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A structural study of M-DNAHoffort, Angela 24 July 2006
In alkaline conditions, a complex called M-DNA is formed between a divalent metal ion, cobalt, nickel or zinc, and duplex DNA. The rate of formation and stability of M-DNA is dependent on many factors, including but not limited to temperature, pH, DNA sequence, and metal or DNA concentrations. It has been hypothesized that the divalent metal ions intercalate into the helix and replace the imino protons involved in the hydrogen bonding of both G-C and A-T base pairs. The complex is thought to have a double helical structure that is similar to B-DNA. The presence of the divalent metal ions and a more compact structure may contribute to M-DNAs remarkable ability to behave as a molecular wire. Because M-DNA is so similar to B-DNA, it adheres to the same rules with regards to self-assembly. The ability of DNA to self-assemble and the electronic conduction of M-DNA are ideal properties for nanotechnology of the future. M-DNA may eventually be used to detect the presence of biologically important small molecules and DNA binding proteins that block the flow of electrons. However, before M-DNA will be widely accepted, it is necessary to obtain an accurate 3-dimensional structure by X-ray crystallography and modelling. <p> In this work interactions between divalent cobalt, nickel or zinc with duplex DNA were studied using two different experimental methods; namely, X-ray crystallography and extended X-ray absorption fine structure spectroscopy. First, crystals of the sequence d[GA(5FU)(5FU)AA(5FU)C] and d[CG(5FU)G(5FU)GCACACG] complexed with divalent metals were grown in M-DNA favouring conditions. Both of the sequences gave crystals that provided diffraction data that were analyzed by molecular replacement using B-DNA models. Unfortunately, the quality of the diffraction was not high enough with either sequence to locate metal binding or to determine a model for M-DNA. Second, X-ray absorption spectroscopy data were analyzed for the Ni2+ K-edge of both Ni2+ M and B-DNA. Several differences between the M and the B-DNA data were noticed and some final bond distances were established.
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2 |
A structural study of M-DNAHoffort, Angela 24 July 2006 (has links)
In alkaline conditions, a complex called M-DNA is formed between a divalent metal ion, cobalt, nickel or zinc, and duplex DNA. The rate of formation and stability of M-DNA is dependent on many factors, including but not limited to temperature, pH, DNA sequence, and metal or DNA concentrations. It has been hypothesized that the divalent metal ions intercalate into the helix and replace the imino protons involved in the hydrogen bonding of both G-C and A-T base pairs. The complex is thought to have a double helical structure that is similar to B-DNA. The presence of the divalent metal ions and a more compact structure may contribute to M-DNAs remarkable ability to behave as a molecular wire. Because M-DNA is so similar to B-DNA, it adheres to the same rules with regards to self-assembly. The ability of DNA to self-assemble and the electronic conduction of M-DNA are ideal properties for nanotechnology of the future. M-DNA may eventually be used to detect the presence of biologically important small molecules and DNA binding proteins that block the flow of electrons. However, before M-DNA will be widely accepted, it is necessary to obtain an accurate 3-dimensional structure by X-ray crystallography and modelling. <p> In this work interactions between divalent cobalt, nickel or zinc with duplex DNA were studied using two different experimental methods; namely, X-ray crystallography and extended X-ray absorption fine structure spectroscopy. First, crystals of the sequence d[GA(5FU)(5FU)AA(5FU)C] and d[CG(5FU)G(5FU)GCACACG] complexed with divalent metals were grown in M-DNA favouring conditions. Both of the sequences gave crystals that provided diffraction data that were analyzed by molecular replacement using B-DNA models. Unfortunately, the quality of the diffraction was not high enough with either sequence to locate metal binding or to determine a model for M-DNA. Second, X-ray absorption spectroscopy data were analyzed for the Ni2+ K-edge of both Ni2+ M and B-DNA. Several differences between the M and the B-DNA data were noticed and some final bond distances were established.
