Porphyrin moieties were rigidly attached to DNA to generate an accurate molecular ruler. Molecular ruler analysis was conducted using steady-state fluorescence, circular dichroism and small angle X-ray scattering spectroscopic techniques, in an attempt to analyse the FRET, exciton coupling and scattering intensity between different porphyrin-porphyrin labelled DNA combinations. A 21-mer test sequence was labelled with a porphyrin in one position on one strand, and seven different positions on seven complementary strands, to overall give seven porphyrin-porphyrin inter-strand combinations. Steady-state fluorescence and circular dichroism spectroscopic analysis of the Soret band revealed individual Watson-Crick bases pair molecular ruler sensitivity. Small angle X-ray scattering attempts between metallated-porphyrin entities did not reveal sufficient scattering at low concentrations, in contrast, an iodinated analogue of the porphyrin system did displayed scattering correlating to different iodine iodine distances. After calibration of the porphyrin system, the moieties were applied to study protein-DNA interactions between Tus, a 36 KDa DNA binding protein, and Ter, a specific 21-mer DNA sequence. Molecular ruler nalysis of the complex required an extended version of the Ter DNA sequence to which modifications were attached. Established FRET pairs FAM and TAMRA were applied to investigate protein-DNA complexation. Native PAGE analysis revealed Tus binds to the extended DNA via a sliding mechanism. Fluorescence analysis of the established FRET pairs identified changes in fluorescence not correlating to changes in FRET, and instead was attributed to emission quenching upon protein binding. Applying the zinc and free base porphyrin version displayed subtle changes in the Soret band circular dichrosim upon complexation, indicating small DNA helical change upon complexation. A 45-mer DNA sequence was designed to form multiple hairpin-duplex conformations with the addition of an appropriate complementary strand. Attaching FRET pairs to the extremes of the DNA sequence enabled multiple DNA conformations, and hence FRET distances to be obtained from one doubly modified DNA sequence. The combinations were characterised by UV-Vis, fluorescence and circular dichroism spectroscopy. Finally, terpyridine labelled DNA sequences selectively formed DNA nanotubes through orthogonal hydrogen bonding and metal complexation interactions. Short DNA strands were designed to self-assemble into long duplexes through a sticky-end approach. Addition of weakly binding metals such as zinc induced the formation of tubular arrays consisting of DNA bundles 50-200 nm wide and 2-50 nm high. TEM displayed additional long distance ordering of the terpyridine-DNA complexes into fibers.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:560571 |
| Date | January 2012 |
| Creators | Burns, Jonathan R. |
| Contributors | Stulz, Eugen |
| Publisher | University of Southampton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://eprints.soton.ac.uk/336428/ |
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