Structural appearance of linker histone H1/siRNA complexes

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 (K(16)) showed also non-cooperative binding with siRNA.

Linker histone H1 stimulates DNA strand exchange between short oligonucleotides retaining high sensitivity to heterology

“…Our data on the oligonucleotides saturation with human H1(0) are closest to this value. Our system is similar to that described recently by Haberland et al43 where it was demonstrated that each H1 molecule can bind one or two ds21mers of siRNA that corresponds to from 42 to 84 bases/H1, also in agreement with our data.…”

“…Somewhat paradoxically, after about four decades of extensive studies of linker histones–DNA interactions there is no generally accepted estimate of the stoichiometry for the binding of H1 to nucleic acids (see discussion in the paper of Mamoon et al19). Existing estimates vary in the rather wide range from 20 to 100 nucleotides per H1 molecule 17–19, 29, 41–43. Apparently, the most straightforward quantitative estimation of the stoichiometric number for H1–dsDNA binding was obtained in the work of Watanabe18 in the experiments on the DNA titration with the fluorescently labeled protein; but due to significant variation of the stoichiometric ratio estimates obtained in other studies the situation remains uncertain and accumulation of additional data obtained with different independent approaches is still required.…”

Development of Streptavidin-Based Nanocomplex for siRNA Delivery

“…Formation of the SSCrP nanocomplex was evaluated using the Gel-Red fluorescence quenching assay as described 10 . Briefly, twenty microliters of the SSCP nanocomplexes containing 1 μM siRNA with varying N/P rations were added to black 96 well plates along with 80 μL Gel Red solution.…”

“…Formation of the SSCP nanocomplex was evaluated using the Gel-Red fluorescence quenching assay as described. 10 Briefly, twenty microliters of the SSCP nanocomplexes containing 1 μM siRNA with varying N/P ratios was added to black 96-well plates along with 80 μL of Gel Red solution. The plates were incubated at room temperature in the dark for 5 min, and fluorescence was measured using a Beckman DTX 880 multimode detector (Beckman Coulter, Inc., Brea, CA) at λ exc 485 nm and λ emi 625 nm.…”