Buffers used were sodium acetate solution for pH 4 and Tris-HCl for pH 7.5. For each x-ray sample, PEI (1 mg/mL) was added to 200 µg of ctDNA (1 mg/mL) in the appropriate 10 mM pH buffer solution to achieve the desired polymer:DNA weight ratio. Sufficient polycation was added to ensure complete condensation of DNA as determined by EtBr displacement assay results. The resulting fibrous PEI-DNA samples were then mixed by vortexing, incubated at room temperature for 30 min and centrifuged (14500 g/min for 5 minutes) to collect the condensate. The condensate was then washed with buffer to remove excess, uncomplexed PEI and moved to a bathing solution of fresh buffer to equilibrate for ~1 hour before X-ray analysis. For pH studies, both DNA and polymer stock solutions were prepared in the same Tris or acetate buffer, and after condensation, the condensate was moved to a fresh buffer of the same pH. For pH response studies, polyplexes were prepared in the same manner in pH 7.5 buffer and equilibrated for 30 min. Condensates were then moved to a fresh 10 mM solution of the pH 4 acetate buffer and equilibrated for 1 or 24 hr before x-ray experiments. These samples are labeled pH 7.5-4 to indicate the pH change after polyplex formation. For time-course studies, samples were also monitored by X-ray over a month to observe changes in DNA packaging with time.