showed that 9 µM lambda DNA solution was condensed by trivalent spermidine only at concentrations greater than ~2 mM. Similarly in our experiments, DNA precipitation only begins at R3 concentrations resulting in N/P ~1.2 with full condensation not observed until N/P 2. Consistent with previous studies on condensed chicken blood or calf thymus DNA, the interhelical spacing between DNA helices (Dint) within these polyplexes depends on both the the length and composition of the polycation. While DNA-DNA spacings can change depending on equilibration time, our measurements of Dint values in polyplexes after one hour (as shown in Figure ) align with those of previous samples equilibrated for two weeks. The shorter equilibration time used here corresponds more closely to those in subsequent DNA damage assays. When comparing peptides, arginines package DNA more compactly than lysine peptides of similar lengths. For example, polyR-DNA displays Dint values over 2 Å smaller than those for polyK-DNA, suggesting a 20% reduction in packaging volume within the polyR condensates. Biological systems exploit this more efficient packaging by arginines. Histones, which use lysines and arginines, package somatic chromatin, while mature sperm cells primarily use argininerich protamines for condensing DNA. This compact packaging in sperm chromatin effectively silences it transcriptionally until fertilization. Polycation length significantly influences packaging density, with a clear decrease in Dint and an increase in DNA packaging density correlating with greater valency as observed when comparing R3, R6, and polyR. Using osmotic stress measurements, we previously showed that this tighter packaging is due to increased attractions in the more highly charged peptides. Of the arginine peptides, all can condense DNA more tightly than polyK, with the exception of R3, which produces a similar Dint value as polyK. Salmon protamine, due to the presence of uncharged amino acids that increase repulsive forces within the condensate, results in a DNA packaging comparable to that of DNA condensed with R5 or R6. Protamine phosphorylation and subsequent dephosphorylation during spermiogenesis is thought to modulate protamine-DNA interactions enabling proper chromatin remodeling. Recently, protamine phosphorylation was also shown to initate parental genome reprogamming consistent with this electrostatic model. Although having little effect on precipitation behavior, a negatively charged phosphoserine in the hexaarginine peptide significantly reduces the resulting DNA packaging density. For example, R3pSR3-DNA has a Dint roughly 4 Å larger than that of R6-DNA, indicating a volume change of roughly 50% within the condensed phase. This marked reduction in packaging induced by the presence of a phosphoserine group could hold biological relevance for the maturation of spermatozoa. It is likely that looser packaging, due to residual phosphates or other post-translational modifications on protamines in sperm chromatin, leaves DNA more vulnerable to free radical insult. Polycation-induced DNA condensation in dilute solutions, similar to our damage assays, leads to the formation of wellordered polyplex nanoparticles (NP). DLS measurements of the resulting hydrodynamic radii (Rh) of the NPs were measured for all polyplexes at N/P 3 (Figure ). Interestingly, we found that the size of the resulting polyplex particles did not correspond to the densities of the DNA packaging. Instead, they mainly depended on the length of the polycation used. The smallest Rh values were recorded for polyplexes created with polyK, polyR, and salmon protamine. Conversely, R3-DNA formed the largest particles, even though its DNA-DNA interhelical spacing was similar to that of polyK-DNA. Both hexaarginine peptides yielded similar Rh values for the polyplexes, despite significantly larger Dint values for R3pSR3-DNA compared to R6-DNA. Notably, the size of the particles in our polyplexes did not correlate with the extent of DNA damage observed. The protective role of DNA compaction is well established. DNA condensed by histones exhibits increased resistance to damage than uncondensed DNA, with heterochromatin showing superior protection over euchromatin. Previous studies have also demonstrated that DNA condensed by polycations is highly protective against damage induced by ionizing radiation. In our study, we also found that DNA condensation offers significant protection within polyplexes (Figure ). Even at low concentrations of AAPH (20 µM), uncondensed DNA undergoes significant oxidation, while the % supercoiled DNA in protamine-DNA is nearly identical to plasmid DNA in the absence of AAPH. Higher free radical concentrations result in more DNA oxidation in both condensed and uncondensed states; however, protamine-DNA displays notable resistance across all concentrations of AAPH. Taking into account the steric effects, it is normally hypothesized that tighter DNA packaging would result in greater protection within polyplexes. Although most condensing agents were able to protect DNA from free radical oxidation (as illustrated in Figure ), neither packaging density nor particle size alone could explain the observed damage trends in polyplexes. Our results suggest that both the packaging density and the length of the polycations play a role in DNA protection. For example, DNA condensed by polyR, which results in tighter DNA packaging, shows less damage from free radicals compared to polyK-DNA. Similarly, R6-DNA exhibits less damage compared to R3pSR3-DNA, presumably due to the tighter DNA packaging by R6. Yet, R3-DNA, despite having a comparable DNA packaging density to polyK-DNA, shows little to no protective effect against free radical insult, implying that steric shielding alone does not explain the protection disparity. Differences in DNA protection may arise from the mobility of polycations within the polyplexes. Although cationic chain exchanges between polyplex-bound and unbound chains have been reported, the internal mobility of polycations remains largely unexplored. To investigate these potential effects, we conducted molecular dynamic (MD) simulations using arginine peptides of varying lengths. Molecular dynamics (MD) simulations revealed several effects of chain length on the interactions between polyarginine and DNA. These insights may elucidate the increased resistance to AAPH-induced DNA damage when associated with longer polyarginine chains. First, our simulations revealed that a greater number of residues from longer chains interact with the DNA at any given time. Consequently, these longer chains occupy a larger surface area on DNA, potentially shielding the DNA from radical attack more effectively. Second, chain length also impacts the stability of polyR-DNA interactions. Shorter polyarginine chains, particularly R3, demonstrated less stable interactions and exhibited highly dynamic behavior within the condensates. Despite similarities in the average number of interactions between R20-DNA and R6-DNA, R6 residues generally showed lower contact fractions compared to R20, indicating slight differences in the interaction dynamics. Additionally, the longer polyarginine chains exhibited a greater number of direct contacts with DNA. As a result, disrupting a DNA-polyarginine interaction would require breaking more residue-residue contacts for longer chains. This finding suggests that longer polyarginine chains may be more strongly bound to DNA, which could enhance their protective effect. In conclusion, we have investigated packaging and DNA damage in polyplexes formed with polycations of different compositions and chain lengths. Using a combination of experimental and MD simulations, we probed the relationship between the polycation chain length, DNA packaging density, and accumulation of damage from free radical oxidation. Our findings suggest that both steric effects and polymer dynamic mechanisms can contribute to DNA protection. Tighter DNA packaging achieved through the use of specific polycations enhances the protective effect, while the length of the polycation also plays a significant role. Simulation results shed light on the interactions between arginine chains of varying length and DNA, highlighting their role in DNA protection through increased surface coverage and more stable interactions. These findings advance our understanding of the factors influencing DNA protection in condensed states and have implications for the design of polycation-based systems for gene delivery and other applications involving DNA condensation.