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Localization of metal ions in DNADinsmore, Michael John 28 April 2008
<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>
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4 |
Localization of metal ions in DNADinsmore, Michael John 28 April 2008 (has links)
<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>
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Oxidative Damage in DNA: an Exploration of Various DNA StructuresNdlebe, Thabisile S. 17 May 2006 (has links)
Research efforts to determine the causes, effects and locations of mutations within the human genome have been widely pursued due to their role in the development of various diseases. The main cause of mutations in vivo is oxidative damage to DNA via oxidants and free radical species. Numerous studies have been performed in vitro to determine how oxidative damage is induced in DNA. Most of these in vitro studies require photosensitizers to initiate the oxidative damage through various mechanisms. For the purposes of this research, all the photosensitizers that were used initiated oxidative damage in DNA through the electron transfer mechanism. In the charge transport studies, an anthraquinone photosensitizer was covalently linked to the 5 end of DNA by a short carbon tether in order to determine the pattern of damage induced along the length of the DNA. Anthraquinone preferentially damages guanine bases. Our first work sought to determine the effects of charge transport through guanine rich quadruplex DNA dimers. The dimers were formed by the combination of two hairpins with duplex overhangs extending beyond the quadruplex region. This enabled the optimal comparison of the effects of charge transport between duplex and quadruplex DNA structures. Another area of research we pursued in this area was to determine the effects of charge transport in M-DNA (a novel DNA conformation that was reported to form in the presence of zinc ions at a pH above 8). Earlier work on M-DNA suggested that it behaved like a molecular wire. Our research attempted to determine the effects of charge transport on this structure in order to show the behavior of a DNA molecular wire as compared to the standard studies performed in this area on normal B-DNA structures. Lastly, in collaboration with Dr. Ramaiah and colleagues we designed some viologen linked acridine photosensitizers which were tested for any ability to cleave GGG bulges. In preliminary studies, these viologen linked acridine derivatives showed preferential cleavage for guanine bases. They were not covalently bound to DNA, although they could potentially form non covalent interactions with DNA such as intercalation and/or groove binding. Our overall research goal was to determine the extent and overall effect of oxidative damage (using different photosensitizers) on the various DNA structures mentioned above.
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The Effect of Sample and Sample Matrix on DNA Processing: Mechanisms for the Detection and Management of Inhibition in Forensic SamplesMoreno, Lilliana I 23 March 2015 (has links)
The presence of inhibitory substances in biological forensic samples has, and continues to affect the quality of the data generated following DNA typing processes. Although the chemistries used during the procedures have been enhanced to mitigate the effects of these deleterious compounds, some challenges remain. Inhibitors can be components of the samples, the substrate where samples were deposited or chemical(s) associated to the DNA purification step. Therefore, a thorough understanding of the extraction processes and their ability to handle the various types of inhibitory substances can help define the best analytical processing for any given sample. A series of experiments were conducted to establish the inhibition tolerance of quantification and amplification kits using common inhibitory substances in order to determine if current laboratory practices are optimal for identifying potential problems associated with inhibition. DART mass spectrometry was used to determine the amount of inhibitor carryover after sample purification, its correlation to the initial inhibitor input in the sample and the overall effect in the results. Finally, a novel alternative at gathering investigative leads from samples that would otherwise be ineffective for DNA typing due to the large amounts of inhibitory substances and/or environmental degradation was tested. This included generating data associated with microbial peak signatures to identify locations of clandestine human graves. Results demonstrate that the current methods for assessing inhibition are not necessarily accurate, as samples that appear inhibited in the quantification process can yield full DNA profiles, while those that do not indicate inhibition may suffer from lowered amplification efficiency or PCR artifacts. The extraction methods tested were able to remove >90% of the inhibitors from all samples with the exception of phenol, which was present in variable amounts whenever the organic extraction approach was utilized. Although the results attained suggested that most inhibitors produce minimal effect on downstream applications, analysts should practice caution when selecting the best extraction method for particular samples, as casework DNA samples are often present in small quantities and can contain an overwhelming amount of inhibitory substances.
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