Biophysical investigation of M-DNA
Wood, David Owen
M-DNA is a complex formed between normal double-stranded DNA and the transition metal ions Zn2+, Ni2+, and Co2+ that is favoured by an alkaline pH. Previous studies have suggested that M-DNA formation involves replacement of the imino protons of G and T bases by the transition metal ions involved in forming the complex. Owing to the conductive properties of this unique DNA conformation, it has potential applications in nanotechnology and biosensing. This work was aimed at improving existing methods and developing new methods of characterizing M-DNA. The effects of base substitutions, particularly those of G and T, were evaluated in light of the proposed structure. Differences between M-DNA conformations induced by Zn2+ and Ni2+ were also investigated with a variety of techniques and compared to the effects of Cd2+ and Mg2+ on double-stranded DNA. M-DNA formation and stability were studied with an ethidium bromide (EtBr) based assay, M-DNA induced fluorescence quenching of DNA labelled with fluorescein and a compatible quenching molecule, isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). Production of monoclonal antibodies against the conformation was also attempted but was unsuccessful. The EtBr-based assay showed Ni(II) M-DNA to be much more stable than Zn(II) M-DNA as a function of pH and in the presence of ethylenediaminetetraacetic acid. Sequence-dependency and the effect of base substitutions were measured as a function of pH. With regards to sequence, d(G)n•d(C)n tracts were found to form the conformation most easily. Base substitutions with G and T analogues that lowered the pKa of these bases were found to stabilize M-DNA more strongly than other base substitutions. A combination of temperature-dependant EtBr and ITC assays showed M-DNA formation to be endothermic, and therefore entropy driven. The SPR studies demonstrated many qualitative differences between Zn(II) and Ni(II) M-DNA formation, allowed characterization of Zn2+, Ni2+, Cd2+, and Mg2+ complexes with single-stranded DNA, and provided unambiguous evidence that M-DNA formation results in very little denaturation of double-stranded DNA. Specifically, the SPR study showed Ni(II) M-DNA to be more stable than Zn(II) M-DNA in the absence of transition metal ions, but also showed that Ni(II) M-DNA required higher concentrations of Ni2+ than Zn2+ to fully form the respective M-DNA conformations. Finally, quenching studies demonstrated Zn(II) M-DNA formation over a pH range from 6.5 to 8.5 provided that a Zn2+:H+ ratio of roughly 105 was maintained. The Keq for this interaction was 1.3 x 10-8 with 1.4 H+ being liberated per base bair of M-DNA formed. These results support the proposed structural model of M-DNA, as lowering the pKa of the bases having titratable protons over the pH range studied facilitated M-DNA formation. The fact that Zn(II) M-DNA formation was observed by fluorescence quenching at any pH provided that a constant ratio of Zn2+:H+ was maintained was consistent with a simple mass-action interaction for M-DNA formation. The differences between Zn(II) and Ni(II) M-DNA formation show that although it requires a higher pH or transition metal ion concentration, Ni(II) M-DNA is more stable than Zn(II) M-DNA once formed. This difference could play an important role in applications of M-DNA which required modulation in the stability of the M-DNA conformation.
DegreeDoctor of Philosophy (Ph.D.)
SupervisorLee, Jeremy S.
CommitteeSen, Dipankar; Qualtiere, Louis; Loh, Lambert; Laferte, Suzanne; Khandelwal, Ramji L.; Bonham, Keith
Copyright DateMay 2005
Surface Plasmon Resonance
DNA-metal ion interactions