Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.646987
Title: Development of techniques and strategies for the characterisation and structure elucidation of oligonucleotides by mass spectrometry
Author: Fisher, Christopher H.
ISNI:       0000 0004 5364 2760
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2014
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Abstract:
In the past, mass spectrometry research has focused strongly on the study of peptides, proteins and other positive mode analytes. Negative ion mode research, particularly oligonucleotide research, has been somewhat neglected over the past two decades, largely due to their propensity for forming salt adducts and complex fragmentation pathways. However, the work in this thesis has focused on the use of negative ion mass spectrometry and ion mobility spectrometry for the characterisation of short, therapeutic-like RNA sequences. nESI-MS/MS-IMS-MS, a novel and robust method for the sequencing of oligonucleotides up to 35 nucleotides in length, is presented. This method utilises IMS separations to de-convolute the complex spectra produced by the fragmentation of oligonucleotides. nESI-MS/MS-IMS-MS has the potential for effective automation of oligonucleotide sequencing, from sample acquisition to final output of sequence. An early version of the program OSeq, a peak picking tool for oligonucleotide MS/MS spectra, is also presented, and is shown to be directly compatible with nESI-MS/MS-IMS-MS data. Oligonucleotide secondary structure is also examined in this thesis, using nESI-IMS-MS. The secondary structures of eight sequences were studied by CD, PAGE and nESI-IMS-MS, with the aim of determining the effectiveness of IMS for the separation of oligonucleotides based upon their secondary structures. Separation of the oligonucleotides was not achieved by IMS; however, evidence is presented for the persistence of structure in the gas-phase. Additionally, mass spectrometry was used to support research into the viruses, hepatitis B and MS2 bacteriophage. Protein and RNA stoichiometries were examined for the hepatitis B hPOL domain. An accurate mass was determined for a 172 nucleotide RNA, and nESI-IMS-MS was utilised for the de-convolution of an amphipol-swamped spectrum for the hPOL subunit, TP. The binding stoichiometry of RNA and two small molecules was examined by nESI-MS, with a view to unravelling MS2 capsid assembly inhibition. Mitoxantrone was shown to bind readily to RNA, and binding was increased by the presence of an adenine bulge in the RNA stem-loop, TR.
Supervisor: Ashcroft, Alison E. ; Stockley, Peter G. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.646987  DOI: Not available
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