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Highly efficient gene silencing activity of siRNA embedded in a nanostructured gyroid cubic lipid matrix.

ABSTRACT: RNA interference (RNAi) is an evolutionarily conserved sequence-specific post-transcriptional gene silencing pathway with wide-ranging applications in functional genomics, therapeutics, and biotechnology. Cationic liposome-small interfering RNA (CL-siRNA) complexes have emerged as vectors of choice for delivery of siRNA, which mediates RNAi. However, siRNA delivery by CL-siRNA complexes is often inefficient and accompanied by lipid toxicity. We report the development of CL-siRNA complexes with a novel cubic phase nanostructure, which exhibit efficient silencing at low toxicity. The inverse bicontinuous gyroid cubic nanostructure was unequivocally established from synchrotron X-ray scattering data, while fluorescence microscopy revealed colocalization of lipid and siRNA in complexes. We attribute the efficient silencing to enhanced fusion of complex and endosomal membranes, facilitated by the cubic phase membrane's positive Gaussian modulus, which may enable spontaneous formation of transient pores. The findings underscore the importance of understanding membrane-mediated interactions between CL-siRNA complex nanostructure and cell components in developing CL-based gene silencing vectors.

SUBMITTER: Leal C

PROVIDER: S-EPMC2991473 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Highly efficient gene silencing activity of siRNA embedded in a nanostructured gyroid cubic lipid matrix.

Leal Cecília C Bouxsein Nathan F NF Ewert Kai K KK Safinya Cyrus R CR

Journal of the American Chemical Society 20101028 47

RNA interference (RNAi) is an evolutionarily conserved sequence-specific post-transcriptional gene silencing pathway with wide-ranging applications in functional genomics, therapeutics, and biotechnology. Cationic liposome-small interfering RNA (CL-siRNA) complexes have emerged as vectors of choice for delivery of siRNA, which mediates RNAi. However, siRNA delivery by CL-siRNA complexes is often inefficient and accompanied by lipid toxicity. We report the development of CL-siRNA complexes with a ...[more]

PMID: 21028803

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Project description:Lyotropic phases of amphiphiles are a prototypical example of self-assemblies. Their structure is generally determined by amphiphile shape and their phase transitions are primarily governed by composition. In this paper, we demonstrate a new paradigm for membrane shape control where the electrostatic coupling of charged membranes to short DNA (sDNA), with tunable temperature-dependent end-to-end stacking interactions, enables switching between the inverted gyroid cubic structure (QII(G)) and the inverted hexagonal phase (HII(C)). We investigated the structural shape transitions induced in the QII(G) phase upon complexation with a series of sDNAs (5, 11, 24, and 48 bp) with three types of end structure ("sticky" adenine (A)-thymine (T) (dAdT) overhangs, no overhang (blunt), and "nonsticky" dTdT overhangs) using synchrotron small-angle X-ray scattering. Very short 5 bp sDNA with dAdT overhangs and blunt ends induce coexistence of the QII(G) and the HII(C) phase, with the fraction of QII(G) increasing with temperature. Phase coexistence for blunt 5 bp sDNA is observed from 27 °C to about 65 °C, where the HII(C) phase disappears and the temperature dependence of the lattice spacing of the QII(G) phase indicates that the sDNA duplexes melt into single strands. The only other sDNA for which melting is observed is 5 bp sDNA with dTdT overhangs, which forms the QII(G) phase throughout the studied range of temperature (27 °C to 85.2 °C). The longer 11 bp sDNA forms coexisting QII(G) and HII(C) phases (with the fraction of QII(G) again increasing with temperature) only for "nonsticky" dTdT overhangs, while dAdT overhangs and blunt ends exclusively template the HII(C) phase. For 24 and 48 bp sDNAs the HII(C) phase replaces the QII(G) phase at all investigated temperatures, independent of sDNA end structure. Our work demonstrates how the combined effects of sDNA length and end structure (which determine the temperature-dependent stacking length) tune the phase behavior of the complexes. These findings are consistent with the hypothesis that sDNAs and sDNA stacks with lengths comparable to or larger than the cubic unit cell length disfavor the highly curved channels present in the QII(G) phase, thus driving the QII(G)-to-HII(C) phase transition. As the temperature is increased, the breaking of stacks due to thermal fluctuations restores increasing percentages of the QII(G) phase.

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Highly efficient gene silencing activity of siRNA embedded in a nanostructured gyroid cubic lipid matrix.

Publications

Highly efficient gene silencing activity of siRNA embedded in a nanostructured gyroid cubic lipid matrix.

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