Interactions of antisense DNa oliginucletide analogs with phospholid membranes (liposomes)

Abstract

Antisense oligonucleotides have the ability to inhibit individual gene expression in the potential treatment of cancer and viral diseases. However, the mechanism by which many oligonucleotide analogs enter cells to exert the desired effects is unknown. In this study, we have used phospholipid model membranes (liposomes) to examine further the mechanisms by which oligonucleotide analogs cross biological membranes. Permeation characteristics of 32P or fluorescent labelled methylphosphonate (MP-oligo), phosphorothioate (S-oligo), alternating methylphosphonate-phosphodiester (Alt-MP) and unmodified phosphodiester (D-oligo) oligodeoxynucleotides were studied using liposomal membranes. Efflux rates (t1/2 values) at 37 degrees C for oligonucleotides entrapped within liposomes ranged from 7-10 days for D-, S- and Alt-MP-oligos to about 4 days for MP-oligos. This suggests that cellular uptake of oligonucleotides by passive diffusion may be an unlikely mechanism, even for the more hydrophobic MP-oligos, as biological effects are observed over much shorter time periods. We also present data that suggest oligonucleotides are unlikely to traverse phospholipid bilayers by membrane destabilization. We show further that MP-oligos exhibit saturable binding (adsorption) to liposomal membranes with a dissociation constant (Kd) of around 20nM. Binding appears to be a simple interaction in which one molecule of oligonucleotide attaches to a single lipid site. In addition, we present water-octanol partition coefficient data which shows that uncharged 12-15 mer MP-oligos are 20-40 times more soluble in water than octanol; the low organic solubility is consistent with the slow permeation of MP-oligos across liposome membranes. These results are thought to have important implications for both the cellular transport and liposomal delivery of modified oligonucleotides.

Conformational change in DNA induced by cationic bilayer membranes

“…Neither 1(12,2) nor CTAB affected the polarization of ethidium bromide in the absence of DNA, indicating that the intercalator does not interact directly with these lipids. These results suggest that ethidium bromide still interacts with DNA even after the addition of 1 (12,2) and that the fluorescence quenching is not due to the release of ethidium bromide molecules from DNA by the lipids. Thus, since the effect was only observed with the bilayer-forming lipid and not with other lipids, interactions between DNA and ethidium bromide seem to be weaker depending on the bilayer structure of the lipids.…”

“…edly suppressed by intercalation into DNA. Although the addition of lipid 1 (12,2) to the DNA-ethidium bromide complex lowered the P value, a substantial level of the value, 0.07, was still observed. The addition of CTAB had no effect on the value.…”

“…tion of the compound into DNA and the spectrum was maximized at 590 nm [16]. The enhanced fluorescence was reduced to nearly the basal level on the addition of the lipid 1 (12,2). Further addition of Triton X-100 to disrupt the bilayer structure, however, led to recovery of the quenched fluorescence to the original level• When various concentrations of lipid 1 (12,2) were added to the DNA-ethidium bromide solution, maximum quenching was observed at a concentration below an equimolar amount of the phosphate groups of DNA (Fig.…”

“…CD spectra of the DNA-lipid complexes revealed that the structure of DNA was changed only by bilayer-forming lipids at temperatures lower than their Tc values. Thus, the conformation of DNA is forced to change by cationic lipids forming the rigid bilayer membrane so that ethidium bromide fluorescence might be reduced, and the conformation can be controlled by selection of the appropriate lipid and temperature.Key words: Bilayer membrane; Cationic lipid; DNA conformation; Ethidium bromide Bilayer membranes formed with cationic lipids have been successfully used as DNA carriers into cultured eukaryotic cells [9][10][11][12][13]. Since these membranes have fluid two-dimensional surface cationic groups neutralizing the anionic phosphate groups of the DNA, DNA interacting with the membranes would exhibit a different conformational change from that interacting with metal ions and proteins.…”

“…Key words: Bilayer membrane; Cationic lipid; DNA conformation; Ethidium bromide Bilayer membranes formed with cationic lipids have been successfully used as DNA carriers into cultured eukaryotic cells [9][10][11][12][13]. Since these membranes have fluid two-dimensional surface cationic groups neutralizing the anionic phosphate groups of the DNA, DNA interacting with the membranes would exhibit a different conformational change from that interacting with metal ions and proteins.…”