Curious how viruses are used to deliver genetic cargoes to cells? Want to know how we can employ this principle in vaccines and fight cancer? Then this CASE PhD studentship at the University of Leeds, in collaboration with AstraZeneca (Cambridge), might be for you.
Background and Objectives
Recent developments in the application of recombinant adeno-associated viruses (rAAVs) and other forms of nucleic acid encapsidation, aimed at delivering genetic material to cells, highlight the urgent need for a comprehensive structural characterization of these particles and their target interactions. Challenges arise around the characterization of the fully assembled particles and the detailed structural changes associated with capsid assembly, manufacture or storage.
In addition to capsid structure determination, there are clear deficits in our understanding of the higher-order structure of the single-stranded (ss)DNA encapsidated in these biopharmaceutical formulations and what determines it. In part this is due to the lack of suitable efficient structural and analytical tools. Development of such tools is particularly important as the field attempts to package nucleic acid exceeding the 4.7kbp of natural genomes. Recently native mass spectrometry (MS) in combination with ion mobility has been used to characterise nucleic acid structural motifs, such as aptasensing sequences (1). Hydrogen/deuterium exchange (HDX)-MS has also recently been demonstrated as a promising new technique to sample the higher-order structure of nucleic acids (2).
Experimental Approach
In this studentship, you will deploy established and develop novel structural biology techniques, with an emphasis on structural mass spectrometry methods. These are capable of providing valuable insights into rAAV capsid structure, ssDNA packaging, and the relationship between these, and changes that occur under biopharmaceutically relevant conditions.
Aim 1: Using a combination of HDX and fast photochemical oxidation of proteins (FPOP – unique in the UK), you will characterize structural changes of the capsid component of intact rAAVs in response to relevant stress conditions e.g. pH, temperature and shear. While HDX is the more established technique and heat-induced swelling has been studied for Dengue viruses (3), FPOP-MS is compatible with samples in complex backgrounds and introduces robust covalent labels that can be analysed by standard tryptic peptide digests and LC-MS/MS workflows. Recent studies have successfully generated preliminary FPOP maps of adenovirus 5 as a proof-of-concept (unpublished work, Leeds).
Aim 2: In collaboration with colleagues at the Diamond synchrotron (R. Rambo) you will use a newly established synchrotron radical footprinting facility to study DNA/RNA higher-order structure and how it relates to sequence and other spatial features. Viral packing has already been studied via synchrotron footprinting, which creates structure/interaction-dependent backbone cleavages, at the NSLSII in Brookhaven, USA, by the Stockley research group (4). This technique will be used here in the UK to characterise the ssDNA packaging mechanism in rAAV.
Aim 3: You will build upon the understanding gained in the application of these techniques to rAAV capsid and content, to explore the relationship between packaged nucleic acid and the resultant capsid structure (5). Empty capsids can be readily distinguished using imaging techniques, but the structural moieties that could be exploited for scalable purification strategies have yet to be characterised. Interactions between ssDNA and viral proteins in capsids are not well understood, nor are the resulting effects on capsid structures and dynamics, and these will be investigated via bespoke bioinformatic tools.
Novelty
The PhD project focuses on mass spectrometry- and synchrotron-based approaches to study viral protein and oligonucleotide structure. You will develop and apply pioneering technology at the three sites (Leeds, AZ/Cambridge and Diamond/Harwell) to target viral integrity, assembly and detailed structural features, with particular emphasis on a set of cutting-edge footprinting and mass spectrometry methods. You will be working with state-of-the-art structural MS equipment including several Waters Synapt HDMS, an Orbitrap Q Exactive UHMR and a unique high-resolution Tofwerk instrument (native MS/ion mobility), as well as HDX and FPOP capabilities and Orbitrap LC-MS/MS to support crosslinking experiments. You will also conduct synchrotron radical footprinting to determine oligonucleotide packing inside viruses.
Training
You will be trained in a wide range of mass spectrometry skills, as well as gaining experience in synchrotron footprinting and other structural and biochemical methods. The Astbury Centre for Structural Molecular Biology at the University of Leeds brings together researchers from across the University – largely from the biological sciences, chemistry and physics – to allow interdisciplinary approaches to be harnessed to understand the molecular basis of life. The Centre has outstanding expertise and research infrastructure in chemical biology, biophysics and all of the major techniques in structural molecular biology. Together, these approaches are combined with analyses of biological function with the ultimate aim of understanding the molecular basis of biological mechanisms in living cells. This PhD project is part of a Collaborative Training Partnership (CTP) between AstraZeneca (Cambridge) and the University of Leeds.
For more information about the project, click on the supervisor's name above to email the supervisor. For more information about the application process or funding, please click on email institution
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