Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.724920
Title: Biophysical studies of oligonucleotides containing duplex stabilising modifications
Author: Marafini, Pietro
ISNI:       0000 0004 6421 4833
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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Abstract:
Chemically modified DNA and RNA probes have been used in numerous diagnostic (e.g. fluorogenic PCR and in situ hybridisation) and therapeutic (e.g. exon-skipping and antisense) applications to increase duplex stability and facilitate mismatch discrimination. There is a constant need for well-studied modifications, and to this end X-pyrene was evaluated as a new cytosine analogue that combines enhanced p-stacking, hydrogen bonding and electrostatic interactions to greatly increase the stability of bulged DNA duplexes and DNA/RNA hybrid duplexes. X-pyrene is highly selective for guanine, and even more importantly duplex stability is reduced dramatically when X-pyrene or a neighbouring base is mismatched. NMR and fluorescence studies indicate that the pyrene moiety stacks within the DNA double helix. To enable easier access to this modification, a reductive amination labelling strategy was developed to produce pyrene-functionalised nucleobase analogues which allowed an easier synthesis of X-pyrene. Amino C6 dT was successfully functionalised with this methodology as well, obtaining the duplex stabilising mono- and di-labelled products. Additionally, a much simpler pyrene-modified thymidine monomer (U-pyrene) was selected for further studies. Single and multiple additions of U-pyrene were found to significantly increase the melting temperature of dsDNA without perturbing the B-DNA duplex, as shown by UV and CD spectroscopy. Interestingly, thermodynamic studies showed that four additions of U-pyrene increased the binding constant of a 14-mer oligonucleotide to its complementary strand 10000-fold when compared to the unmodified control. Two oligonucleotides containing a single addition of U-pyrene were also crystallised and their structures were studied by X-ray diffraction. To the author's knowledge these represent the first examples of X-ray crystal structures of a chemically modified pyrene oligonucleotide. They also proved to be of great interest as they highlight the capacity of U-pyrene to facilitate the production of interesting nanostructures.
Supervisor: Brown, Tom Sponsor: ATDBio Ltd
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.724920  DOI: Not available
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