Due to the ribose-phosphate backbone oligonucleotides are hydrophilic and dissolve well in water or buffer. Adding a lipophilic modification to the oligonucleotide shifts its solubility, making it amphiphilic, so that it may be directed to lipophilic target sites such as membranes, lipids or hydrophobic proteins. The lipophilic moiety also enhances passage through lipid membranes and uptake into cells or tissues.
The utility of lipophilic modifications on oligonucleotides has been widely explored for drug delivery, in antisense, siRNA, and aptamer research, and for immunostimulatory oligos. They also found use in nanobiotechnology.
Enhanced delivery and uptake of DNA antisense oligonucleotides or siRNA has been demonstrated for cholesterol-modified oligonucleotides. The highly lipophilic character of the steroid cholesterol leads to a higher uptake into cells and thereby an increased activity.
The hydrophobicity of the cholesterol can be exploited to attach oligonucleotide-conjugates to membranes or vesicles. A feature that also has been used to build artificial DNA-nanostructures associated with lipid membranes.
1. Modification of antisense phosphodiester oligodeoxynucleotides by a 5' cholesteryl moiety increases cellular association and improves efficacy. Krieg AM, Tonkinson J, Matson S, Zhao Q, Saxon M, Zhang LM, Bhanja U, Yakubov L, Stein CA; Proc. Natl. Acad. Sci USA (1993), 90, 1048-1052.
2. Silencing of microRNAs in vivo with ‘antagomirs‘. Krützfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, Stoffel M; Nature (2005), 438, 685 - 689.
3. Synthetic lipid membrane channels formed by designed DNA nanostructures. Langecker M, Arnaut V, Martin TG, List J, Renner S, Mayer M, Dietz H, Simmel FC; Science (2012), 338. 932 – 936.
α-Tocopherol is a naturally occurring vitamin (vitamin E group). Conjugated to an oligonucleotide it adds a lipophilic character. As tocopherol can be recognised and transported by body specific proteins and carriers, the tocopherol modification may also serve as a means to route delivery of the conjugated oligonucleotide within the organism.
The tocopherol derivative Trolox also belongs to the vitamin E group. In comparison to tocopherol, Trolox is more hydrophilic and further shows strong antioxidant properties. Due to the improved intercellular effect and cellular uptake, new opportunities for oligo conjugates with these lipophilic modifications arise in the field of antisense oligonucleotides.
Furthermore, in combination with a fluorescent dye, Trolox is able to "stabilise" the dye and protect it against fading.
1. Efficient in vivo delivery of siRNA to the liver by conjugation of alpha-tocopherol. Nishina K, Unno T, Uno Y, Kubodera T, Kanouchi T, Mizusawa H, Yokota T; Mol Ther. (2008), 16:734–740.
Fatty acids bound to oligonuclotides render the conjugate amphiphilic so that the conjugates can be attached to lipid phases, bilayers or vesicle. The oligo-moiety is thus presented to the aqueous phase, while the fatty acid dips into the lipid phase. An increased uptake through cell membranes is also observed.
1. DNA modification of live cell surface. Borisenko GG, Zaitseva MA, Chuvilin AN, Pozmogova GE; Nucleic Acids Res. (2009), 37, e28.
2. Lipid modification of GRN163, an N3' -> P5' thio-phosphoramidate oligonucleotide, enhances the potency of telomerase inhibition. Herbert BS, Gellert GC, Hochreiter A, Pongracz K, Wright WE, Zielinska D, Chin AC, Harley CB, Shay JW, Gryaznov SM; Oncogene (2005), 24, 5262–5268.
3. General method for modification of liposomes for encoded assembly on supported bilayers. Yohina-Ishii, Miller GP, Kraft ML, Kool ET, Boxer SG; J. Am. Chem. Soc. (2005), 9, 1356- 1357.