Linear, single-stranded oligonucleotides (ODN) with non-methylated cytosine-guanine (CpG) motifs are immunomodulatory since they resemble bacterial DNA and serve as “danger signals”. These CpG-ODNs promote predominately a TH1-response, with secretion of IL-12 and IFN-γ, and B-cell, natural killer (NK)-cell and dendritic cell (DC) activation and have a broad potential as therapeutic agents, i.e. for cancer gene therapy and for the treatment of allergic diseases. Distinct groups of CpG-ODNs were characterized differing in structure and function: one group promotes B-cell proliferation, monocyte stimulation and secretion of IgM and IL-6; another activates plasmacytoid DC to produce IFN-α/-β and thus γδT-cells and NK-cells to express CD69 and secrete IFN-γ. And a third group exhibits combined properties of stimulating IL-6 and IgM secretion from B cells as well as IFN-α production from pDCs. Phosphorothioate (PS) modifications, usually introduced to enhance stability, result in several side-effects, like prolongation of the blood clotting time, non-specific binding to various proteins and acute toxicities in primates. In addition, a recent publication showed severe side-effect like significant organ enlargements and morphological changes in mice [Heikenwalder et al., Nat Med. 10:187, 2004]. We generated short covalently-closed dumbbell-like structures (dSLIM) to stabilize the DNA without the otherwise necessary PS-modification. Moreover, the covalently closed constructs do not signal for apoptosis, as high intracellular concentrations of open DNA ends would do. These dSLIM molecules are stable in serum and during long-term storage regarding both DNA integrity and biological function. Their broad activity, like increasing surface expression of CD80/B7.1, CD40, HLA-DR/MHC-II and CD54/ICAM-1 and enhancing production of a wide range of cytokines (IL-6, IFN-α, IFN-γ, IL-12, IL-2), was strictly dependent on molecule structure and size. Increasing or decreasing of stem size lead to reduced potency of the dumbbell-shaped dSLIM molecule. This was observed for a decreased size of the loops as well. But the most intriguing result was the significantly reduced toxicity of dSLIM compared to PS-ODN: After repeated injection of dSLIM or PS-ODN, respectively, into mice the mice receiving PS-ODN developed enlargement of liver, spleen and lymph nodes whereas dSLIM did not induce such changes. In addition, damage of liver and spleen - such as necrotic hepatocytes or hyperplasia - was observed in PS-ODN treated but not in dSLIM treated mice. Nevertheless, both dSLIM and PS-ODN induced a comparable IL-12 production in these mice in vivo. The significant differences of side-effect were true for PS-ODN and dSLIM molecules with various nucleotide sequences. In conclusion, we present the new class of potent immunomodulators (dSLIM) with significantly reduced side-effects.