Structural investigations of RNA through the application of Raman, Raman optical activity and surface enhanced spectroscopies

• Main Author: Hobro, Alison J.
• Published: University of Manchester 2008
• Subjects: 571.4
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491481

RNA molecules are involved in a wide range of cellular processes from genetic coding to catalytic activities, and determination of their secondary and tertiary structures is essential for the understanding of their functions. The work presented in this thesis shows the structural information that can currently be obtained using Raman and ROA spectroscopies for nucleic acid components and RNA molecules. Raman and ROA spectra can be used to identify particular building blocks, for example, with some marker bands characteristic for mono- and tri-nucleotides. In oligonucleotides and RNA sequences, Raman is particularly sensitive to the environment of the bases, while ROA is most sensitive to the conformation of the ribose-phosphate backbone. Therefore this complementary information can be used to identify single base changes and the introduction of a bulge sequence in the EMCV IRES Domain I, as well as allow tentative identification of structural features within the Adenovirus VA RNAr. Moreover, perturbation induced changes can also be monitored using Raman spectroscopy. Results presented here show these studies can be used to identify changes in specific parts of the molecule with particular temperatures, as well as identifying particular changes in secondary and/or tertiary structure with other perturbations, such as Mg2+ concentrations. The main limitation for ROA studies of RNA is the requirement for relatively high concentrations and long data collection times which can limit the range of RNA molecules that can be studied. Surface enhanced spectroscopic techniques can reduce the sample concentration and data collection requirements through the interaction of a molecule with surface plasmons associated with a nanoscale· roughened metal surface. The second half of this thesis concentrates on the implications of surface enhancement for Raman (SERS) and ROA (SEROA) studies of RNA. The application of SERS to RNA appears to be more complex than for other analyte species because the enhancement process is strongly time dependent. This, together with the spectral profile, can be influenced by a number of factors, including the nature of the aggregating agent and the concentration of the reagents involved. The effects of changes of these variables on the spectral profile and the time dependence of SERS enhancement are presented and discussed in the context of physical changes occurring during the experiment. Overall, Raman and ROA studies of RNA have provided information about the secondary and tertiary structures of RNA molecules despite their varying conformations and transitions, a matter that can be complicated for high-resolution techniques, such as X-ray crystallography. Surface enhanced Raman studies of RNA can, with careful preparation, provide meaningful results whilst reducing sample concentration requirements. However, the processes involved in generating surface enhanced ROA spectra appear to be significantly more complex and, for nucleic acid components, SEROA has not been successfully measured.