<p class=MsoNormal style='text-align:justify;text-indent:.5in;line-height:150%'><span
style='mso-bidi-font-weight:bold'>M-DNA is a novel complex formed between DNA
and transition metal ions under alkaline conditions.<span
style='mso-spacerun:yes'> </span>The unique properties of M-DNA were
manipulated in order to rationally place metal ions at specific regions within
a double-stranded DNA helix.<span style='mso-spacerun:yes'>
</span>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.<span
style='mso-spacerun:yes'> </span>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.<span style='mso-spacerun:yes'> </span>The pH at which M-DNA formation
occurs is further decreased by the incorporation of 4-thiothymine (s<sup>4</sup>T).<span
style='mso-spacerun:yes'> </span>DNA oligomers with a mixed sequence composed
of </span>half d(AT) and the other half d(TG)d(CA)<span style='mso-bidi-font-weight:
bold'> showed that only 50% of the DNA is able to incorporate Zn<sup>2+</sup>
ions at pH 7.9.<span style='mso-spacerun:yes'> </span>This suggests that only
regions corresponding to the tracts of <span class=GramE>d(</span>TG)d(CA) are
being transformed.<span style='mso-spacerun:yes'> </span><o:p></o:p></span></p>
<p class=MsoNormal style='text-align:justify;text-indent:.5in;line-height:150%'><span
style='mso-fareast-language:ZH-CN'>Duplex DNA monolayers were self-assembled on
gold through <span class=GramE>a</span> 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.<span
style='mso-spacerun:yes'> </span>The film thickness, density, elemental
composition and ratios for samples were analyzed and compared.<span
style='mso-spacerun:yes'> </span>The DNA surface coverage, calculated from
both XPS and electrochemical measurements, was <span class=GramE>approximately
1.2 x 10<sup>13 </sup>molecules/cm<sup>2</sup></span><sub> </sub>for
B-DNA.<span style='mso-spacerun:yes'> </span>All samples showed distinct peaks
for C 1s, O 1s, N 1s, P 2p and S 2p as expected for a thiol-linked DNA.<span
style='mso-spacerun:yes'> </span></span><span style='mso-bidi-font-weight:
bold'>On addition of Zn<sup>2+</sup> to form M-DNA the C 1s, P 2p and S 2p
showed only small changes </span><span style='mso-fareast-language:ZH-CN'>while
both the N 1s and O 1s spectra changed considerably.<span
style='mso-spacerun:yes'> </span>This result is consistent with Zn<sup>2+</sup>
interacting with oxygen on the phosphate backbone as well as replacing the
imino protons of thymine (T) and guanine (G) in M-DNA.<span
style='mso-spacerun:yes'> </span>Analysis of the Zn 2p spectra also
demonstrated that the concentration of Zn<sup>2+</sup> present under M-DNA
conditions is consistent with Zn<sup>2+</sup> binding to both the phosphate
backbone as well as replacing the imino protons of T or G in each base
pair.<span style='mso-spacerun:yes'> </span>After the M-DNA monolayer is
washed with a buffer containing only Na<sup>+</sup> the Zn<sup>2+</sup> bound
to the phosphate backbone is removed while the Zn<sup>2+</sup> bound internally
still remains. </span><span style='mso-bidi-font-weight:bold'>Variable angle x-ray
photoelectron spectroscopy (VAXPS) was also used to examine monolayers
consisting of mixed sequence oligomers.<span style='mso-spacerun:yes'>
</span>Preliminary results suggest that under M-DNA conditions, the zinc to
phosphate ratio changes relative to the position of the <span class=GramE>d(</span>TG)d(CA)
tract being at the top or bottom of the monolayer.<span
style='mso-spacerun:yes'> </span><span style='mso-spacerun:yes'> </span><o:p></o:p></span></p>
<p class=MsoNormal style='text-align:justify;text-indent:.5in;line-height:150%'><span
style='mso-bidi-font-weight:bold'>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.<span
style='mso-spacerun:yes'> </span>T</span>he effectiveness of using the IrCl<sub>6</sub><sup>2-/3-
</sup>redox couple to investigate DNA monolayers and the potential advantages
of this system over the standard Fe(CN)<sub>6</sub><sup>3-/4-</sup> redox
couple are demonstrated.<span style='mso-spacerun:yes'> </span>B-DNA
monolayers were converted to M-DNA by incubation in buffer containing 0.4 mM Zn<sup>2+</sup>
at pH 8.6 and studied by cyclic voltammetry (CV), electrochemical impedance
spectroscopy (EIS) and chronoamperometry (CA) with IrCl<sub>6</sub><sup>2-/3-</sup>.<span
style='mso-spacerun:yes'> </span><sup><span style='mso-spacerun:yes'> </span></sup>Compared
to B-DNA, M-DNA showed significant changes in CV, EIS and CA spectra.<span
style='mso-spacerun:yes'> </span>However, only small changes were observed
when the monolayers were incubated in Mg<sup>2+ </sup>at pH 8.6 or in Zn<sup>2+</sup>
at pH 6.0.<span style='mso-spacerun:yes'> </span>The heterogeneous
electron-transfer rate (<i style='mso-bidi-font-style:normal'>k</i><sub>ET</sub>)
between the redox probe and the surface of a bare gold electrode was determined
to be 5.7 x 10<sup>-3</sup> cm/s.<span style='mso-spacerun:yes'> </span>For a
B-DNA modified electrode, the <i style='mso-bidi-font-style:normal'>k</i><sub>ET</sub>
through the monolayer was too slow to be measured.<span
style='mso-spacerun:yes'> </span>However, under M-DNA conditions, a <i
style='mso-bidi-font-style:normal'>k</i><sub>ET</sub> of 1.5 x 10<sup>-3</sup>
cm/s was reached.<span style='mso-spacerun:yes'> </span>As well, the percent
change in resistance to charge transfer (R<sub>CT</sub>), measured by EIS, <span
class=GramE>was</span> used to illustrate the dependence of M-DNA formation on
pH.<span style='mso-spacerun:yes'> </span>This result is consistent with Zn<sup>2+</sup>
ions replacing the imino protons on thymine and guanine residues.<span
style='mso-spacerun:yes'> </span>Also, at low pH values, the percent change in
R<sub>CT</sub> seems to be greater for <span class=GramE><span
style='mso-bidi-font-weight:bold'>d(</span></span><span style='mso-bidi-font-weight:
bold'>TG)<sub>15</sub>d(CA)<sub>15</sub> compared to oligomers with mixed
d(AT) and d(TG)d(CA) tracts.<span style='mso-spacerun:yes'> </span></span>The
IrCl<sub>6</sub><sup>2-/3- </sup>redox couple was also effective in
differentiating between single-stranded and double-stranded DNA during
dehybridization and rehybridization experiments.<span
style='mso-spacerun:yes'> </span><span style='mso-bidi-font-weight:bold'><o:p></o:p></span></p>
Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:SSU.etd-04252008-111143 |
Date | 28 April 2008 |
Creators | Dinsmore, Michael John |
Contributors | Warrington, Rob C., Sammynaiken, Ramaswami, Martz, Lawrence W., Lee, Jeremy S., kraatz, Heinz-Bernhard, Khandelwal, Ramji L., Geyer, C. Ronald, Yu, Hua-Zhong (Hogan) |
Publisher | University of Saskatchewan |
Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
Language | English |
Detected Language | English |
Type | text |
Format | application/pdf |
Source | http://library.usask.ca/theses/available/etd-04252008-111143/ |
Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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