Biophysical studies of oligonucleotides containing duplex stabilising modifications

Thesis

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 π-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.

Authors: Marafini, P

• DOI: 10.5287/ora-545dpypep
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: pyrene; RNA; nucleic acids; DNA; crystallography; labelling of oligonucleotides
• Subjects: Biophysics; Chemical Biology; Chemistry; Biochemistry