Exciprobe Molecular Bio-Systems for Nucleic Acid Detection in Homogeneous and Heterogeneous Format

Rapid and reliable identification of genetic variations in humans could be crucial to the prediction of adverse drug responses at earlier stages of therapeutic treatment, but also could be vital to the recognition of variable patient susceptibility to certain disease states. In recent years considerable amount of efforts have been made towards the development of new techniques for SNP detection and analysis in both homogenous and heterogeneous formats in order to facilitate new strategies with improved efficiency and a high level of accuracy.The research presented in this thesis was focused on two main aspects. The first aspect was to initiate the development of the excimer- and/or exciplex based systems for potential application in the heterogeneous format, which could be used in the future for SNP analysis. At this proof-of-concept stage, gold nanoparticles (GNPs) were used as a solid-phase platform. In order to assess whether the exci-probes probes were capable of working on the surface of GNPs, two types of functionalised targets (i.e. Thiol-5'-GCCAAACACAGAATCG3' and Biotin-5'-GCCAAACACAGAATCG3') were immobilised on the GNP surface. It was confirmed in the independent experiments carried out in the homogeneous format that although the presence of either 5'-thiol or 5'-biotin anchor group might notably decrease the stability of the hybridised duplexes, these modifications did not prevent these systems to emit excimer fluorescence in solution phase upon hybridisation. However, fluorescence experiments performed in the heterogeneous format between the DNA-GNPs conjugates and 5'- and 3'-pyrene-modified probe systems showed rather disappointing results, presumably, due to a considerable quenching effect induced by GNPs. This led to a conclusion that GNPs-assembled hybrid systems may not be the best hybridization bio-assays in terms of detection of excimer or exciplex signal detection.The second aspect of this research was to explore some novel uni-molecular exci-probes (supplied by Link. Technologies. Ltd) bearing the new fluorescent reporter groups to achieve a suitable wavelength shift in excitation and emission for both monomer and excimer bands. The excimer-based and exciplex-based uni-molecular probe systems (i.e. 5PhacP4-3P3-3, 5CyacP4-3P3-3, 5triPhacP4-3P3-3, 5PhacP4-3N2-3 and 5CyacP4-3N2-3) demonstrated a notable red-shift of both excitation and emission bands, showing that the proof-of-principle stage has been achieved in terms of development of novel reporter groups that could match the fluorescence characteristics of commercially available instrumentation. However, these probes showed only limited success in terms of intensity of the emitted excimer-and/or-exciplex fluorescence and require further development.