Synthesis of alkylating oligonucleotide derivatives containing cholesterol or phenazinium residues at their 3′‐terminus and their interaction with DNA within mammalian cells

Boutorin, A.S.; Gus'kova, L.V.; Иванова, Е. М.; Kobetz, N.D.; Зарытова, В. Ф.; Ryte, A. S.; Yurchenko, L. V.; Vlassov, V. V.

doi:10.1016/0014-5793(89)81023-3

Cited by 149 publications

(74 citation statements)

References 9 publications

(3 reference statements)

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“…In particular, for the same number density and lipid composition, 5Ј-Poly T / 3Ј-Poly A 24-mers gave a much higher yield of lipid and content mixing than a fully overlapping complementary sequence pair (22). Related results have been reported using more complex, double cholesterol-anchored-DNA conjugates (23,24) [singly anchored cholesterol-DNA conjugates, which have been proposed for drug delivery purposes (25), partition only transiently to lipid bilayers or vesicles in contrast to the anchor shown in Fig. 4].…”

mentioning

confidence: 73%

Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides

Chan

Lengerich

Boxer

2009

Proc. Natl. Acad. Sci. U.S.A.

273

287

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Synthetic lipid-oligonucleotide conjugates inserted into lipid vesicles mediate fusion when one population of vesicles displays the 5 -coupled conjugate and the other the 3 -coupled conjugate, so that anti-parallel hybridization allows the membrane surfaces to come into close proximity. Improved assays show that lipid mixing proceeds more quickly and to a much greater extent than content mixing, suggesting the latter is rate limiting. To test the effect of membrane-membrane spacing on fusion, a series of conjugates was constructed by adding 2-24 noncomplementary bases at the membrane-proximal ends of two complementary sequences. Increasing linker lengths generally resulted in progressively reduced rates and extents of lipid and content mixing, in contrast to higher vesicle docking rates. The relatively flexible, single-stranded DNA linker facilitates docking but allows greater spacing between the vesicles after docking, thus making the transition into fusion less probable, but not preventing it altogether. These experiments demonstrate the utility of DNA as a model system for fusion proteins, where sequence can easily be modified to systematically probe the effect of distance between bilayers in the fusion reaction.DNA machine ͉ synthetic biology F usion of lipid membranes plays a central role in many biological processes. For example, in the case of synaptic transmission, the reaction is tightly regulated by a number of proteins of which the SNAREs [soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptors] have been shown to play a crucial role (1-3). In vitro studies have demonstrated the sufficiency of SNAREs to mediate fusion when reconstituted into a number of model systems, including freestanding lipid vesicles (4-7), supported lipid bilayers (8-10), and tethered vesicles (11). Generally these studies suggest that SNAREs are able to bring two membranes into close apposition by forming a trans complex in a docking reaction which is highly energetically favorable (12). This is illustrated in Fig. 1A, which is based on the x-ray structures of the soluble domains of SNARE proteins in complex (13). Several steps along the mechanism to achieve membrane fusion following this basic docking step have been proposed, and some of these are illustrated in Fig. 2, which also serves to illustrate assays commonly used to assess their role. In this step-wise mechanism, the bilayers first dock, then adopt an intermediate, hemifused state in which the outer leaflets have merged, but the inner leaflets and contents remain distinct. A transition into full fusion then involves mixing of both leaflets and content exchange.Despite substantial research on the role of SNARE proteins in the fusion process, many questions remain regarding the physical mechanism. Studies using simpler model systems can provide insight into the fundamental mechanisms of the rearrangement of bilayers and lead to a better understanding of the biological process (14, 15). We have prepared a series of DNA-lipid conjugates in which DNA o...

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mentioning

confidence: 73%

Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides

Chan

Lengerich

Boxer

2009

Proc. Natl. Acad. Sci. U.S.A.

