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Permanent link (DOI): https://doi.org/10.7939/R3FJ29K7W

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Novel Spectroscopic Probes for Detecting DNA Damage Open Access

Descriptions

Other title
Subject/Keyword
Fluorescence
Terbium
Chimeric RNA DNA probe
LNA probe
Hypochromism
2-aminopurine
Nucleic acid detection
Hybridization
Ultraviolet light
Molecular beacon
DNA damage
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
El-Yazbi, Amira F
Supervisor and department
Loppnow, Glen (Chemistry)
Examining committee member and department
Krull, Ulrich (Chemistry, U of T)
Le, X.C. Chris (Chemistry)
Gibbs-Davis, Julianne (Chemistry)
Campbell, Robert (Chemistry)
Department
Department of Chemistry
Specialization

Date accepted
2013-03-26T08:50:34Z
Graduation date
2013-06
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Absorption of UV light by nucleic acids can result in the formation of molecular lesions leading to mutagenesis, carcinogenesis, and cell death. Thus, understanding DNA damage is important for elucidating the molecular mechanisms of disease. Much effort has been focused on developing methods for detecting DNA damage. However, almost all of the proposed methods consist of multi-step procedures, are limited to a specific type of damage, require expensive instruments, and/or suffer from a high level of interference. In this thesis, I present some novel simple, mix-and-read fluorescent assays for the detection of DNA damage. The goal is to design probes that are superior to conventional fluorescent molecular beacons (MBs) in detecting DNA damage. The first approach was to design MBs with modified DNA backbones. For this purpose, locked nucleic acid (LNA) and chimeric RNA-DNA (chMB) MBs were designed. The results show that chMBs are more sensitive and selective for DNA damage than LNA MBs that have comparable selectivity to conventional MBs. However, these probes show a signal that is inversely proportional to the amount of damage. Therefore, the second approach was to design probes that give signals directly proportional to the amount of damage. For this purpose, probes with 2-aminopurine (2AP) were designed. Such probes show no fluorescence for undamaged DNA and fluorescence for damaged DNA. 2AP probes offer high sensitivity and selectivity comparable to MBs, but are expensive, especially with an increasing number of 2APs in the probe to increase sensitivity. Thus, the hypochromism probe was designed. For this probe, the absorbance signal increases with increasing amount of damage. Results show that the hypochromism probe is more selective and more than ten times cheaper than conventional MBs, but less sensitive. The need for a sensitive, selective and inexpensive probe was the motivation to design the Tb3+/hairpin probe. Single-stranded DNA greatly enhances the Tb3+ emission, but duplex DNA does not. Undamaged DNA targets will hybridize with the hairpin with no emission. The Tb3+/DNA hairpin probe proves to be the cheapest, most sensitive and selective probe for the quantification of DNA damage of all the probes presented here.
Language
English
DOI
doi:10.7939/R3FJ29K7W
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
A. El-Yazbi, G.R. Loppnow, Can. J. Chem. 89 (2011) 402-408.A.F. El-Yazbi, G.R. Loppnow, Anal. Chim. Acta. 726 (2012) 44-49.A.F. El-Yazbi, G.R. Loppnow, Can. J. Chem., published on the web 10 December 2012, 10.1139/cjc-2012-0417.

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