title: Toroid formation in charge neutralized flexible or semi-flexible biopolymers: potential pathway for assembly of DNA carriers.
abstract: BACKGROUND
  The theoretical state diagram for semi-flexible macromolecules such as DNA predicts that a tightly wound toroid can be a stable structure. Experimentally, toroids roughly 100 nm in diameter are routinely observed for DNA in the presence of multivalent cations at low DNA concentration. Theory also predicts toroids can form between non-DNA semi-flexible polymers and multivalent counterions. This phenomenon provides a means to co-package DNA with functionalized anionic polymers to create gene delivery systems.
  
  
  RESULTS
  We show using electron microscopy that non-DNA polymers (polylysine, polyglutamic acid, and dextran sulfate) form toroids when mixed with multi- or polyvalent ions of opposite charge. The non-DNA toroids are similar in diameter to ones made with DNA. The results using dextran sulfate, a semi-flexible polymer, are explained by current theory. However, theory predicts that high flexibility in polypeptides should discourage their incorporation into stable toroids. To explain these latter observations we propose that charge neutralization facilitates secondary structure formation, which confers stiffness, thereby allowing stable toroids for the polypeptides studied. We measured the secondary structure of the toroid-forming polypeptides using circular dichroism (CD). The CD spectrum indicates the polypeptides undergo transitions from non-ordered structures (random coil) to ordered secondary structures (either alpha-helix or beta-sheet) upon charge neutralization which supports the hypothesis. The type of secondary structure is dependent on the type of multivalent counterion used to form the toroids. Formation of the polypeptide toroids confers resistance to heat denaturation of the resulting polypeptide secondary structure. The CD spectrum of DNA in a toroid also is changed from that of uncomplexed DNA, but all of the counterions used to form DNA toroids created structures with similar CD spectra in the DNA region (250-290 nm).
  
  
  CONCLUSIONS
  The toroid structure obtained using DNA is observed in other semi-flexible non-DNA polymers such as dextran sulfate, and also in flexible polymers such as polylysine and polyglutamic acid upon charge neutralization with multivalent counterions. In the flexible polymers we propose that this phenomenon is due to induction of secondary structure upon charge neutralization, which decreases polymer flexibility, i.e. increases polymer stiffness, to enable toroid formation. These results have significant implications for the co-assembly of non-DNA anionic polymers with DNA to create nanoscopic gene carriers.
authors.affiliation: Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, CA 94143-0446, USA.
authors.forename: Mary X
authors.initials: MX
authors.lastname: Tang
authors.forename: Weijun
authors.initials: W
authors.lastname: Li
authors.forename: Francis C
authors.initials: FC
authors.lastname: Szoka
mesh_terms: D001704:Biopolymers / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D046911:Macromolecular Substances; D009690:Nucleic Acid Conformation; D017433:Protein Structure, Secondary
chemical_list: D001704:Biopolymers; D046911:Macromolecular Substances; D004247:DNA