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J. Biol. Chem., Vol. 279, Issue 32, 33071-33078, August 6, 2004

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Necessity of Oligonucleotide Aggregation for Toll-like Receptor 9 Activation^*

Christina C. N. Wu, Jongdae Lee, Eyal Raz, Maripat Corr, and Dennis A. Carson

From the 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

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.

Received for publication, October 23, 2003 , and in revised form, May 17, 2004.

^* This work was supported in part by National Institutes of Health Grants AI56453, AI40682, AR44850, AR07567, and GM23200. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: 9500 Gilman Dr., #0663, La Jolla, CA 92093-0663. Tel.: 858-534-5442; Fax: 858-534-5399; E-mail: c5wu{at}ucsd.edu.

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