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Merck
  • High-throughput chemical modification of oligonucleotides for systematic structure-activity relationship evaluation.

High-throughput chemical modification of oligonucleotides for systematic structure-activity relationship evaluation.

Bioconjugate chemistry (2014-11-15)
Daniel Zewge, Francis Gosselin, Denise M Kenski, Jenny Li, Vasant Jadhav, Yu Yuan, Sandhya S Nerurkar, David M Tellers, W Michael Flanagan, Ian W Davies
초록

Chemical modification of siRNA is achieved in a high-throughput manner (96-well plate format) by copper catalyzed azide-alkyne cycloadditions. This transformation can be performed in one synthetic operation at up to four positions with complete specificity, good yield, and acceptable purity. As demonstrated here, this approach extends the current synthetic options for oligonucleotide modifications and simultaneously facilitates the systematic, rapid biological evaluation of modified siRNA.

MATERIALS
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Sigma-Aldrich
Imidazole, ACS reagent, ≥99% (titration)
Sigma-Aldrich
Water, PCR Reagent
Sigma-Aldrich
Water, for embryo transfer, sterile-filtered, BioXtra, suitable for mouse embryo cell culture
Sigma-Aldrich
Trichloroacetic acid, suitable for electrophoresis, suitable for fixing solution (for IEF and PAGE gels), ≥99%
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Trichloroacetic acid, BioXtra, ≥99.0%
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Triethylamine, BioUltra, ≥99.5% (GC)
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Azidomethyl phenyl sulfide, 95%
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Methylamine solution, 40 wt. % in H2O
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Methylamine solution, 2.0 M in THF
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Methanol, anhydrous, 99.8%
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Methylamine solution, 2.0 M in methanol
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Trichloroacetic acid, ≥99.0% (titration)
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Trichloroacetic acid, ACS reagent, ≥99.0%
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Acetonitrile solution, contains 0.1 % (v/v) trifluoroacetic acid, suitable for HPLC
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Acetonitrile solution, contains 0.05 % (v/v) trifluoroacetic acid
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Triethylamine, for amino acid analysis, ≥99.5% (GC)
Supelco
Water, for TOC analysis
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Acetonitrile solution, contains 0.1 % (v/v) formic acid, suitable for HPLC
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Benzyl azide solution, ~0.5 M in dichloromethane, ≥95.0% (HPLC)
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Methanol, NMR reference standard
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Methylamine solution, 33 wt. % in absolute ethanol ((denatured with 1% toluene))
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Methanol, HPLC Plus, ≥99.9%, poly-coated bottles
Imidazole, European Pharmacopoeia (EP) Reference Standard
Pure Water Density Standard, UKAS ISO/IEC17025 and ISO Guide 34 certified, density: 0.9982 g/mL at 20 °C, density: 0.9970 g/mL at 25 °C
Pure Water Density Standard, UKAS ISO/IEC17025 and ISO Guide 34 certified, density: 0.9982 g/mL at 20 °C, density: 0.9970 g/mL at 25 °C
Ondansetron impurity E, European Pharmacopoeia (EP) Reference Standard
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Imidazole, ReagentPlus®, 99%, Redi-Dri, free-flowing
Sigma-Aldrich
Methanol, ACS reagent, ≥99.8%
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Methanol, Absolute - Acetone free
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Methanol, BioReagent, ≥99.93%