Steady-state and time-resolved fluorescence spectroscopies have been used to investigate the photophysical properties of the fluorescent DNA base analogue, 2-aminopurine (2AP). For the first time, the fluorescence decay of 2AP is shown to be biexponential. In aqueous solution, 2AP has lifetimes of 11.0ns and 13.5ns, which are shown to decrease in less polar solvents. These two lifetimes are attributed to the N7-H and N9-H tautomers of 2AP. The fluorescence responses of ten 2AP labelled DNA duplexes have been recorded. The decays are fitted by four discrete components and all are dominated by a ~100ps component, which is consistent with efficient quenching of the 2AP by electron transfer from guanine. The fluorescence response of 2AP within the duplexes is sensitive to both the nature of the bases in its immediate vicinity and the extended nucleotide sequence. 2AP labelled DNA has been used as a probe of base flipping by the M.HhaI methyltransferase enzyme. The DNA was labelled such that duplexes with the 2AP adjacent to, opposite and at the target site for base flipping have been studied. Duplexes with the 2AP outside of the M.Hhal recognition sequence have also been investigated. When 2AP is the target base for flipping its fluorescence decay shows a significant response to enzyme binding. It is shown that this change in photophysical behaviour can be used as a definitive indicator of the base flipping mechanism. A similar response is also shown for the M.TaqI base flipping enzyme. Placing the 2AP at positions adjacent to and opposite the target base for flipping demonstrate the amazing selectivity, of HhaI for its target base and demonstrate the effectiveness of the enzyme’s stabilisation of the DNA duplex during base flipping.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:659907 |
| Date | January 2005 |
| Creators | Neely, Robert K. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/12712 |
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