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Oligonucleotides conjugated with short chemically defined polyethylene glycol chains are efficient antisense agents.

Shokrzadeh N, Winkler AM, Dirin M, Winkler J - Bioorg. Med. Chem. Lett. (2014)

Bottom Line: We examined the use of short PEG ligands on the in vitro effect of antisense agents.Circular dichroism showed that the tethering of PEG12-chains to phosphodiester and phosphorothioate oligonucleotides had no influence on their secondary structure and did not reduce the affinity to the counter strand.In an in vitro tumor model, a luciferase reporter assay indicated unchanged gene silencing activity compared to unmodified compounds, and even slightly superior target down regulation was found after treatment with a phosphorothioate modified conjugate.

View Article: PubMed Central - PubMed

Affiliation: University of Vienna, Department of Pharmaceutical Chemistry, Althanstraße 14, 1090 Vienna, Austria.

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Related in: MedlinePlus

Effect of PEGylated phosphorothioate antisense oligonucleotides in a luciferase reporter assay after transfection with lipofectamine 2000 (A) and after naked application (B). For transfections, the indicated oligonucleotides were mixed with the psiCHECK-2-Gal-1 plasmid encoding renilla luciferase fused to the human cDNA of galectin-1, and the firefly luciferase for normalization. The mixture was co-transfected into the breast cancer cell line MCF-7 and after 48 h incubation, the luminescence levels were recorded in a plate reader. For the evaluation of unassisted cellular uptake, the plasmid was first transfected into the cells, and 24 h later the oligonucleotides were added into fresh medium. Luminescence levels were analyzed after a 48 h incubation period. Values are reported as renilla normalized to firefly luciferase signals (n = 3, error bars are SEM). In co-transfections, the phosphorothioate conjugate 5 showed higher efficacy compared to the isosequential oligonucleotide 4 (100 nM, p < 0.05), but no influence of the PEGylation on gene silencing activity was found after naked application (gymnotic uptake).
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f0010: Effect of PEGylated phosphorothioate antisense oligonucleotides in a luciferase reporter assay after transfection with lipofectamine 2000 (A) and after naked application (B). For transfections, the indicated oligonucleotides were mixed with the psiCHECK-2-Gal-1 plasmid encoding renilla luciferase fused to the human cDNA of galectin-1, and the firefly luciferase for normalization. The mixture was co-transfected into the breast cancer cell line MCF-7 and after 48 h incubation, the luminescence levels were recorded in a plate reader. For the evaluation of unassisted cellular uptake, the plasmid was first transfected into the cells, and 24 h later the oligonucleotides were added into fresh medium. Luminescence levels were analyzed after a 48 h incubation period. Values are reported as renilla normalized to firefly luciferase signals (n = 3, error bars are SEM). In co-transfections, the phosphorothioate conjugate 5 showed higher efficacy compared to the isosequential oligonucleotide 4 (100 nM, p < 0.05), but no influence of the PEGylation on gene silencing activity was found after naked application (gymnotic uptake).

Mentions: Breast cancer cells MCF-7 were treated with both phosphodiesters (1 and 3) and phosphorothioates (4 and 5) with and without 3′-PEG ligand (Fig. 2). After co-transfection with lipofectamine 2000, all tested oligonucleotides resulted in significant gene silencing (Fig. 2A, p < 0.05). Neither statistically significant enhancement nor decrease of the effect was detected by the PEGylation of the phosphodiester backbone compound. In contrast, the PEGylated phosphorothioate 5 showed higher gene silencing activity than its non-PEGylated counterpart 4 after transfection in a concentration of 100 nM (p < 0.05).


Oligonucleotides conjugated with short chemically defined polyethylene glycol chains are efficient antisense agents.

Shokrzadeh N, Winkler AM, Dirin M, Winkler J - Bioorg. Med. Chem. Lett. (2014)

Effect of PEGylated phosphorothioate antisense oligonucleotides in a luciferase reporter assay after transfection with lipofectamine 2000 (A) and after naked application (B). For transfections, the indicated oligonucleotides were mixed with the psiCHECK-2-Gal-1 plasmid encoding renilla luciferase fused to the human cDNA of galectin-1, and the firefly luciferase for normalization. The mixture was co-transfected into the breast cancer cell line MCF-7 and after 48 h incubation, the luminescence levels were recorded in a plate reader. For the evaluation of unassisted cellular uptake, the plasmid was first transfected into the cells, and 24 h later the oligonucleotides were added into fresh medium. Luminescence levels were analyzed after a 48 h incubation period. Values are reported as renilla normalized to firefly luciferase signals (n = 3, error bars are SEM). In co-transfections, the phosphorothioate conjugate 5 showed higher efficacy compared to the isosequential oligonucleotide 4 (100 nM, p < 0.05), but no influence of the PEGylation on gene silencing activity was found after naked application (gymnotic uptake).
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Related In: Results  -  Collection

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f0010: Effect of PEGylated phosphorothioate antisense oligonucleotides in a luciferase reporter assay after transfection with lipofectamine 2000 (A) and after naked application (B). For transfections, the indicated oligonucleotides were mixed with the psiCHECK-2-Gal-1 plasmid encoding renilla luciferase fused to the human cDNA of galectin-1, and the firefly luciferase for normalization. The mixture was co-transfected into the breast cancer cell line MCF-7 and after 48 h incubation, the luminescence levels were recorded in a plate reader. For the evaluation of unassisted cellular uptake, the plasmid was first transfected into the cells, and 24 h later the oligonucleotides were added into fresh medium. Luminescence levels were analyzed after a 48 h incubation period. Values are reported as renilla normalized to firefly luciferase signals (n = 3, error bars are SEM). In co-transfections, the phosphorothioate conjugate 5 showed higher efficacy compared to the isosequential oligonucleotide 4 (100 nM, p < 0.05), but no influence of the PEGylation on gene silencing activity was found after naked application (gymnotic uptake).
Mentions: Breast cancer cells MCF-7 were treated with both phosphodiesters (1 and 3) and phosphorothioates (4 and 5) with and without 3′-PEG ligand (Fig. 2). After co-transfection with lipofectamine 2000, all tested oligonucleotides resulted in significant gene silencing (Fig. 2A, p < 0.05). Neither statistically significant enhancement nor decrease of the effect was detected by the PEGylation of the phosphodiester backbone compound. In contrast, the PEGylated phosphorothioate 5 showed higher gene silencing activity than its non-PEGylated counterpart 4 after transfection in a concentration of 100 nM (p < 0.05).

Bottom Line: We examined the use of short PEG ligands on the in vitro effect of antisense agents.Circular dichroism showed that the tethering of PEG12-chains to phosphodiester and phosphorothioate oligonucleotides had no influence on their secondary structure and did not reduce the affinity to the counter strand.In an in vitro tumor model, a luciferase reporter assay indicated unchanged gene silencing activity compared to unmodified compounds, and even slightly superior target down regulation was found after treatment with a phosphorothioate modified conjugate.

View Article: PubMed Central - PubMed

Affiliation: University of Vienna, Department of Pharmaceutical Chemistry, Althanstraße 14, 1090 Vienna, Austria.

Show MeSH
Related in: MedlinePlus