| Popis: |
The genome of eukaryotic cells is constantly under attack. The DNA damage products resulting from such attacks are generally termed lesions, which can be generated by endogenous cellular processes or exogenous agents. Determining the structural impact these lesions have on DNA is critical for elucidating the mechanisms by which they exert their toxicity. Specifically, it is known that aryl radical species react with the C8-site of deoxyguanosine (dG) to form C8-aryl-dG adducts. Similar bulky DNA adducts may be formed through the covalent attachment of electrophilic species to the C8-site of dG. Typically, this leads to alteration of the overall structure and context of DNA, and such changes may lead to the propagation of a modified genetic code. Many of these adducts possess conformational heterogeneity within DNA, which has been proposed to account for various mutagenic outcomes. In order to study the impact of C8-aryl-dG lesions, authentic oligonucleotide standards are required. Standard solid-phase DNA synthetic procedures are inefficient at generating C8-aryl-dG modified oligonucleotides. This is due to the inherent acid sensitivity of these adducts, which results in larger rates of hydrolysis. Thus, it was necessary to develop an efficient solid phase synthesis of oligonucleotides containing acid sensitive C8-aryl-dG adducts. Such a method was here optimized and is presented in this thesis. Aside from altering the stability of the glycosidic bonds, C8-aryl dG adducts also display fluorescence emission. These fluorescent nucleosides have been shown to be sensitive to their solvent environment, base stacking and H-bonding interactions. This has provided a diagnostic handle within DNA, and allowed C8-aryl-dG adducts to function as “probing adducts”. Specifically, C8-aryl-dG adducts are here utilized in different systems to report on: (1) various duplex perturbations, which stem from adduct formation within the NarI recognition sequence, (2) duplex–quadruplex exchange reactions within the thrombin binding aptamer, a common strategy used in DNA-based diagnostics, and (3) G-quadruplex conformational heterogeneity within the human telomeric sequence, which has implications in cancer and aging. The work presented in this thesis establishes 8-aryl-dG adducts as effective fluorescent probes, and supports their use in molecular diagnostic systems aimed at studying various DNA structure-function relationships. |
