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Localization of metal ions in DNA

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. <o:p></o:p>
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. <o:p></o:p>
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)15d(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. <o:p></o:p>

M-DNA

X-ray photoelectron spectroscopy

DNA monolayers

Biosensors

Electrochemical Impedance Spectroscopy

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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Localization of metal ions in DNA

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