Formation of stable cationic lipid/DNA complexes for gene transfer.

Stable cationic lipid/DNA complexes were formed by solubilizing cationic liposomes with 1% octylglucoside and complexing a DNA plasmid with the lipid in the presence of detergent. Removal of the detergent by dialysis yielded a lipid/DNA suspension that was able to transfect tissue culture cells up to 90 days after formation with no loss in activity. Similar levels of gene transfer were obtained by mixing the cationic lipid in a liposome form with DNA just prior to cell addition. However, expression was completely lost 24 hr after mixing. The transfection efficiency of the stable complex in 15% fetal calf serum was 30% of that obtained in the absence of serum, whereas the transient complex was completely inactivated with 2% fetal calf serum. A 90-day stability study comparing various storage conditions showed that the stable complex could be stored frozen or as a suspension at 4 degrees C with no loss in transfection efficiency. Centrifugation of the stable complex produced a pellet that contained approximately 90% of the DNA and 10% of the lipid. Transfection of cells with the resuspended pellet and the supernatant showed that the majority of the transfection activity was in the pellet and all the toxicity was in the supernatant. Formation of a stable cationic lipid/DNA complex has produced a transfection vehicle that can be stored indefinitely, can be concentrated with no loss in transfection efficiency, and the toxicity levels can be greatly reduced when the active complex is isolated from the uncomplexed lipid.

Large electrostatic differences in the binding thermodynamics of a cationic peptide to oligomeric and polymeric DNA.

Results presented here demonstrate that the thermodynamics of oligocation binding to polymeric and oligomeric DNA are not equivalent because of long-range electrostatic effects. At physiological cation concentrations (0.1-0.3 M) the binding of an oligolysine octacation KWK6-NH2 (+8 charge) to single-stranded poly(dT) is much stronger per site and significantly more salt concentration dependent than the binding of the same ligand to an oligonucleotide, dT(pdT)10 (-10 charge). These large differences are consistent with Poisson-Boltzmann calculations for a model that characterizes the charge distributions with key preaveraged structural parameters. Therefore, both the experimental and the theoretical results presented here show that the polyelectrolyte character of a polymeric nucleic acid makes a large contribution to both the magnitude and the salt concentration dependence of its binding interactions with simple oligocationic ligands.

Cationic facial amphiphiles: a promising class of transfection agents.

A promising class of compounds for DNA transfection have been designed by conjugating various polyamines to bile-acid-based amphiphiles. Formulations containing these compounds were tested for their ability to facilitate the uptake of a beta-galactosidase reporter plasmid into COS-7 cells. Dioleoyl phosphatidyl ethanolamine (DOPE) formulations of some of the compounds were several times better than Lipofectin at promoting DNA uptake. The most active compounds contained the most hydrophilic bile acid components. The activity is clearly not related to affinity for DNA: the hydrophobic bile acid conjugates were found to form stable complexes with DNA at lower charge ratios than the hydrophilic conjugates. We suggest that the high activity of the best compounds is related to their facial amphiphilicity, which may confer an ability to destabilize membranes. The success of these unusual cationic transfection agents may inspire the design of even more effective gene delivery agents.

Seed and vascular expression of a high-affinity transporter for cationic amino acids in Arabidopsis.

In most plants amino acids represent the major transport form for organic nitrogen. A sensitive selection system in yeast mutants has allowed identification of a previously unidentified amino acid transporter in Arabidopsis. AAT1 encodes a hydrophobic membrane protein with 14 membrane-spanning regions and shares homologies with the ecotropic murine leukemia virus receptor, a bifunctional protein serving also as a cationic amino acid transporter in mammals. When expressed in yeast, AAT1 mediates high-affinity transport of basic amino acids, but to a lower extent also recognizes acidic and neutral amino acids. AAT1-mediated histidine transport is sensitive to protonophores and occurs against a concentration gradient, indicating that AAT1 may function as a proton symporter. AAT1 is specifically expressed in major veins of leaves and roots and in various floral tissues--i.e., and developing seeds.

Binding studies of cationic thymidyl deoxyribonucleic guanidine to RNA homopolynucleotides.

Deoxyribonucleic guanidine is a potential antisense agent that is generated via the replacement of the negative phosphodiester linkages of DNA [--O--(PO2-)--O--] with positively-charged guanidinium (g) linkages [--NH--C(==NH2+)--NH--]. A pentameric thymidyl deoxyribonucleic guanidine molecule [d(Tg)4T-azido] has been shown to base pair specifically to poly(rA) with an unprecedented affinity. Both double and triple strands consisting of one and two equivalents of d(Tg)4T-azido paired with one equivalent of poly(rA) are indicated by thermal denaturation experiments. At an ionic strength of 0.22, the five bases of d(Tg)4T-azido are estimated to dissociate from a double helix with poly(rA) at > 100 degrees C! The effect of ionic strength on thermal denaturation is very pronounced, with stability greatest at low ionic strengths. The method of continuous variation indicates that there is an equilibrium complex with a molar ratio of d(Tg) to r(Ap) or d(Ap) of 2:1. Based on this evidence, models of the structures of d(Tg)9T-azido bound to r(Ap)9A are proposed.