DNA and siRNA must be packaged for protection prior to transfection in vivo when they are administered to humans or other animals. Dr. A. J. Mixson's laboratory at the University of Maryland, Baltimore Medical School has developed a family of branched histidine-lysine (HK) peptides that confer improved transfection in mice compared to naked nucleic acid. The branched polymer denoted H3K4b has a superior ability to transfect siRNA in vitro compared to H3K(+G)4b. H3K(+G)4b, made by the addition of two glycines to each of the original H3K4b branches, is presumably a more flexible polymer, and it allows for better transfection of plasmid DNA than H3K4b. Biophysical characterization of the HK-DNA and HK-siRNA complexes is aimed at understanding how the structures of both peptides affect their biological activity. This characterization was performed using ethidium bromide exclusion from nucleic acid, DNase I plasmid degradation, dynamic light scattering, circular dichroism, isothermal titration calorimetry and atomic force microscopy. Results from the characterization suggest that H3K4b forms condensed regions of packaged plasmid with some repeating accessibility of the DNA, compared to a more even coating of plasmid by H3K(+G)4b. Based on these results a "coating versus clumping" model was developed to relate the transfection efficiency of each peptide to its binding of plasmid DNA. A specific model for packaging of siRNA with these peptides was not developed, but we believe that characteristics that lead to effective transfection of plasmid are not key to siRNA delivery. A better understanding of characteristics important to peptide-nucleic acid complex formation may lead to the development of improved transfection agents.