Abstract
Methods and protocols for automated synthesis and purification of immune modulatory oligonucleotides (IMOs), a novel class of Toll-like receptor 9 (TLR9) agonists, are described. IMOs containing two short identical sequences of 11-mers with phosphorothioate linkages can be synthesized in parallel synthetic strategy. A C3-linker that mimics the natural inter-nucleotide distance was commonly used for joining the two segments of IMOs. NittoPhase solid support bearing a symmetrical C3-linker (glycerol) and nucleoside-β-cyanoethyl-N,N-diisopropylphosphoramidites were used for IMO synthesis. The parallel synthesis was carried out in a 3′→ 5′ direction with removal of the final dimethoxytrityl (DMT) protecting group. After synthesis, the IMO was cleaved and deprotected by treating with aqueous ammonia. The product was purified on anion-exchange HPLC, desalted, lyophilized, and characterized by anion-exchange HPLC, capillary gel electrophoresis, polyacrylamide gel electrophoresis, and MALDI-TOF mass spectral analysis.
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Acknowledgments
The authors thank Dr. Sudhir Agrawal for support, encouragement, and suggestions.
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Putta, M.R., Yu, D., Kandimalla, E.R. (2011). Synthesis, Purification, and Characterization of Immune-Modulatory Oligodeoxynucleotides that Act as Agonists of Toll-Like Receptor 9. In: Goodchild, J. (eds) Therapeutic Oligonucleotides. Methods in Molecular Biology, vol 764. Humana Press. https://doi.org/10.1007/978-1-61779-188-8_18
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DOI: https://doi.org/10.1007/978-1-61779-188-8_18
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