|dc.description.abstract||Deoxyribonucleic acid (DNA) forms the basis of all known living organisms.
Despite the essential role played by DNA, its dynamic system and functional behaviour
are still not completely understood. The work presented in this thesis aims to explore
the structural dynamics of DNA systems, using fluorescence-based approaches, and to
attempt to develop a technique for the measurement of fluorescence decays of biological
molecules on the ultrafast (femtosecond) timescale.
Absorption of UV radiation by DNA is known to lead to mutations and damage to
DNA structure and functionality. For the majority of absorbed photons, the excitation
energy dissipates harmlessly as heat, but in some instances this energy transfers to
regions of DNA that are more susceptible to damage. 2-Aminopurine (2AP), a fluorescent
analogue of the native DNA base adenine, can be incorporated into DNA with minimal
perturbation to the DNA structure, and can be used to investigate inter-base electronic
energy transfer. By selectively exciting the native DNA base in 2AP-containing
dinucleotides and utilising 2AP fluorescence as an energy acceptor, the mechanism of
electronic energy transfer has been investigated. Analysis of the resulting fluorescence
lifetimes of 2AP has revealed that energy transfer preferentially excites conformations
in which the bases are highly stacked, and the fluorescence of 2AP is highly quenched.
This has led to a re-evaluation of energy transfer efficiencies between the natural bases
and 2AP, and has shown that transfer efficiencies cannot be determined correctly from
steady-state fluorescence measurements.
To investigate the influence of base dynamics on the quenching of 2AP
fluorescence in DNA, time-resolved fluorescence measurements were carried out on
2AP-containing systems in frozen solution at 77 K. These studies included dinucleotides,
single–strand oligonucleotides and their corresponding duplexes. In all cases,
comparison of the fluorescence decay parameters measured at room temperature with
those measured at 77 K showed that elimination of base dynamics prevented rapid
quenching, on the 10s of ps timescale or faster, although quenching on the 100s of ps
timescale persisted for 2AP in single strands and duplexes.
The multi-exponential fluorescence decay of 2AP in DNA and its high sensitivity to
local environment is commonly exploited to investigate DNA-enzyme interactions.
Transposases are enzymes involved in the movement of sections of DNA (transposons)
within the genome. The Mos1 transposase catalyses the movement of a transposon via
a cut-and-paste mechanism involving several intermediate complexes. Understanding
the complex mechanism by which the transposase can remove and insert a section of
DNA would allow these enzymes to be used as biomolecular tools. The structure of the
intermediate Mos1 strand-transfer complex (STC) has been investigated by
incorporating 2AP into several regions of the transposon and analysing the fluorescence
decay. The involvement of a base-flipping-like mechanism has been identified in the
mechanism of strand transfer for the Mos1 transposon.
The time-resolved fluorescence measurements performed in this thesis are
limited to time resolution of ~20 ps and longer using TSCPC. However, an abundance of
photophysical events in DNA occur on the femtosecond timescale. Development of a
methodology utilising fluorescence gating techniques (such as sum-frequency
generation or diffraction from a transient grating) have been attempted, in order to
construct an experimental system that enables the broadband detection of ultrafast
fluorescence decays. Despite the lack of immediate success in recording the fluorescence
decay from a sample, due to technical issues and time-constraints, initial
characterisation of the set-up was performed and the prospect of broadband detection
was demonstrated. Overall, this thesis gives insight into some of the dynamic processes
taking place in DNA and presents work performed to develop a system that would allow
the extension of these studies to processes occurring on the fs timescale.||en
|dc.contributor.sponsor||Engineering and Physical Sciences Research Council (EPSRC)||en
|dc.publisher||The University of Edinburgh||en
|dc.relation.hasversion||A bend, flip and trap mechanism for transposon integration E. R. Morris, H. Grey, G. McKenzie, A. C. Jones and J. M. Richardson, Elife, 2016, 5, 1–23. DOI: 10.7554/eLife.15537 URL: http://dx.doi.org/10.7554/eLife.15537||en
|dc.title||Photophysical studies of 2-Aminopurine in DNA||en
|dc.type||Thesis or Dissertation||en
|dc.type.qualificationname||PhD Doctor of Philosophy||en