The project will be a collaboration with Prof Glenn Burley (University of Strathclyde)
The strong and specific binding of small molecules and proteins to DNA has relevance across many areas, from fundamental biophysics, through chemical biology to nanotechnology. In each application, the physical principles governing the ability of a molecule to select and bind strongly to a specific sequence of DNA bases need to be understood. As functional intermolecular interactions of DNA occur in the solution phase at room temperature it is equally essential that the structural dynamics of the molecules are measured to fully reveal the binding mechanism.
Recently, the NTH group has developed ultrafast 2D-IR spectroscopy as a tool for measuring biomolecular interactions. 2D-IR spectroscopy spreads the infrared spectrum of a molecule over two frequency axes, similar to 2D-NMR methods, allowing unravelling of complex spectra of mixtures and complexes such that a) base specific information on DNA structural dynamics in solution can be obtained b) spectral features associated with binding of small molecules to DNA can be observed c) the ultrafast time resolution of 2D-IR spectroscopy can be harnessed to temperature-jump initiation to observe the melting of DNA-ligand complexes in real time.
In this project we will take the next steps in understanding biomolecular interactions, using 2D-IR to study the binding of DNA aptamers to target molecules. Aptamers are short sequences of single-stranded DNA, identified by selective amplification from a random pool of sequences, which bind specifically to small molecules or proteins. Aptamer binding is accompanied by formation of secondary structures including loops and G-quadruplexes that are thought to facilitate specific intermolecular contacts. Although aptamers possess considerable potential for use in drug design or for biomarker identification, the mode of selection means that many of the details of their structure, dynamics or binding interactions are yet to be revealed. 2D-IR is ideally placed to deliver this knowledge via direct measurement of intermolecular contacts and the use of T-jump spectroscopy to observe the separation of aptamer-target complexes in real time, giving atomistic insight into binding mechanisms. This information will provide vital experimental validation of computational aptamer design methods.
The project will feature three sections:
1) 2D-IR spectroscopy of aptamer binding to small molecules. Using commercially-available aptamers for adenine tri-phosphate we will identify spectroscopic signatures of aptamer structures (loops and G-quadruplexes), providing the basis for later time resolved experiments.
2) 2D-IR spectroscopy of aptamer binding to protein targets. Using readily available proteins (lysozyme and thrombin) we will identify spectroscopic signatures of aptamer-protein contacts and observe the structural dynamics of aptamers and proteins separately and in combination. This will provide new insight into the structure and dynamics of the complexes.
3) T-jump 2D-IR spectroscopy will observe melting of aptamer-target complexes in real time. Use of small molecule and protein targets will reveal changes in secondary structure of the aptamer in response to complex melting, mapping out the potential energy landscape of the unfolded aptamer and the mechanisms of binding.
The work will be performed in the York Centre for Photochemistry and at the Rutherford Appleton Laboratory where NTH holds facility Programme Access to 2D-IR spectrometers.
The NTH group is one of two globally using T-jump 2D-IR spectroscopy to study DNA dynamics in solution and unique in applying it to biomolecular complexes. This work is therefore expected to generate high impact publications and feed into parallel projects developing high-throughput 2D-IR analysis of DNA binding systems.
The student will receive training in advanced time-resolved spectroscopy and handling of biological samples. Transferable skills associated with multidisciplinary research will be gained. Regular visits to the Rutherford Appleton Laboratory will add experience of using world-leading laser systems and of planning and leading experimental campaigns.
All Chemistry research students have access to our innovative Doctoral Training in Chemistry (iDTC): cohort-based training to support the development of scientific, transferable and employability skills: https://www.york.ac.uk/chemistry/postgraduate/idtc/
The Department of Chemistry holds an Athena SWAN Gold Award and is committed to supporting equality and diversity for all staff and students. The Department strives to provide a working environment which allows all staff and students to contribute fully, to flourish, and to excel: https://www.york.ac.uk/chemistry/ed/
. This PhD project is available to study full-time or part-time (50%).
This PhD will formally start on 1 October 2020. Induction activities will start on 28 September.