title: Peptide conjugation at the 5'-end of oligodeoxynucleotides abrogates toll-like receptor 9-mediated immune stimulatory activity.
abstract: Bacterial and synthetic DNA containing unmethylated CpG motifs act as ligands of Toll-like receptor 9 (TLR9). Our earlier studies showed that 5'-accessibility of synthetic oligodeoxynucleotides containing CpG motif (ODN) is required for TLR9-mediated immune stimulatory activity. Blocking the 5'-end of ODN through conjugation to a variety of moieties reduces immune stimulatory activity (Bioconjugate Chem. 2002, 13, 966-974). In the present study, we conjugated a model peptide, a 28-amino-acid-long beta-amyloid peptide, to either the 5'- or the 3'-end of an ODN via C3 and C6 alkyl linkers. We compared the immune stimulatory activity of the resulting conjugates with that of a parent ODN without conjugation in TLR9-transfected cells, mouse spleen cell cultures, and in vivo in mice. ODN with the peptide conjugated at the 3'-end via C3 and C6 linkers had immune stimulatory activity similar to that of the parent ODN in both in vitro and in vivo in mice. On the contrary, conjugation of peptide at the 5'-end of the ODN significantly abrogated immune stimulatory activity. In conclusion, the results presented here demonstrate that peptide/protein conjugation to ODN is optimal at the 3'-end with either C3 or C6 linker and conjugation at the 5'-end leads to significant loss of TLR9-mediated immune stimulation.
authors.affiliation: Idera Pharmaceuticals, Inc., Cambridge, Massachusetts 02139, USA.
authors.forename: Mallikarjuna Reddy
authors.initials: MR
authors.lastname: Putta
authors.forename: Fu-Gang
authors.initials: FG
authors.lastname: Zhu
authors.forename: Daqing
authors.initials: D
authors.lastname: Wang
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Bhagat
authors.forename: Meiru
authors.initials: M
authors.lastname: Dai
authors.forename: Ekambar R
authors.initials: ER
authors.lastname: Kandimalla
authors.forename: Sudhir
authors.initials: S
authors.lastname: Agrawal
mesh_terms: D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000276:immunology; D000818:Animals; D002478:Cells, Cultured; D006801:Humans; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000276:immunology*; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010455:Peptides / Q000737:chemistry* / Q000276:immunology*; D013154:Spleen / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D051217:Toll-Like Receptor 9 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D016229:Amyloid beta-Peptides; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D010455:Peptides; C495370:Tlr9 protein, mouse; D051217:Toll-Like Receptor 9