Energy-structure correlations of plasmid DNA in different topological forms

Abstract

Differential scanning microcalorimetry (DSC), UV absorption and circular dichroism (CD) have been used to study structure and stability of linear (lin), open circular (oc), supercoiled (cd) and relaxed circular duplex (rd) DNA and calf thymus (CT) DNA. Investigations were made in low salt buffer and in the presence of 7.2 M NaClO4. The chaotropic action of perchlorate promotes a reduction of the overall stability of DNA, which permits a direct determination of the transition enthalpies of all four DNA configurations. The stabilities against thermal denaturation have been found to increase in the series lin approximately oc less than cd less than rd. These relative stabilities can be rationalized on the basis of the linkage between supercoiling and secondary structural changes in topologically constrained duplex DNA. On the basis of these studies, a model of the melting process could be suggested that is consistent with the energetic and spectroscopic data.

Differential scanning calorimetric studies of the thermal stability of plasmid DNA complexed with cationic lipids and polymers

“…This reduction in negative supercoiling would reduce the T M of the complex because fewer negative supercoils would be available to counter the positive supercoiling effect of increasing temperature. 6 This interaction would be qualitatively similar to the topological effects of intercalators such as ethidium bromide, which can induce positive supercoils into plasmids 15 but can also stabilize LIN/OC DNA by binding to G-C rich regions. 16 The DSC thermograms of DNA with ethidium bromide shown in Figure 1 are in fact quite comparable to the effects seen with PEI and PLL when added at low ratios to the DNA, in support of this hypothesis.…”

“…Recent improvements in the sensitivity and temperature range (up to 1258C) of modern pressurized differential scanning calorimeters, 5 however, allow the evaluation of the thermal stability of supercoiled (SC) DNA (which can unfold above 1008C) without the use of chaotropic agents to lower its denaturation temperature. 6 Therefore, DSC is a potentially unique tool with which to evaluate the stability of plasmid DNA in nonviral gene therapy vehicles.…”

“…The SC and closed circular forms of DNA unwind at a higher temperature than linear and nicked circular forms of the same sequence. 6 This is a result of the topological constraint imposed upon the molecule when in a closed circular form (with and without supercoiling), which reduces the cooperativity of base pair melting and the gain in entropy upon structural disruption. 7 Thermally induced changes will also introduce positive writhing into the plasmid to compensate for the unwinding effect of helix melting.…”

Dynamic binding capacity of plasmid DNA in histidine–agarose chromatography

“…Likewise, the observed changes in sc pDNA binding capacity can be attributed to temperatureinduced, pre-denaturation conformational changes. This hypothesis is supported by a calorimetric and spectroscopic study which describes that an increase in temperature promotes the removal of negative superhelical turns in sc pDNA molecules (Thumm et al, 1988). Since this relaxation is accompanied by a decrease in the number of dissociated base pairs (Thumm et al, 1988), the interaction of pDNA with the histidine ligands via the bases decreases, thus lowering the binding capacity.…”

“…This hypothesis is supported by a calorimetric and spectroscopic study which describes that an increase in temperature promotes the removal of negative superhelical turns in sc pDNA molecules (Thumm et al, 1988). Since this relaxation is accompanied by a decrease in the number of dissociated base pairs (Thumm et al, 1988), the interaction of pDNA with the histidine ligands via the bases decreases, thus lowering the binding capacity. It should also be stressed that a typical pDNA preparation contains sc topoisomers with multiple degrees of supercoiling (Murphy et al, 2003).…”