This thesis focuses on understanding the interactions of cyanine dyes with DNA, through the use of both experimental and computational techniques. Although cyanine dyes are widely used as nucleic acids stains in fluorescence applications, the nature of the association process is not always clear. The cyanine dye PicoGreenRTM (PG) has proven to be extremely useful in monitoring DNA damage but its structure is proprietary, making it impossible to understand its specific interactions with DNA. However, its structure is known to resemble that of another cyanine dye, thiazole orange (TO). We anticipated that the N-propyl pyridinium derivative of TO (PTO) would also intercalate in DNA and that its extra positive charge, relative to TO, would aid in the association. Our studies have focused on the associations of PG, TO and PTO with DNA.
Chapter 3 deals with the association of the dyes with single-stranded DNA homopolymers. The combination of spectroscopic techniques and molecular dynamics (MD) calculations provides a unique understanding of the associations of TO and PTO with single-stranded DNA homopolymers. There is highly specific binding of TO and PTO to poly(dG) and poly(dA), while poly(dC) and poly(dT) bind the dyes very weakly and appear to promote dye aggregation. Due to its proprietary structure, PG could not be studied computationally. However, the experimental spectral results suggest that PG associates differently with single-stranded DNA than do TO and PTO.
There are two major findings in Chapter 4. Firstly, the dyes associate strongly with double-stranded DNA, as demonstrated by the experimental spectral results and the MD calculations. Experimental evidence supports not only monomeric dye intercalation in DNA, but also dimeric dye intercalation. The results of the MD simulations suggest that TO and PTO bind to DNA without sequence specificity. The second major finding was that a new type of stable dye/DNA complex is formed when single strands of poly(dA) and poly(dT) are hybridized in the presence of PG or PTO, which cannot be obtained by addition of the dye to poly(dA)·poly(dT). For all three dyes, complete DNA renaturation did not occur during thermal cycling of dye/double-stranded calf thymus DNA. These results suggest that intercalating cyanine dyes can interfere with DNA hybridization to double-stranded DNA.
Chapter 5 is concerned with a more practical aspect of the association of cyanine dyes with DNA: using thiazole orange to report UVC-induced DNA damage. A variety of spectroscopic techniques, as well as agarose gel electrophoresis, were examined for their ability to detect UVC-induced DNA damage. The most sensitive methodology of all of those tested was fluorescence spectroscopy using TO. All of the spectroscopic techniques involving TO suggest that TO intercalation is susceptible to UVC-induced DNA damage. The computational studies suggest that the presence of cyclobutadipyrimidines, and not 8-oxo-2'-deoxyguanosine, is a factor in the experimentally observed reduction in dye intercalation. These studies have been a proof-of-concept, to demonstrate the usefulness of this fluorescence technique in detecting high levels of DNA damage, comparable to those used in food irradiation.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/29680 |
| Date | January 2007 |
| Creators | Mikelsons, Larisa |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 309 p. |
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