AAPH-induced DNA damage in uncondensed DNA and polyplexes. The DNA cleavage activity of AAPH was studied using gel electrophoresis of supercoiled pBR322 plasmid DNA. In all gel lanes, the lower band is due to supercoiled (SC) plasmid DNA while the upper band is due to open circular DNA (OC). It is believed that a single strand break is sufficient to convert SC DNA to the more relaxed OC DNA. When both strands are cleaved, a linear (L) DNA band appears between the SC and OC bands. Previous work has shown that AAPH, a water-soluble azo compound, generates peroxyl and alkoxyl free radicals upon degradation capable of inducing DNA oxidative damage. We first determined the effect of temperature on the generation of free radicals by AAPH. Figure shows the effect of incubating 200 ng of uncondensed pBR322 plasmid in 10 mM Tris-HCl buffer (pH 7.5) with varying concentrations of AAPH for 1 hr at either 37 ºC or 60 ºC. Representative agarose gels obtained are shown in Figure . In the absence of AAPH, pBR322 plasmid DNA at both temperatures is primarily in the supercoiled (SC) form (Lane 1). At both temperatures, we observe a dose-dependent increase in the appearance of damaged (OC and L) forms of pBR322 plasmid when incubated with AAPH. To quantify the damage, we also plot the percentage of intact SC DNA as a function of increasing AAPH concentration for triplicate gels in Figure . Significantly greater DNA damage is observed at higher temperature for equivalent AAPH concentrations. Incubation of pBR322 plasmid at 60 ºC in the absence of AAPH (lane 1) does not result in the loss of DNA supercoiling compared to 37 ºC, However, 50 µM AAPH at 60 ºC is sufficient to convert all supercoiled DNA to linear and open coil forms. In contrast, AAPH concentrations greater than 1 mM are required to completely eliminate the supercoiled band when plasmid DNA is treated with AAPH at 37 ºC. Having demonstrated the capacity to induce significant DNA strand breaks at low (10-200 µM) AAPH concentrations at 60 ºC, these conditions were used to examine the effect of condensation on the protection of DNA against free radical damage. Next, we evaluated the protective effect of protamine condensation on DNA damage by AAPH-generated free radicals. The results for pBR322 plasmid DNA in the absence or presence of salmon protamine are shown in Figure for three different AAPH concentrations. The percent undamaged (SC) DNA is plotted in Figure . In the absence of AAPH (Fig. , lane 1), pBR322 is primarily in the supercoiled form. As expected, increasing the AAPH concentration results in a significant decrease in SC DNA for uncondensed DNA. After incubation with 20 µM AAPH for 1 hr, uncondensed plasmid was readily oxidized by radicals generated by AAPH, resulting in the vast majority (>70%) of the DNA being in the open coiled form (Figure , lane 2). By 500 µM AAPH, a clear linear DNA band is observed giving evidence for double-strand breaks due to oxidation. Condensation by salmon protamine conferred significant protection to DNA from free radical damage. For these studies, all protamine-DNA polyplexes were formed at N/P ratio 3 to ensure complete condensation of the DNA. After mixing, polyplexes were incubated for 30 min at room temperature prior to treatment with AAPH. After AAPH treatment for 1 hr, polyplexes were decondensed by competitive dissociation with dextran sulfate and immediately subjected to agarose electrophoresis. At 20 µM AAPH, the percent SC DNA in protamine-condensed plasmid DNA is not significantly different (p = 0.569) from the control DNA that was not exposed to AAPH. A dose dependent increase in DNA damage was observed for protamine-condensed samples. However, at all AAPH concentrations studied, significantly higher amounts of supercoiled DNA are found for protamine-condensed DNA than those of naked DNA. For example, after 1 hr of incubation with 100 µM AAPH, less than 10% of the uncondensed DNA is intact, compared to ~73% of the DNA remaining SC when condensed with protamine. Results in Figure were averaged from five gels and results are given as mean ± standard deviation. Higher concentrations of protamine, or higher N/P ratios, did not significantly alter the degree of damage observed in condensed samples. for the DNA against AAPH-induced damage. Protamine and polyK condensed DNA is protected similarly to R6-DNA. Statistical analysis of the results in Figure shows that polyR-condensed DNA shows the highest levels of DNA protection that is significantly greater (P < 0.001) than that observed for any other polycation studied. Although undamaged DNA from polylysine and protamine condensed DNA is observed to be higher than that of R6 condensed DNA, the observed difference is not significantly greater at P < 0.001 level. Atomistic molecular dynamics simulations. To examine the molecular-level behavior that explains the relationship between a polyarginine chain's length and the protection that it offers to DNA, we performed atomistic molecular dynamics simulations of a DNA helix in the presence of either R3, R6, or R20, where the R20 chain served as a model for polyR. A sufficient number of polyarginine chains were added at random locations in each simulation box to result in an N/P ratio of 3. Many peptide chains spontaneously moved toward and became bound to the DNA as shown in Figure . However, in each simulation, some arginine chains remained free in solution, suggesting the DNA helices are saturated with the arginine oligomers in the simulations. We calculated the average number of polyarginine residues and chains that were in direct contact (within 3 Å) with DNA during the last 50 ns of each simulation (Table ). In all three simulations, enough arginine residues are in direct contact with DNA to neutralize at least 90% of the DNA charge. Thus, the polyplex has a net positive charge when considering the total charge of the chains bound to DNA. Chain length does have a modest impact on the number of residues bound to DNA, with R3 having fewer residues in contact with DNA (36 ± 3) than R6 (40 ± 2) and R20 (41 ± 1). This result also correlates with the solvent-accessible surface area (SASA) of the DNA in the three simulations. A similar number of arginine residues in the R6 and R20 simulations are bound to DNA, and the DNA SASA is similar for these systems. In the R3 simulation, however, fewer arginine residues are in contact with the DNA, and the SASA of the DNA is larger, potentially increasing the risk of DNA damage from exposure to damaging radicals in solution.