Principal Supervisor: Dr Anna Peacock - School of Chemistry
Co-supervisor: James Tucker (Chemistry), Timothy Dafforn (Biosciences)
PhD project title: Design and construction of new biobricks for synthetic biology, based on coiled coil DNA conjugates
University of Registration: University of Birmingham
An important component of synthetic biology is the development of a tool box of well understood biobricks with predictable properties and functions, which can be combined into larger architectures to develop new functional biosystems. The use of short DNA strands in DNA origami and nanotechnology is now well understood, taking advantage of predictable design rules. Similarly, so is the field of de novo design of peptide sequences which fold into well-defined miniature protein scaffolds. Though a range of folds have been explored, the rules for coiled coil formation (including varying number of strands, parallel, antiparallel, homo- and hetero-structures) are best understood.
Both DNA and peptide design therefore offer exciting opportunities to control and even trigger, association events and motion at the biomolecular level. They have both independently found applications to do this, however, to the best of our knowledge no reports exist which explore the design rules for conjugates containing both coiled coil peptide and DNA components. These conjugates would represent a new class of biobricks, in which either the peptide or DNA handle can be exploited, by introducing a switching mechanism between the two. This PhD project will therefore develop a library of peptide-DNA conjugates in an effort to evaluate the priority order of the respective design principles. Once these are established, switching mechanisms will be introduced into the design with the option to trigger enhancing or disabling one component, will be explored.
This project will involve automated peptide and DNA synthesis, their subsequent coupling, and studying their folding and association in solution. Peptides based on the (IAALEQK)x or (IAAIEQK)x heptad repeats are predicted to form two and three stranded coiled coils, respectively. When coupled to an appropriate DNA strand for formation of duplex DNA, the former will be expected to form the complementary two stranded coiled coil. However, use of the latter peptide sequence may in fact force the DNA to adopt an alternative triple helix structure. By varying the number of peptide heptad repeats, and the length of the DNA strand, we aim to understand the tipping point at which one biocomponent dominates. Similar strategies will be adopted for other coiled coil and DNA architectures. Introduction of switching sites for either enhancing or disabling the ability of one component to dominate will be explored next. Here the fact that both the peptide and DNA components are synthesised, opens up a large range of potential switching units that can be successfully introduced into the design. These include, though are not limited to, photoswitchable units (e.g. azobenzene or anthracene) into the backbone or side chains, or metal regulated units (e.g. polypyridyl switching groups, artificial metal enhanced nucleobases, engineered structural metal ion sites within the coiled coil). For example, Hg(II) ions binding to engineered cysteine sites in the interior of the coiled coil has been shown to control coiled coil oligomeric state. Alternatively DNA length can be increased by photoactivated dimerization of anthracene-DNA strands, thereby enhancing DNA hybridization. These various strategies will be investigated to control biomolecular recognition and folding.
- Berwick, M. R.; Lewis, D. J.; Pikramenou, Z.; Jones, A. W.; Cooper, H. J.; Wilkie, J.; Britton, M. M.; Peacock, A. F. A. “De Novo Design of Ln(III) Coiled Coils for Imaging Applications” J. Am. Chem. Soc., 2014, 136, 1166-1169.
- Zastrow, M.; Peacock, A. F. A.; Stuckey, J.; Pecoraro, V. L. “Hydrolytic Catalysis and Structural Stabilization in a Designed Metalloprotein” Nature Chem., 2012, 4, 118-123.
- Peacock, A. F. A. “Incorporating metals into de novo proteins” Curr. Opin. Chem. Biol., 2013, 17, 934-939.
BBSRC Strategic Research Priority: Industrial Biotechnology and Bioenergy
Techniques that will be undertaken during the project:
Training and experience will be gained in protein design, peptide and DNA synthesis, biophysical characterisation techniques including, but not limited to, analytical ultracentrifugation, gel electrophoresis, ultraviolet-visible and circular dichroism spectroscopy.
Contact: Dr Anna Peacock, University of Birmingham