title: Necessity of oligonucleotide aggregation for toll-like receptor 9 activation.
abstract: Toll-like receptor 9 (TLR9), a member of the interleukin-1 (IL-1) family of pathogen-associated molecular pattern receptors, is activated by unmethylated CpG-containing sequences in bacterial DNA or synthetic oligonucleotides (ODNs) in the endosomal compartment. The stimulation of an IL-1 response is thought to require the aggregation of its receptor. By analogy, we postulated that the potency of a TLR9 ligand should depend first on its ability to enter cells and gain access to TLR9 and second on its capacity to form a multimeric complex capable of cross-linking these receptors. Previously, we selected from a random library a series of phosphodiester ODNs with enhanced ability to permeate cells. Here, we studied the structural requirements for these penetrating ODNs to elicit a functional TLR9 response, as assessed by cytokine production from bone marrow-derived mouse mononuclear cells. The presence of a prototypic murine immunostimulatory DNA hexameric sequence (purine-purine-CG-pyrimidine-pyrimidine) in the ODNs was not sufficient for stimulation. In addition, the TLR9-activating ODNs had to have the ability to form aggregates and often to form secondary structures near the core CpG motifs. Multimerization was promoted by the presence of a guanine-rich 3'-terminus. The phosphodiester ODNs with CpG motifs that did not aggregate antagonized the effects of the multimeric TLR9 activators. These findings suggest that an optimal TLR9 agonist needs to contain a spatially distinct multimerization domain and a receptor binding CpG domain. This concept may prove useful for the design of new TLR9-modulating agents.
authors.affiliation: Division of Rheumatology Allergy and Immunology, Department of Medicine and the Sam and Rose Stein Institute for Research on Aging, University of California San Diego, La Jolla, California 92093-0663, USA. c5wu@ucsd.edu
authors.forename: Christina C N
authors.initials: CC
authors.lastname: Wu
authors.forename: Jongdae
authors.initials: J
authors.lastname: Lee
authors.forename: Eyal
authors.initials: E
authors.lastname: Raz
authors.forename: Maripat
authors.initials: M
authors.lastname: Corr
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Carson
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000943:Antigens, Differentiation / Q000502:physiology; D001483:Base Sequence; D001854:Bone Marrow Cells / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000187:drug effects* / Q000502:physiology*; D018664:Interleukin-12 / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D053594:Myeloid Differentiation Factor 88; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D011956:Receptors, Cell Surface / Q000172:deficiency / Q000187:drug effects* / Q000502:physiology*; D011971:Receptors, Immunologic / Q000172:deficiency / Q000502:physiology; D013329:Structure-Activity Relationship; D051217:Toll-Like Receptor 9
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000943:Antigens, Differentiation; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D009838:Oligodeoxyribonucleotides; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; C495370:Tlr9 protein, mouse; D051217:Toll-Like Receptor 9; D018664:Interleukin-12; D004247:DNA