Structural appearance of linker histone H1/siRNA complexes.
Efficient non-viral vectors for the in vivo siRNA transfer are still being searched for. Comparing the differences of the structural appearance of siRNA and pDNA one would assume differences in the assembling behaviour between these polyanions when using polycationic vectors such as nuclear proteins. The spontaneous assembly of nuclear proteins such as histone H1 (H1) with pDNA as polyanion which has intensively been investigated over the last decade, showed a particulate structure of the resulting complexes. For an efficient in vivo use small almost monomolecular structures are searched for. Using siRNA as the polyanion might enforce this structural prerequisite lacking unwanted aggregation processes, exploiting the molecular size of siRNA. We therefore investigated the structure of H1/siRNA complexes. Five commonly used methods characterizing the resulting assemblies such as retardation gels, static and dynamic light scattering, reduction of ethidium bromide fluorescence, analytical ultracentrifugation, and electron microscopy were used. From analytical ultracentrifugation we learned that under physiological salt conditions the siRNA-H1 binding was not cooperative, even though the gel analysis showed disproportionation which would be an indication for a cooperative binding mode. H1 formed very small and stable complexes with siRNA at a molar ratio of 1:1 and 1:2. In order to find out if the observed structural appearance of the H1/siRNA complexes is due to unspecific charge effects only or to special features of H1, polylysine was included in the study. Low molecular weight polylysine (K(16)) showed also non-cooperative binding with siRNA.
Department of Otorhinolaryngology, Molecular Biological Research Laboratory, Charité University Medicine Berlin, Berlin, Germany. annekathrin.haberland@charite.de
Annekathrin
A
Haberland
Sergei
S
Zaitsev
Norbert
N
Waldöfner
Bettina
B
Erdmann
Michael
M
Böttger
Wolfgang
W
Henke
D000042:Absorption; D000818:Animals; D002417:Cattle; D004247:DNA / Q000378:metabolism / Q000648:ultrastructure; D024202:Electrophoretic Mobility Shift Assay; D004996:Ethidium; D005453:Fluorescence; D006657:Histones / Q000737:chemistry*; D008027:Light; D010316:Particle Size; D010957:Plasmids / Q000378:metabolism / Q000648:ultrastructure; D011107:Polylysine / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000648:ultrastructure; D012542:Scattering, Radiation; D044967:Serum; D014461:Ultracentrifugation
D006657:Histones; D034741:RNA, Small Interfering; D011107:Polylysine; D004247:DNA; D004996:Ethidium