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DNA molecular machines: Light activated shuttling on a DNA scaffold

Principal Supervisor: Professor James Tucker - School of Chemistry

Co-supervisor: Anna Peacock - Chemistry, Timothy Dafforn - Biosciences

PhD project title: DNA molecular machines: Light activated shuttling on a DNA scaffold

University of Registration: University of Birmingham

Project outline:

Scientists are investing significant effort into developing small molecular machines capable of translational or rotational motion at the molecular level in response to an external stimulus, with potential future applications in molecular electronics and data storage. These systems are often based on supramolecular architectures such as the catenane (two mechanically interlocked macrocycles) or rotaxanes (a threaded macrocycle with stoppers) that contain individual components held together by a topological bond. Docking stations which utilise recognition motifs are required in order to control molecular motion. However, the large majority of docking stations traditionally used, are not very sophisticated or selective. In contrast, biomolecular recognition in nature is extremely sophisticated and can be highly selective. This project therefore looks to couple biomolecular recognition, using protein and DNA segments as the building blocks, and architectures with potential applications as molecular machines.

Proteins such as transcription factors can bind to DNA with a high degree of sequence selectivity. Often direct contact is made between the DNA target site and a relatively short protein segment, commonly an alpha-helix, despite the protein being much larger. A number of examples exist which demonstrate that this small protein fragment alone can still display similar sequence selective DNA binding, and therefore these fragments and DNA represent attractive building blocks for assembling molecular machine type architectures.

In this project peptide sequences based on the DNA transcription factor GCN4 will be synthesised. Dimers of these have been shown to bind sequence selectively to DNA, therefore both dimers and fully cyclised versions will be prepared and their binding to DNA containing a target site, explored. Synthetic DNA containing two target sites with different affinity, and where one of these contains non-native functionality, will be prepared. For example an azobenzene unit introduced into the backbone of a high affinity target site (target site B) will allow trans-cis isomerization to be triggered by light, causing a large structural conformational change, thereby changing the affinity of the dimerised/cyclised peptide unit, resulting in translational motion to the second lower affinity target site (target site A). This can be revered on triggering the cis-trans isomerisation on heating or use of a different wavelength of light.

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In addition to the field of molecular machines, systems such as this could find potential applications for controlling the spatial alignment and positioning of an array of enzyme units, the order and proximity of which could be important in the ultimate biosynthetic pathway which is accessible, and which could in turn be triggered (turned on, off or altered) in response to an external stimulus.

References: 

  1. Duprey, JLHA; Carr-Smith, J; Horswell, SL; Kowalski, J; Tucker, JHR. “Macrocyclic Metal Complex–DNA Conjugates for Electrochemical Sensing of Single Nucleobase Changes in DNA”, J. Am. Chem. Soc, 2016, 138, 746-749
  2. Bullen, G. A.; Tucker, J. H. R.; Peacock, A. F. A. “Exploiting anthracene photodimerization within peptides: light induced sequence-selective DNA binding” Chem. Comm., 2015, 51, 8130-8133.
  3. 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.
  4. 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.
  5. 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:

Students will receive training in peptide, DNA and organic synthesis, as well as various biophysical techniques including, but not limited to, analytical ultracentrifugation, gel electrophoresis, ultraviolet-visible and circular dichroism spectroscopy.

Contact: Professor James Tucker, University of Birmingham