Stacking of short DNA induces the gyroid cubic-to-inverted hexagonal phase transition in lipid–DNA complexes

Abstract

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 (QIIG) and the inverted hexagonal phase (HIIC). We investigated the structural shape transitions induced in the QIIG 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 QIIG and the HIIC phase, with the fraction of QIIG increasing with temperature. Phase coexistence for blunt 5 bp sDNA is observed from 27 °C to about 65 °C, where the HIIC phase disappears and the temperature dependence of the lattice spacing of the QIIG 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 QIIG phase throughout the studied range of temperature (27 °C to 85.2 °C). The longer 11 bp sDNA forms coexisting QIIG and HIIC phases (with the fraction of QIIG again increasing with temperature) only for “nonsticky” dTdT overhangs, while dAdT overhangs and blunt ends exclusively template the HIIC phase. For 24 and 48 bp sDNAs the HIIC phase replaces the QIIG 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 QIIG phase, thus driving the QIIG-to-HIIC phase transition. As the temperature is increased, the breaking of stacks due to thermal fluctuations restores increasing percentages of the QIIG phase.

Self-organization of nucleic acids in lipid constructs

“…Lower degree of curvature and larger water channels are eventually more favorable for the inclusion of rigid, rod-like siRNA molecules. Combined with previous studies involving DNA length and bicontinuous cubic phase stabilization, 50 we argue that there is an optimum length and amount of nucleic acids that can be incorporated into cubic phase matrices without changing their internal structure. The Cryo-EM images are consistent with the idea of the GMO-PEG lipid units preferentially locating at the outer rims of cubosomes (lipid only particles) and cuboplexes (lipid-siRNA complex particles).…”

“…50 They used “sticky” adenine (A)–thymine (T) (dAdT) overhangs for long DNA demonstration. As control groups, no overhangs (blunt) and “nonsticky” (dTdT) overhangs were used respectively.…”

Distinct solubility and cytotoxicity regimes of paclitaxel-loaded cationic liposomes at low and high drug content revealed by kinetic phase behavior and cancer cell viability studies

“…Complexes were formed by mixing the cationic lipid with negatively charged DNA at a positive charge ratio of 5. Two types of DNA were used: a large luciferase plasmid DNA and a short double-stranded DNA 5 base pairs long, with 2 extra unpaired thymine residues at the 3′ end of each strand to minimize end-to-end DNA sticking [52]. Liposomes and DNA were each diluted in DMEM before subsequent mixing.…”

Cationic liposome–nucleic acid nanoparticle assemblies with applications in gene delivery and gene silencing

“…Remarkably, this structure was found to improve TE in hard-totransfect mouse embryonic fibroblast cells. More recently, a novel bicontinuous gyroid cubic phase (figure 2b, labelled Q G,siRNA II ) with siRNA incorporated within its two water channels was reported [36][37][38]. This phase was discovered in mixtures of siRNA with CLs containing a cubic phase forming lipid (GMO: 1-monooleoyl-glycerol) and DOTAP or the multivalent cationic lipid MVL5.…”