EASTBIO Revealing nuclear architecture – Multiplexed labelling of RNA, DNA and proteins using super-resolution microscopy

The aim of this project is to develop and use bioorthogonal tools to simultaneously label DNA and RNA, and to visualise them together with proteins, namely SAF-A, using super-resolution microscopy. These new tools will help to answer fundamental biological questions of how DNA, RNA and SAF-A interact in the cell nucleus and how SAF-A levels influence nuclear architecture in neurons. This truly multidisciplinary PhD project will provide skills in organic chemistry (e.g. synthesis of fluorophores, modified nucleosides, and tetrazines), cell culture and molecular biology techniques, as well as super-resolution imaging.

Project Details:
SAF-A (Scaffold Attachment Factor A, also known as HNRNPU) is a key nuclear protein with gene mutations and aberrant expression linked to neurological disorders such as epilepsy and autism.1 We have recently 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 RNA3 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.

The Lilienkampf and Gilbert Labs have shown that RNA and DNA can be independently labelled using bioorthogonal chemistry tools. 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 whereas SAF-A can be labelled using a SNAP-tag or with fluorescent antibodies (methods available in the Gilbert lab). 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 labelled using azide and tetrazine-based fluorescent probes alongside a fluorescent antibody for SAF-A. 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 direct stochastic optical reconstruction microscopy (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 interference) affects nuclear architecture (and DNA damage response) in neurons.

Application Process:
To apply for an EASTBIO PhD studentship http://www.eastscotbiodtp.ac.uk/, follow the instructions below:
Check FindaPhD https://www.findaphd.com/phds/program/bbsrc-eastbio-doctoral-training-partnership-call-for-applications-for-2021/?p1048 for our available projects and contact potential supervisors before you apply.

After you have discussed the projects of interest to you with the project supervisors, download and complete our Equality, Diversity and Inclusion survey https://edinburgh.onlinesurveys.ac.uk/eastbio-dtp-equality-diversity-inclusion-form-2021 and then fill in the EASTBIO Application Form and submit the application form plus your academic transcripts to Dr Annamaria Lilienkampf ([Email Address Removed]) (Links to the forms can be found here: http://www.eastscotbiodtp.ac.uk/how-apply-0)

Send the EASTBIO Reference Form to your two academic/professional referees, and ask them to submit these directly to Dr Annamaria Lilienkampf ([Email Address Removed]). (Link to the form can be found here: http://www.eastscotbiodtp.ac.uk/how-apply-0)

If you are nominated by the supervisor(s) of the EASTBIO PhD project you wish to apply for, they will provide a Supervisor Support Statement.

All EASTBIO (online) interviews will be in the week 8-12 February 2021 with awards made the following week.

The School of Chemistry holds a Silver Athena SWAN award in recognition of our commitment to advance gender equality in higher education. The University is a member of the Race Equality Charter and is a Stonewall Scotland Diversity Champion, actively promoting LGBT equality. The University has a range of initiatives to support a family friendly working environment. See our University Initiatives website for further information. University Initiatives website: https://www.ed.ac.uk/equality-diversity/help-advice/family-friendly