Unveiling the structure of RNA using EPR spectroscopy

Abstract

This thesis describes work on the application of Electron Paramagnetic Resonance (EPR) to long non-coding RNA (lncRNA), which are transcribed from non-protein coding regions present in the human genome. As little is known about lncRNA secondary and tertiary structures, investigating the correlation between their conformation and their function is of great interest. EPR is a spectroscopy technique used to analyse and identify chemical species defined as paramagnetic and since RNA is not, the labelling of RNA with radical probes is necessary. This is implemented through a technique called site directed spin labelling (SDSL), which consists in the transcription of RNA with suitably modified nucleotides triphosphate (NTP) in specific positions to which probes are attached.

Labelling is implemented using Cu(I)-catalysed azide-alkyne cycloaddition (CuAAC) and strain promoted alkyne-azide cycloaddition (SPAAC). These require two reactant species: an alkyne and an azide. Therefore, the aim is to synthesise spin labels and nucleotides suitably modified with these functional groups to allow spin labelling via click reaction. In St Andrews, the problems of labelling RNA for Förster resonance energy transfer (FRET) studies have been a focus of Professor Carlos Penedo. His group has worked on an enzymatic method for stepwise transcription and labelling of a long non-coding RNA, specifically a 71 nt pbuE bacterial riboswitch, using the T7 RNA Polymerase and Escherichia coli RNA polymerase (RNAP). Once the transcribed RNA has been obtained (modified at specific sites), the FRET labelling has been carried out incorporating fluorescent probes using click chemistry methods. In this way it was possible to study the riboswitch conformations using FRET spectroscopy. Following this approach, the aim of this thesis is to adapt the concept for producing spin-labelled RNA in sufficient quantity for EPR measurements. My aim is to demonstrate a post-transcriptional spin labelling approach to allow the incorporation spin labels at specific positions. I tested which modified-for-click RNA uridine (U) nucleotides can be incorporated via transcription, and I tested how feasible the click reaction is for spin labelling the RNA. Further, I developed the stepwise strategy for allowing the site directed spin labelling of long RNA at specific positions. Success in these approaches, and extension to other modified nucleotides, should lead to a flexible and high yielding method for producing spin-labelled RNA without limit on length (as for chemical approaches) or position (as for ligation).

Date of Award	12 Jun 2023
Original language	English
Awarding Institution	University of St Andrews
Supervisor	Janet Eleanor Lovett (Supervisor)