Synthesis, hybridization properties and antiviral activity of lipid-oligodeoxynucleotide conjugates

Abstract

Triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (2) was coupled to the 5' terminus of oligodeoxynucleotides via hydrogen phosphonate solid support DNA synthesis methodology. Duplex DNA oligomers with a single 5'-phospholipid melted at lower temperatures than the corresponding unmodified duplex, but duplexes bearing lipids at each 5' end had higher Tms. In uptake experiments with L929 cells, 8-10 times more lipid-DNA became cell-associated than did unmodified DNA. Unmodified antisense diesters were inactive in a VSV antiviral assay in L929 cells (at up to 200 microM). Attachment of a lipid to the oligomer, however, led to a greater than 90% at 150 microM (greater than 80% at 100 microM) reduction in viral protein synthesis. The antiviral activity depended on the sequence of the oligodeoxynucleotide, but some compounds having little or no base complementarity to the viral target were also effective. Phosphorothioate derivatives reduced viral protein synthesis by 20-30% at 100 microM in the VSV assay. The lipid-DNA compounds were not toxic to the cells at up to 100 microM.

Supported lipid bilayers, tethered lipid vesicles, and vesicle fusion investigated using gravimetric, plasmonic, and microscopy techniques

“…While, in parallel efforts, Yoshina-Ishii and Boxer used chemically activated lipid head groups to couple short single-stranded DNA to lipid vesicles for similar purposes, 12 we used self-inserting lipophilic cholesterol-modified DNA, 38 initially developed to enhance the cellular uptake of antisense DNA by lipidation. 39 Cholesterol-DNA was found to self-incorporate into the vesicle membrane, which apparently facilitated the preparation of DNA-tagged vesicles. However, membrane attachment by self-incorporation of cholesterol-modified DNA suffered from a severe drawback.…”

MODIFIED OLIGONUCLEOTIDES: Synthesis and Strategy for Users

“…Examples include conjugation of oligonucleotides to viral fusogenic peptides (37,38), staphylococcal nuclease and other enzymes (39,40), neoglycopeptides (41), phospholipids (42), thiolated polysaccharides (43), vitamin E (44), lipophilic groups such as cholesterol (45)(46)(47), and nonradioactive detection labels (48)(49)(50).…”

Inhibition of gene expression by anti-sense oligodeoxynucleotides

“…This process appears to be saturable, size-dependent and possibly receptor-mediated [20,21]. Improvements in direct uptake into the cell can be achieved by conjugating ODN to a lipophilic moiety, such as poly-L-lysin [22,23], to cholesterol or lipid group [24]; or by substituting the charged phosphodiester groups on the sugarphosphate backbone with non-charged groups such as methyl phosphonate or phosphorothioates [25]. Increased membrane permeabilization, with the pore-forming agent streptolysin O [26], has also been reported to faciliate uptake where the extent of anti-sense inhibition was dependent on the concentration of ODN present during permeabilization and diminished to control levels over the ensuing 48 h.…”