Synthesis, Purification, and Characterization of Immune-Modulatory Oligodeoxynucleotides that Act as Agonists of Toll-Like Receptor 9

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.