Synthesis and studies on modified oligonucleotides for diagnostic and therapeutic applications

Thesis

This thesis describes two different but related areas: intercalative thiazole orange (TO) probes and artificial DNA backbones. Thiazole orange (TO) is an asymmetric cyanine dye that is used to detect nucleic acids by fluorescence due to its intercalating characteristics, which results in an "on-off" fluorescent switch on binding to complementary nucleic acids. Modified nucleic acid backbones improve the half-lives of therapeutic oligonucleotides in vivo by conferring resistance to nuclease enzymic digestion. These two areas are both potentially important role in terms of diagnostic and therapeutic oligonucleotide properties.

The use of TO in various fluorogenic probe systems is described in this thesis. Phosphoramidite monomers based on a thymidine nucleoside with alkyne and amine modifications at the 5-position of the base, and with various lengths of amine linker (C3, C6) were designed for the orthogonal labelling of oligonucleotides at thymine sites. A single tube labelling technique was developed to functionalise the amino group via amide bond formation and the alkyne by CuAAC click chemistry (1,4-disubstituted 1,2,3- triazole formation). These new monomers were tested in oligonucleotide probes designed to facilitate fluorescence resonance energy transfer (FRET) between TO and a FRET acceptor/reporter dye (e.g. ROX) on binding to their complementary DNA or RNA targets. The steady state and time-resolved fluorescence properties of these modified oligonucleotides were investigated. Thermal duplex melting experiments revealed a beneficial increase in melting temperature (Tm) due to the presence of single or multiple thiazole orange dyes, whereas the reporter dye caused duplex destabilisation. These new combination probes were used to detect a SARS-COV-2 sequence in a LAMP (Loop- mediated isothermal amplification) assay where they provided good specificity and high sensitivity.

The synthesis and properties of oligonucleotides modified with triazole-locked nucleic acids (TL-LNA) were studied with antisense applications in mind. Triazole backbones are charge neutral five-atom connections that have previously been shown to be duplex destabilising, hence inappropriate for antisense applications. I show that the thermal stability of oligonucleotides containing the TL backbone bound to complementary RNA targets is improved when the triazole is combined with a locked nucleic acid sugar on the 3 ́-side of the triazole ring. TL-LNA nucleoside phosphoramidite dimers were synthesised to make the required oligonucleotide sequences. LNA-TLs of the kind produced in this study have more favourable properties than previous TL modifications.

Authors: Bai, C

• DOI: 10.5287/ora-kzzoyb0gg
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: synthesis; modified oligonucleotides
• Subjects: Nucleic acid probes