Characterization of structure and mechanism of transfection-active peptide-DNA complexes.
We studied a number of physicochemical parameters of transfection-active peptide-DNA complexes including size, aggregation behaviour and circular dichroism (CD) spectra. These data were brought in relationship to the transfection activity of these peptides in order to better understand the mechanism of peptide-mediated gene transfer. A DNA binding oligolysine (K(16)) and a peptide comprising K(16) with an added peptide loop containing the arbitrary sequence RAD not known as a receptor ligand were used. Whereas the K(16)-DNA complex at 88% charge neutralization of the DNA phosphates collapsed into small toroidal particles with a diameter of 200 nm by dynamic light scattering, K(16)-cRAD did not. Instead, large aggregates were observed. CD spectra showed that the K(16)-DNA complexes were in a -psi state observed at liquid crystalline phases. Increasing positive charge by addition of further K(16) or disturbing the -psi state by introducing the RAD-peptide loop resulted in increasing instability indicated by aggregation and loss of the -psi CD spectrum of the complexes. Transfection experiments indicated that the aggregated material was the transfection-active component.
Dallüge
Roman
R
Franz Volhard Clinic at the Max Delbrück Center for Molecular Medicine, Wiltbergstrasse 50, D-13125 Berlin, Germany.
Haberland
Annekathrin
A
Zaitsev
Sergei
S
Schneider
Marc
M
Zastrow
Heidi
H
Sukhorukov
Gleb
G
Böttger
Michael
M
D002460:Cell Line; D002738:Chloroquine; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics; D018625:Microscopy, Atomic Force; D008968:Molecular Conformation; D008970:Molecular Weight; D010316:Particle Size; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D012965:Sodium Chloride; D014162:Transfection
D010455:Peptides; D012965:Sodium Chloride; D002738:Chloroquine; D004247:DNA; D008156:Luciferases