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Structural appearance of linker histone H1/siRNA complexes

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Abstract

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 (K16) showed also non-cooperative binding with siRNA.

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Abbreviations

DMSO:

Dimethylsulfoxide

DSS:

Dithiobis(succinimidylsuberate)

Ethbr:

Ethidium bromide

FITC:

Fluoresceine isothiocyanate

H1:

Histone H1

K16 :

(Lysine)16

N/P-ratio:

Nitrogen/phosphate ratio

PDMDAAC:

Poly(N,N′-dimethyldiallylammonium)chloride

pDNA:

Plasmid DNA

pCMV Luc:

Plasmid luciferase carrying a cytomegalie-virus-promotor

siRNA:

Small interferring RNA

TBE:

Tris–borate–EDTA buffer

TBTU:

O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate

TCA:

Trichloroacetic acid

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Acknowledgements

The work of A. Haberland and W. Henke was supported by HAL Allergy, Haarlem, NL and RiNA network for RNA technologies financed by the City of Berlin, the German Federal Ministry of Education and Research and the European Regional Development Fund. Our extra special thanks go to Dr. Birgit Mazurek (director of the Molecular Biological Research Laboratory, Dept. of Otorhinolaryngology, Charité University Medicine Berlin, Germany) for useful and encouraging discussions and her general support. We are grateful to Dr. Matthias Truss (Children’s Hospital, Laboratory for Molecular Biology, Charité-CCM University Medicine Berlin, Ziegelstr. 5–9, 10098 Berlin, Germany) for supplying an enormous amount of control siRNA, enabling the physicochemical measurements. We are also grateful to Prof. Dr. J. Behlke (Max-Delbrück-Centrum for Molecular Medicine, Berlin-Buch, 13125 Berlin, Germany) who supplied the service for carrying out the experiments of analytical ultracentrifugation. The work of Dr. Waldöfner was supported by The European Regional Development Fund (ERDF)—Project NanoMed No. EFRE 20002006/2ue/2.

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Authors and Affiliations

  1. Molecular Biological Research Laboratory, Department of Otorhinolaryngology, Charité University Medicine Berlin, Charitéplatz 1, 10117, Berlin, Germany

    Annekathrin Haberland & Wolfgang Henke

  2. Institute of Laboratory Medicine and Pathobiochemistry, Charité-University Medicine Berlin, Augustenburgerplatz 1, 13353, Berlin, Germany

    Annekathrin Haberland

  3. Institute of Environmental Medicine, University of Pennsylvania, School of Medicine, 3620 Hamilton Walk, Philadelphia, PA, USA

    Sergei Zaitsev

  4. MagForce® Nanotechnologies AG, c/o Center of Biomedical Nanotechnology (CBN) Spandauer Damm 130, 14050, Berlin, Germany

    Norbert Waldöfner

  5. Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany

    Bettina Erdmann & Michael Böttger

Authors
  1. Annekathrin Haberland
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  2. Sergei Zaitsev
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  3. Norbert Waldöfner
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  4. Bettina Erdmann
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  5. Michael Böttger
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  6. Wolfgang Henke
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Correspondence to Annekathrin Haberland.

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Haberland, A., Zaitsev, S., Waldöfner, N. et al. Structural appearance of linker histone H1/siRNA complexes. Mol Biol Rep 36, 1083–1093 (2009). https://doi.org/10.1007/s11033-008-9282-8

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  • DOI: https://doi.org/10.1007/s11033-008-9282-8

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