Localization of metal ions in DNA

• Main Author: Dinsmore, Michael John
• Other Authors: Warrington, Rob C.
• Format: Others
• Language: en
• Published: University of Saskatchewan 2008
• Subjects: M-DNA; X-ray photoelectron spectroscopy; DNA monolayers; Biosensors; Electrochemical Impedance Spectroscopy
• Online Access: http://library.usask.ca/theses/available/etd-04252008-111143/

<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 (s⁴T).<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 Zn²⁺ 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 10¹³molecules/cm²</span>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 Zn²⁺ 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 Zn²⁺ 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 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.<span style='mso-spacerun:yes'>  </span>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. </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 IrCl₆^2-/3-redox couple to investigate DNA monolayers and the potential advantages of this system over the standard Fe(CN)₆^3-/4- 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 Zn²⁺ at pH 8.6 and studied by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and chronoamperometry (CA) with IrCl₆^2-/3-.<span style='mso-spacerun:yes'>  </span>^{<span style='mso-spacerun:yes'> </span>}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 Mg²⁺at pH 8.6 or in Zn²⁺ at pH 6.0.<span style='mso-spacerun:yes'>  </span>The heterogeneous electron-transfer rate (<i style='mso-bidi-font-style:normal'>k</i>_ET) between the redox probe and the surface of a bare gold electrode was determined to be 5.7 x 10^-3 cm/s.<span style='mso-spacerun:yes'>  </span>For a B-DNA modified electrode, the <i style='mso-bidi-font-style:normal'>k</i>_ET 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>_ET of 1.5 x 10^-3 cm/s was reached.<span style='mso-spacerun:yes'>  </span>As well, the percent change in resistance to charge transfer (R_CT), 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 Zn²⁺ 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_CT 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)₁₅d(CA)₁₅ compared to oligomers with mixed d(AT) and d(TG)d(CA) tracts.<span style='mso-spacerun:yes'>  </span></span>The IrCl₆^2-/3-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>