EASTBIO Using chemistry to reveal nuclear architecture – Multiplexed labelling of RNA, DNA and proteins

Project Details:

SAF-A (Scaffold Attachment Factor A) is a key nuclear protein with gene mutations and aberrant expression linked to neurological disorders such as epilepsy and autism.1 The Gilbert lab has shown that SAF-A oligomerises in the presence of ATP and chromatin-associated RNA form a dynamic nuclear mesh, which maintains euchromatin in a decompacted configuration, with low SAF-A protein levels causing genomic instability.2 To fundamentally understand how SAF-A interacts with DNA and RNA to form a nuclear mesh and how aberrant protein scaffold levels affect nuclear architecture, it is necessary to simultaneously label and visualise SAF-A, RNA and DNA at high resolution. This multidisciplinary project aims to develop a multiplexed imaging platform to investigate the localisation of proteins (SAF-A), RNA and DNA in a mammalian nucleus.

The Lilienkampf and Gilbert Labs have shown that RNA andDNA can be independently labelled using bioorthogonal chemistries. Metabolic labelling of RNA and DNA with chemically modified Uridine and Deoxyuridine (EdU), respectively, enabled subsequent visualisation by wide-field light microscopy using bioorthogonal tetrazine/alkene and azide/alkyne “click-chemistries” with fluorescently tagged probes. In this PhD project, we will now develop an imaging platform to enable simultaneous detection of RNA, DNA and proteins by using super-resolution microscopy.

To investigate nuclear organisation and kinetics, metabolically tagged DNA and RNA will be fluorescently abelled using azide and tetrazine-based fluorescent probes, whereas SAF-A can be labelled using a SNAP-tag or with fluorescent antibodies (methods available in the Gilbert lab). We will investigate a combination of different fluorophores.

Once successful multiplexed labelling is achieved in normal cells using wide field or confocal microscopy, the imaging platform will be transferred for super-resolution imaging, using D-STORM, taking advantage of methods we have recently developed. Finally, these novel imaging tools will be used to investigate the localisation and interaction of SAF-A, RNA and DNA in neuronal cells (such as LUHMES) and ultimately used to investigate how SAF-A depletion (by means of auxin-inducible degron system or by RNA nterference) affects nuclear architecture (and DNA damage response) in neurons.

Application Process:

To apply for an EASTBIO PhD studentship, follow the instructions below:

·        Informal enquiries should be addressed to Dr Annamaria Lilienkampf. To apply, please send a cover letter outlining your previous research experience and reasons for applying, alongside an up-to-date CV to [Email Address Removed]

·        After you have discussed the projects of interest to you with Dr Annamaria Lilienkampf., download and complete our Equality, Diversity and Inclusion survey and then fill in the EASTBIO Application Form and submit to Dr Llienkampf.

·        Send the EASTBIO Reference Form to your two academic/professional referees, and ask your referees to submit your references directly to Dr Annamaria Lilienkampf [Email Address Removed]

We anticipate that our first set of interviews will be held 6^th – 10^th February 2023.

If you have further queries about the application/recruitment process please contact EASTBIO

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