The application of click reactions in the synthesis of modified oligonucleotides

Thesis

'Click' chemistry has been widely applied in nucleic acid studies, the most common reactions being the copper-catalysed and strain promoted alkyne-azide cycloadditions (CuAAC and SPAAC respectively). This thesis describes the use of these complementary reactions in the synthesis of modified oligonucleotides. Both reactions are used to ligate alkyne and azide-modified oligonucleotides together to form larger strands on the solid phase. The CuAAC reaction can produce ODNs containing a biocompatible triazole linkage that mimics the DNA phosphodiester linkage, however its use in vivo is limited by the cytotoxicity of Cu(I). In contrast, the SPAAC reaction does not require catalysis, but more chemically complex cyclooctynes are needed. Multiple sequential ligations by the SPAAC reaction can be carried out on solid-phase without a template oligonucleotide, allowing the efficient synthesis of oligonucleotides up to ca. 190 bases in length, and greatly simplifying purification. Click chemistry was used to synthesize novel nucleic acid fluorescent probes. Using the SPAAC reaction, fluorescently labelled oligonucleotides were ligated to obtain permanently cross linked stable duplexes, which were evaluated for their FRET properties. A new nucleic acid probe system was developed containing thiazole orange (TO) or benzothiazole orange (BO), and a second fluorophore was directly attached to the same nucleobase by CuAAC. The thermal stability and steady state fluorescence of the oligonucleotides containing these modifications were studied. These probes can be multiplexed for detection of point mutations in DNA. This was exemplified by targeting R516G mutation in the CFTR gene by simultaneously monitoring two channels in real-time PCR.

Authors: Qiu, J

• DOI: 10.5287/ora-zbobpv5k0
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford