title: Synthesis, purification, and characterization of immune-modulatory oligodeoxynucleotides that act as agonists of Toll-like receptor 9.
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.
authors: Idera Pharmaceuticals, Inc., Cambridge, MA, USA. mputta@iderapharma.com
authors: Mallikarjuna Reddy
authors: MR
authors: Putta
authors: Dong
authors: D
authors: Yu
authors: Ekambar R
authors: ER
authors: Kandimalla
mesh_terms: D000276:Adjuvants, Immunologic / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000494:pharmacology; D001424:Bacterial Infections / Q000188:drug therapy / Q000276:immunology / Q000382:microbiology; D001483:Base Sequence; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D019075:Electrophoresis, Capillary; D005990:Glycerol / Q000737:chemistry; D006801:Humans; D007155:Immunologic Factors / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000494:pharmacology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000494:pharmacology; D054735:Phosphorothioate Oligonucleotides / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D051217:Toll-Like Receptor 9 / Q000819:agonists* / Q000276:immunology / Q000378:metabolism; D014777:Virus Diseases / Q000188:drug therapy / Q000276:immunology / Q000821:virology
chemical_list: D000276:Adjuvants, Immunologic; D007155:Immunologic Factors; D009838:Oligodeoxyribonucleotides; D054735:Phosphorothioate Oligonucleotides; D051217:Toll-Like Receptor 9; D005990:Glycerol