Physical Sciences for Health CDT
Thesis project - "Expansion Microscopy as a tool to investigate DNA repair"
Dr Rob Neely, School of Chemistry
Professor George Bassel, School of Biosciences
Dr Jo Morris, Institute of Cancer and Genomic Studies
Dr Steve Thomas, Institute of Cardiovascular Sciences
Dr Iain Styles, School of Computer Science
The aim of this project is to investigate the development of Expansion Microscopy (ExM) and its application to the quantitative study of complex clusters of proteins assembled on DNA.
The DNA damage response (DDR) is a crucial process enabling detection and effective repair of DNA damage. Where this response is defective, mutations and genome wide aberrations result from reduced capability to correctly repair lesions. There are numerous types of DNA lesions, necessitating multiple, distinct pathways of DNA repair each incorporating numerous proteins. Aging is linked to DNA damage accumulation. This is thought to be a combination of reduced repair capacity in addition to normal endogenous DNA damage. Where patients present with inherited DDR defects, they are susceptible to diseases of premature aging. Such inherited defects are linked with predisposition to cancer, neurodegenerative disease and immune defects.
Despite much progress into the understanding of DDR and the implication of defects within this response, a major challenge exists; elucidating the proteins involved, DNA-protein and protein-protein interactions which drive correct choice of repair pathway, and how these are altered as a result of inherited DDR defects.
Light microscopy is commonplace in the study of biological systems; however, its resolution is limited to 250nm in the lateral direction and 400-750nm in the axial direction by the diffraction limit. This means any features smaller than or closer in proximity than this limit cannot be effectively resolved. Some instrumental, chemical and computational methods known as super resolution methods have been developed to overcome this limit, but are marred with complexity and specific requirements of samples, fluorophores and their environment.
Expansion Microscopy (ExM) is a method to aid with the visualisation of 3D biological specimens with nanoscale resolution of ~65nm on traditional microscopes. To achieve this, specimens (e.g. cells, tissue slices) are labelled (e.g. with a fluorescent antibody), and then embedded into a polyelectrolyte gel and the labels become anchored to the gel by an anchoring moiety. The gel can be expanded by dialysing in water. A digestion step is performed prior to this to remove any biological features which could hinder expansion to ensure specimens are expanded uniformly in all dimensions. Specimens can be expanded by ~4.5 times. By physically increasing the size of the specimen, sub diffraction limit molecular detail can be visualised.
This project will apply and extend the methodology that underpins ExM to visualise DNA repair mechanisms.
In order to achieve this, we will develop approaches to characterise the expansion of the gel to determine expansion factor and the uniformity of the expansion. This will allow the development of a quantitative framework and will be used to fine tune the expansion of the gel and the achievable effective resolution.
We will develop a method for immobilising and labelling DNA in the swellable polyelectrolyte gel by application of custom anchoring molecules and labelling procedures. Given the complex nature of nucleic acids, 3D computational analysis will be used to confirm the fidelity of expansion of DNA by comparing pre- and post-expansion images. Expanded images will be compared to those obtained via super resolution methods.
We will then incorporate visualisation of proteins involved in DNA repair process to determine their relative location, abundance on the DNA and response to drugs. These features will be resolved at unprecedented resolution and quantification will be enabled by application of state-of-the-art computational image analysis.