Bacterial and synthetic DNAs containing CpG dinucleotides in specific sequence contexts activate the vertebrate immune system through Toll-like receptor 9 (TLR9). In the present study, we used a synthetic nucleoside with a bicyclic heterobase [1-(2 -deoxy--D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; R] to replace the C in CpG, resulting in an RpG dinucleotide. The RpG dinucleotide was incorporated in mouse-and human-specific motifs in oligodeoxynucleotides (oligos) and 3 -3-linked oligos, referred to as immunomers. Oligos containing the RpG motif induced cytokine secretion in mouse spleen-cell cultures. Immunomers containing RpG dinucleotides showed activity in transfected-HEK293 cells stably expressing mouse TLR9, suggesting direct involvement of TLR9 in the recognition of RpG motif. In J774 macrophages, RpG motifs activated NF-B and mitogen-activated protein kinase pathways. Immunomers containing the RpG dinucleotide induced high levels of IL-12 and IFN-␥, but lower IL-6 in time-and concentrationdependent fashion in mouse spleen-cell cultures costimulated with IL-2. Importantly, immunomers containing GTRGTT and GARGTT motifs were recognized to a similar extent by both mouse and human immune systems. Additionally, both mouse-and humanspecific RpG immunomers potently stimulated proliferation of peripheral blood mononuclear cells obtained from diverse vertebrate species, including monkey, pig, horse, sheep, goat, rat, and chicken. An immunomer containing GTRGTT motif prevented conalbumin-induced and ragweed allergen-induced allergic inflammation in mice. We show that a synthetic bicyclic nucleotide is recognized in the C position of a CpG dinucleotide by immune cells from diverse vertebrate species without bias for flanking sequences, suggesting a divergent nucleotide motif recognition pattern of TLR9.T he immune system of vertebrates recognizes synthetic oligodeoxynucleotides, bacterial and plasmid DNAs containing unmethylated CpG dinucleotides in specific sequence contexts (CpG motifs) (1-5). This recognition is mediated by a molecular pattern recognition receptor, Toll-like receptor 9 (TLR9) (6). Upon activation by CpG DNA, TLR9 initiates signaling pathways that activate several transcription factors, including NF-B and AP-1 (3-5). The immunological events that occur after CpG DNA activation of the immune system include B cell proliferation, increased expression of MHC class II antigens, increased synthesis of RNA and DNA, and the release of a number of cytokines and chemokines (7-9). The potent T helper 1 immune response produced by CpG activation of the innate immune system supports the broad therapeutic application of CpG DNA against cancer, infectious diseases, asthma, and allergies and as adjuvants in immunotherapy. Several studies have demonstrated the potential effectiveness of CpG DNAs as therapies in animal models, and a number of CpG DNAs are currently being tested in clinical trials (4, 10).We previously showed that two CpG DNAs attached through their 5Ј ends failed to stimulate the immune syst...