nanopatterned and allow direct targeting of Complement activation to specific cells within a population of cell types to demonstrate targeted cell killing.
In the first 30 months of this project we have consolidated as a team including me, 2 PhD students and 1 Postdoc. Together, we have determined which DNA nanotemplate is optimal for complement activation and successfully demonstrated the first instance of DNA-mediated immune system activation. We are currently determining how different antibody geometries affect complement activation by exploring multiple nanopatterns based on hexagonal lattices, including the valency, height and geometry of the activating molecules. We have also performed high-resolution cryo-electron tomography of DNA-mediated complement activation and can clearly see stepwise binding, initiation, activation and termination of the entire immune pathway.
As a separate project, we are determining the structures of other immune-system activators, and have discovered that these are stimulated by pH changes and oxidative stresses induced in wound- and infection environments. These complexes are involved in immune defense and autoimmune disease progression, but also found in neurological disorders such as Alzheimers. As such, the structure-function relationship of these molecules will have far-reaching consequences for structural biologists, immunologists and neurologists.
Developing small-molecule complement activators has also progressed, but not in the direction initially envisaged. We were unable to derive agonistic aptamers based on nucleic acids. However, we have had success deriving peptide-based activators. We are currently pursuing these are potential immunotheraputics.
Developing DNA nanotechnology as a nanopatterning platform has also enabled us to form collaborations with various groups in immunology, organic chemistry, biotechnology and microscopy groups.
Using DNA to regulate complement activation is a novel method to gain control over a complex biological system. Our data show that we can now modify DNA to display biomolecules that are able to activate complement. This is a novel mechanism to control the spatial organization of antibody-binding sites to enable us to understand complement activation at both a structural and functional level.
We have identified small-molecule activators of complement, and are in the process of functionalizing DNA and other nanopatterning molecules to demonstrate cell-killing properties. This will lead to novel immunotherapeutics for the treatment of diseases such as cancer.