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 a catenane (two mechanically interlocked macrocycles) or a rotaxane (a threaded macrocycle with stoppers) which contain individual components held together by a topological bond. Molecular motion in such systems commonly relies on interactions between chemical components that are often difficult to synthesise, adapt and scale up. Biomolecular recognition on the other hand, which is also highly selective, offers a solution to these challenges. This project therefore looks to utilise biomolecular recognition, using protein and DNA segments as the building blocks, to devise new molecular machines that are easy to make, modular and scalable.

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.

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] Ali, A.; Bullen, G. A.; Cross, B.; Dafforn, T. R.; Little, H. A.; Manchester, J.; Peacock, A. F. A.; Tucker, J. H. R. “Light-controlled thrombin catalysis and clot formation using a photoswitchable G-quadruplex DNA aptamer” Chem. Commun., 2019, 55, 5627-5630.

[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.

Techniques

• Nucleic acid and peptide synthesis, modification & characterisation
• DNA and peptide engineering
• Bioconjugation chemistry
• Spectroscopy (e.g. NMR, UV, CD, fluorescence)
• Microscopy (AFM, fluorescence microscopy, cryo-EM)
• Mass spectrometry, HPLC
• Molecular dynamics simulations