Novel Spectroscopic Probes for Detecting DNA Damage

• Author / Creator

  El-Yazbi, Amira F

• 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.

• Subjects / Keywords

  • 2-aminopurine
  • Hypochromism
  • Fluorescence
  • Ultraviolet light
  • Terbium
  • Molecular beacons
  • LNA probe
  • Hybridization
  • Nucleic acid detection
  • DNA damage
  • Chimeric RNA DNA probe

• Graduation date

  Spring 2013

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3FJ29K7W

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.