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Title: Studies on chemically modified oligonucleotides and on DNA triplexes
Author: Moraru Allen, Ana-Ariana
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1997
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The interactions between modified oligonucleotides and DNA binding proteins can provide important information on the mode of binding and the structure of the enzyme-substrate complex. Studies were carried out to determine the interaction of DNA with O6-methyl-G-transferase, a DNA repair enzyme which removes the alkyl group from guanine residues which are alkylated on the O6-position. A series of modified oligonucleotides have been synthesised containing O6-methylguanine, O6-ethylguanine and N6-methyl-2,6-diaminopurine. The oligonucleotides were obtained in high purity. UV melting (thermal denaturing) experiments have been carried out on duplexes containing the modified bases paired with cytosine and thymine respectively. These oligonucleotides were prepared in order to study their interactions with a mutant O6-methyl-G-transferase enzyme lacking DNA repair activity. Gel shift experiments suggest that the above oligonucleotides are recognised by the enzyme. Attempts were made to crystallise the enzyme-DNA complex. Triplex DNA is formed by the specific binding of a DNA strand in the major groove of a preformed DNA duplex. It has been suggested that such structures might have a biological role, especially in gene regulation, and that there is potential for therapeutic applications in which gene expression is repressed by triplex formation. One difficulty encountered in the studies of DNA triplexes is their low thermodynamic stability. In order to overcome this problem oligonucleotides with appropriate triplex forming sequences have been synthesised with the three strands linked by hexaethyleneglycol or 1,8-octanediol chains. These molecules are capable of folding back on themselves to give intramolecular triplexes with significantly increased stabilities compared to intermolecular triplexes. UV melting studies have been carried out to determine the thermal stability of these triplexes, in the presence and in the absence of the DNA triplex binding drug coralyne. Circular dichroism and NMR studies were carried out in collaboration with Dr A. Lane at the National Institute of Medical Research, London.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available