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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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Chemical conjugation scheme of short PEG chains to antisense oligonucleotides. (A) During oligonucleotide synthesis, aminohexyl linkers were attached to antisense oligonucleotides at the 3′- or 5′-terminus. After cleavage and deprotection, the amines were reacted with PEG12-NHS reagent in an aqueous buffer solution. Using a surplus of the PEG ligand, the reaction proceeded nearly quantitatively after 2 h. (B) The attachment reaction was monitored by taking samples with subsequent analyses on a denaturing polyacrylamide gel. The gel shows a representative image of the reaction of a 3′-aminohexyl phosphorothioate antisense oligonucleotide to give conjugate 5 (from left to right; 0 min, 30 min, 120 min reaction time).
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f0020: Chemical conjugation scheme of short PEG chains to antisense oligonucleotides. (A) During oligonucleotide synthesis, aminohexyl linkers were attached to antisense oligonucleotides at the 3′- or 5′-terminus. After cleavage and deprotection, the amines were reacted with PEG12-NHS reagent in an aqueous buffer solution. Using a surplus of the PEG ligand, the reaction proceeded nearly quantitatively after 2 h. (B) The attachment reaction was monitored by taking samples with subsequent analyses on a denaturing polyacrylamide gel. The gel shows a representative image of the reaction of a 3′-aminohexyl phosphorothioate antisense oligonucleotide to give conjugate 5 (from left to right; 0 min, 30 min, 120 min reaction time).

Mentions: These linkers were then used as attachment points for NHS-activated PEG12 units (Scheme 1). For a nearly quantitative yield after 2 h reaction time, a 50fold surplus of the NHS-PEG ligand was added from a stock solution in anhydrous DMF to the oligonucleotide dissolved in sodium borate buffer. The unreacted PEG ligand was removed from the mixture by gel filtration, and the PEGylated oligonucleotide was purified by preparative gel electrophoresis. Analyses on gel electrophoresis (Scheme 1) and HPLC confirmed product purities.


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)

Chemical conjugation scheme of short PEG chains to antisense oligonucleotides. (A) During oligonucleotide synthesis, aminohexyl linkers were attached to antisense oligonucleotides at the 3′- or 5′-terminus. After cleavage and deprotection, the amines were reacted with PEG12-NHS reagent in an aqueous buffer solution. Using a surplus of the PEG ligand, the reaction proceeded nearly quantitatively after 2 h. (B) The attachment reaction was monitored by taking samples with subsequent analyses on a denaturing polyacrylamide gel. The gel shows a representative image of the reaction of a 3′-aminohexyl phosphorothioate antisense oligonucleotide to give conjugate 5 (from left to right; 0 min, 30 min, 120 min reaction time).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263527&req=5

f0020: Chemical conjugation scheme of short PEG chains to antisense oligonucleotides. (A) During oligonucleotide synthesis, aminohexyl linkers were attached to antisense oligonucleotides at the 3′- or 5′-terminus. After cleavage and deprotection, the amines were reacted with PEG12-NHS reagent in an aqueous buffer solution. Using a surplus of the PEG ligand, the reaction proceeded nearly quantitatively after 2 h. (B) The attachment reaction was monitored by taking samples with subsequent analyses on a denaturing polyacrylamide gel. The gel shows a representative image of the reaction of a 3′-aminohexyl phosphorothioate antisense oligonucleotide to give conjugate 5 (from left to right; 0 min, 30 min, 120 min reaction time).
Mentions: These linkers were then used as attachment points for NHS-activated PEG12 units (Scheme 1). For a nearly quantitative yield after 2 h reaction time, a 50fold surplus of the NHS-PEG ligand was added from a stock solution in anhydrous DMF to the oligonucleotide dissolved in sodium borate buffer. The unreacted PEG ligand was removed from the mixture by gel filtration, and the PEGylated oligonucleotide was purified by preparative gel electrophoresis. Analyses on gel electrophoresis (Scheme 1) and HPLC confirmed product purities.

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