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Title: Studying the human telomeric intramolecular quadruplex using fluorescence resonance energy transfer
Author: Green, J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2004
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The work described here involves the use of fluorescence measurements to investigate the intramolecular quadruplex formed by the human telomeric repeat. By monitoring the efficiency of energy transfer between fluorophores either side of the structure, it is possible to follow the opening of the quadruplex in real time. In many of the experiments described here this opening was induced by the addition of a strand complementary to the quadruplex-forming sequence, to form a conventional duplex structure. Initial studies focused on the rate of opening of the human telomeric intramolecular quadruplex using peptide nucleic acid (PNA), an artificial nucleic acid mimic with an unchanged back-bone. It was observed that, for micromolar PNA, the rate of opening was independent of the concentration of PNA. This is probably due to the rate of opening being limited by a rearrangement of the quadruplex-forming strand that must occur before hybridisation can take place. The activation energy of opening was obtained by repeating the opening at several temperatures. One reason for the choice of a fluorescence-based technique is the great sensitivity with which such measurements can be performed. This allows the use of much lower sample concentration than would be possible with other measurements, such as circular dichroism or UV absorbance. With the correct equipment it is possible to detect the emission from single fluorescent species. These sorts of measurements remove the averaging that occurs when measuring many molecules at the same time. The use of single molecule detection was applied to the study of the human telomeric intramolecular quadruplex, for which there are two published structures. Two species were found in solution, and molecular modelling was used to propose assignments of these to known structures for this quadruplex. Finally, the interaction between the human telomeric intramolecular quadruplex and a peptide-hemicyanine conjugate ligand was investigated. In the presence of a complementary DNA strand the quadruplex opens and forms a duplex. It was observed that in the presence of sodium ions the ligand slowed down this hybridisation in a manner that allowed the determination of the binding constant between the quadruplex and the ligand. This was repeated at several temperatures in order to obtain thermodynamic and kinetic parameters for binding and hybridisation respectively. Furthermore, in the presence of potassium the ligand was observed to have no effect on the rate of hybridisation. The results of these experiments were used to suggest a structural model for binding.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available