Localization of metal ions in DNA
| dc.contributor.advisor | Lee, Jeremy S. | en_US |
| dc.contributor.committeeMember | Warrington, Rob C. | en_US |
| dc.contributor.committeeMember | Sammynaiken, Ramaswami | en_US |
| dc.contributor.committeeMember | Martz, Lawrence W. | en_US |
| dc.contributor.committeeMember | kraatz, Heinz-Bernhard | en_US |
| dc.contributor.committeeMember | Khandelwal, Ramji L. | en_US |
| dc.contributor.committeeMember | Geyer, C. Ronald | en_US |
| dc.contributor.committeeMember | Yu, Hua-Zhong (Hogan) | en_US |
| dc.creator | Dinsmore, Michael John | en_US |
| dc.date.accessioned | 2008-04-25T11:11:43Z | en_US |
| dc.date.accessioned | 2013-01-04T04:29:49Z | |
| dc.date.available | 2009-04-28T08:00:00Z | en_US |
| dc.date.available | 2013-01-04T04:29:49Z | |
| dc.date.created | 2008 | en_US |
| dc.date.issued | 2008 | en_US |
| dc.date.submitted | 2008 | en_US |
| dc.description.abstract | M-DNA is a novel complex formed between DNA and transition metal ions under alkaline conditions. The unique properties of M-DNA were manipulated in order to rationally place metal ions at specific regions within a double-stranded DNA helix. Investigations using thermal denaturation profiles and the ethidium fluorescence assay illustrate that the pH at which M-DNA formation occurs is influenced heavily by the DNA sequence and base composition. For instance, DNA with a sequence consisting of poly[d(TG)•d(CA)] is completely converted to M-DNA at pH 7.9 while DNA consisting entirely of poly[d(AT)] remains in the B-DNA conformation until a pH of 8.6 is reached. The pH at which M-DNA formation occurs is further decreased by the incorporation of 4-thiothymine (s4T). DNA oligomers with a mixed sequence composed of half d(AT) and the other half d(TG)•d(CA) showed that only 50% of the DNA is able to incorporate Zn2+ ions at pH 7.9. This suggests that only regions corresponding to the tracts of d(TG)•d(CA) are being transformed. Duplex DNA monolayers were self-assembled on gold through a Au-S linkage and both B- and M-DNA conformations were studied using X-ray photoelectron spectroscopy (XPS) in order to better elucidate the location of the metal ions. The film thickness, density, elemental composition and ratios for samples were analyzed and compared. The DNA surface coverage, calculated from both XPS and electrochemical measurements, was approximately 1.2 x 1013 molecules/cm2 for B-DNA. All samples showed distinct peaks for C 1s, O 1s, N 1s, P 2p and S 2p as expected for a thiol-linked DNA. On addition of Zn2+ to form M-DNA the C 1s, P 2p and S 2p showed only small changes while both the N 1s and O 1s spectra changed considerably. This result is consistent with Zn2+ interacting with oxygen on the phosphate backbone as well as replacing the imino protons of thymine (T) and guanine (G) in M-DNA. Analysis of the Zn 2p spectra also demonstrated that the concentration of Zn2+ present under M-DNA conditions is consistent with Zn2+ binding to both the phosphate backbone as well as replacing the imino protons of T or G in each base pair. After the M-DNA monolayer is washed with a buffer containing only Na+ the Zn2+ bound to the phosphate backbone is removed while the Zn2+ bound internally still remains. Variable angle x-ray photoelectron spectroscopy (VAXPS) was also used to examine monolayers consisting of mixed sequence oligomers. Preliminary results suggest that under M-DNA conditions, the zinc to phosphate ratio changes relative to the position of the d(TG)•d(CA) tract being at the top or bottom of the monolayer. Electrochemistry was also used to investigate the properties of M-DNA monolayers on gold and examine how the localization of metal ions affects the resistance through the DNA monolayer. The effectiveness of using the IrCl62-/3- redox couple to investigate DNA monolayers and the potential advantages of this system over the standard Fe(CN)63-/4- redox couple are demonstrated. B-DNA monolayers were converted to M-DNA by incubation in buffer containing 0.4 mM Zn2+ at pH 8.6 and studied by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and chronoamperometry (CA) with IrCl62-/3-. Compared to B-DNA, M-DNA showed significant changes in CV, EIS and CA spectra. However, only small changes were observed when the monolayers were incubated in Mg2+ at pH 8.6 or in Zn2+ at pH 6.0. The heterogeneous electron-transfer rate (kET) between the redox probe and the surface of a bare gold electrode was determined to be 5.7 x 10-3 cm/s. For a B-DNA modified electrode, the kET through the monolayer was too slow to be measured. However, under M-DNA conditions, a kET of 1.5 x 10-3 cm/s was reached. As well, the percent change in resistance to charge transfer (RCT), measured by EIS, was used to illustrate the dependence of M-DNA formation on pH. This result is consistent with Zn2+ ions replacing the imino protons on thymine and guanine residues. Also, at low pH values, the percent change in RCT seems to be greater for d(TG)15•d(CA)15 compared to oligomers with mixed d(AT) and d(TG)•d(CA) tracts. The IrCl62-/3- redox couple was also effective in differentiating between single-stranded and double-stranded DNA during dehybridization and rehybridization experiments. | en_US |
| dc.identifier.uri | http://hdl.handle.net/10388/etd-04252008-111143 | en_US |
| dc.language.iso | en_US | en_US |
| dc.subject | M-DNA | en_US |
| dc.subject | X-ray photoelectron spectroscopy | en_US |
| dc.subject | DNA monolayers | en_US |
| dc.subject | Biosensors | en_US |
| dc.subject | Electrochemical Impedance Spectroscopy | en_US |
| dc.title | Localization of metal ions in DNA | en_US |
| dc.type.genre | Thesis | en_US |
| dc.type.material | text | en_US |
| thesis.degree.department | Biochemistry | en_US |
| thesis.degree.discipline | Biochemistry | en_US |
| thesis.degree.grantor | University of Saskatchewan | en_US |
| thesis.degree.level | Doctoral | en_US |
| thesis.degree.name | Doctor of Philosophy (Ph.D.) | en_US |