Characterisation of ss-RNA Structures using Vibrational Spectroscopies

  • Sarah Dowd

Student thesis: Phd


Antisense-Oligonucleotides (ASOs) are a next generation of therapeutic pharmaceutical, synthesised to be complementary to short lengths of mRNAs that are implicated in diseases such as Huntingtons and Spinal Muscular Atrophy. ASOs reduce or alter the expression of target mRNA. The secondary structure of ASOs is intrinsically linked to their binding affinity and efficacy. Secondary structure unfolding and folding can occur as a result of pharmaceutical (temperature and concentration changes) and environmental stresses (buffer and pH changes). Structural characterisation of ASOs using crystallography and Nuclear Magnetic Resonance (NMR) can be both expensive and challenging. Vibrational spectroscopies (Raman Spectroscopy, Fourier-Transform Infrared Spectroscopy, Raman Optical Activity and Circular Dichroism) have the potential to be quick methods for relating small changes in oligonucleotide secondary structure to stability or efficacy, prior to experiments with Small Angle X-Ray Scattering (SAXS) / NMR for more detailed structural analysis. Vibrational spectroscopies were used on increasingly complex single-stranded RNA (ss-RNA) structures. Raman is used to look at distinct spectral features of Nucleotides; examples include the sugar pucker of the Ribose ring and Imidazole ring peaks in Purines. A collaboration to generate Density Functional Theory (DFT) data has been used to improve on assignments of RNA nucleotides. Raman peaks are then identified for mono-, di- and tri- phosphate RNA nucleotides as well as Poly-RNA samples (Polyuridylic acid) and the hybridisation of a short 12-mer oligo. This library is used to assign the identity of an additional nucleotide within an N+1-mer of an AstraZeneca / Ionis Pharmaceuticals ASO: AZD9150. Combining Raman Optical Activity with Raman spectroscopy has previously been used to characterise secondary and tertiary RNA structural motifs, including a U-C mismatch and a hairpin (Hobro, 2007). In two AstraZeneca / Ionis Pharmaceuticals ASOs (AZD4785 and AZD Compound A), nucleotide and phosphate backbone modifications (such as Locked Nucleic Acids) affect spectroscopic assignments by inducing new secondary structure conformations. Raman has been used to look at experimental conditions related to pharmaceutical stresses (Hobro, 2008); concentration, buffer exchange and pH changes were observed to cause structural changes in AZD4785. SAXS is used to look at a fibril like hybridisation of ASOs at a high concentration; following this, NMR NOESY data is used to demonstrate how heat can prevent this conformation, potentially increasing drug efficacy. Raman, ROA and SAXS were used to observe the structure of ss-RNA within the Cowpea Mosaic Virus and detect changes following modification of the RNA to encode for GFP. Transmission Electron Microscopy and SAXS were used to compare viral capsids of a CPMV virus and a Virus Like Particle. The potential of plant viruses to be used to produce unmodified RNA on a large scale, as well as act as a delivery system for ASOs, is discussed. References: Hobro A. J. and Blanch E. W. et al. (2007) - Nucleic Acids Res, 35, 4, 1169 to 1177. Hobro A. J. and Blanch E. W. et al. (2008) - Vibrational Spectroscopy, 48, 1, 37 to 43
Date of Award1 Aug 2020
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorAndrew Doig (Supervisor), Jason Micklefield (Supervisor) & Royston Goodacre (Supervisor)


  • GFP
  • Ionis pharmaceuticals
  • Circular dichroism
  • mRNA
  • pharmaceuticals
  • vibrational spectroscopy
  • NMR
  • DFT
  • Assignments
  • FT-IR
  • library
  • virus protein coat
  • Icosahedral
  • Virus
  • Viral capsids
  • Nucleotides
  • Nucleotide
  • Structural Motifs
  • Density functional theory
  • Oligonucleotide
  • Transmission electron microscopy
  • RNA Structure
  • Structural
  • Raman
  • Raman Optical Activity
  • Spectroscopic
  • ASO
  • Antisense Oligonucleotide
  • single stranded RNA
  • Single-stranded RNA
  • Oligomer
  • AstraZeneca
  • Spectroscopy
  • AZD 4785
  • SAXS
  • VLP
  • Virus Like Particle
  • Cowpea Mosaic Virus
  • AZD 9150
  • AZD9150
  • Small angle x-ray scattering
  • CPMV
  • AZD4785

Cite this