273

287

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“…These telomerase inhibitory effects of the oligonucleotides, however, were significantly more profound in vitro in the presence of a transfection reagent. Several modifications of oligonucleotides, such as lipid or cellpenetrating peptide conjugates, have been suggested to improve oligonucleotide in vitro activity (Boutorin et al, 1989;Letsinger et al, 1989;Shea et al, 1990;Farooqui et al, 1991;Reed et al, 1991;Stein et al, 1991;MacKellar et al, 1992;Oberhauser and Wagner, 1992;Polushin and Cohen, 1994;Mishra et al, 1995). In particular, cholesterol modification of phosphorothioate oligonucleotides has been shown to improve inhibition of HIV replication (Letsinger et al, 1989;Farooqui et al, 1991;Stein et al, 1991).…”

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confidence: 99%

Lipid modification of GRN163, an N3′ → P5′ thio-phosphoramidate oligonucleotide, enhances the potency of telomerase inhibition

et al. 2005

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The vast majority of human cancers express telomerase activity, while most human somatic cells do not have detectable telomerase activity. Since telomerase plays a critical role in cell immortality, it is an attractive target for a selective cancer therapy. Oligonucleotides complementary to the RNA template region of human telomerase (hTR) have been shown to be effective inhibitors of telomerase and, subsequently, cancer cell growth in vitro. We show here that a lipid-modified N3 0 -P5 0 thiophosphoramidate oligonucleotide (GRN163L) inhibits telomerase more potently than its parental nonconjugated thio-phosphoramidate sequence (GRN163). Cells were treated with both the first-(GRN163) and secondgeneration (GRN163L) oligonucleotides, including a mismatch control, with or without a transfection enhancer reagent. GRN163L inhibited telomerase activity effectively in a dose-dependent manner, even without the use of a transfection reagent. The IC 50 values for GRN163 in various cell lines were on average sevenfold higher than for GRN163L. GRN163L inhibition of telomerase activity resulted in a more rapid loss of telomeres and cell growth than GRN163. This report is the first to show that lipid modification enhanced the potency of the novel GRN163 telomerase inhibitor. These results suggest that the lipidconjugated thio-phosphoramidates could be important for improved pharmacodynamics of telomerase inhibitors in cancer therapy.

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“…Many studies have examined the ability of cholesteryl-conjugated oligonucleotides to associate with cells and effects on efficacy. These studies examined the uptake and effects of cholesteryl-rnodified antisense oligonucleotides in transformed fibroblasts or lymphocytes (Reed etal., 1995;Zhang et al, 1995;Boutorin et al, 1989;Kreig et aI., 1993). In this report we have investigated antiviral activity and cellular uptake in normal human cells, which seem to display different characteristics with respect to uptake and subcellular distribution in that most of the oligonucleotide remains in the cytoplasm in uninfected cells.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Human Cytomegalovirus DNA Replication with a Phosphorothioate Cholesteryl-Modified Oligonucleotide is Mediated by Rapid Cellular Association and Virus-Facilitated Nuclear Localization

Zhang¹,

Smith²,

Smyth³

et al. 1997

Antivir Chem Chemother

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SummaryWe have previously shown that an antisense phosphorothioate (PS) oligodeoxynucleotide has potent anti-human cytomegalovirus (HCMV) activity (GS Pari, AK Field &JA Smith, Antimicrobial Agents and Chemotherapy 1995, 39: 1157-1161). We have now used a modified PS oligonucleotide havingthree 2'-O-methyl nucleotides at the 3' end and four2'-O-methyl nucleotides at the 5' end, containing a cholesteryl moiety linked to the 3' end by a novel thiono-triester linkage. This compound, UL36ANTI-M, is superior to the PS (UL36ANTI) version with respect to antiviral potency, melting temperature and nuclease resistance. Also, we show that cellular association for this oligonucleotide is rapid, occurring within 15 min after treatment and is about 12-fold higher when compared to UL36ANTI. This increased rate of cellular association also correlates with antiviral properties in that a 15 min incubation with UL36ANTI-M was sufficient to achieve 75% inhibition of viral DNA replication and complete inhibition was achieved after only a 1 h pretreatment. In addition confocal microscopic examination showed a change in subcellular distribution from perinuclear to nuclear for oligonucleotides in HCMV-infected human fibroblasts.However, the total amount of cell-associated oligonucleotide was unchanged in infected cells.

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Synthesis of alkylating oligonucleotide derivatives containing cholesterol or phenazinium residues at their 3′‐terminus and their interaction with DNA within mammalian cells

Cited by 149 publications

References 9 publications

Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides

Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides

Lipid modification of GRN163, an N3′ → P5′ thio-phosphoramidate oligonucleotide, enhances the potency of telomerase inhibition

Inhibition of Human Cytomegalovirus DNA Replication with a Phosphorothioate Cholesteryl-Modified Oligonucleotide is Mediated by Rapid Cellular Association and Virus-Facilitated Nuclear Localization

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