The Influence of Base Sequence on the Immunological Properties of Defined Oligonucleotides

To assess the influence of base sequence on the immunostimulatory activities of DNA, cell binding and mitogenicity of a series of 30-mer phosphodiester oligonucleotides were tested using murine spleen cells. These compounds consisted of either a single base or a six base CpG motif in the context of 5' and 3' flanking sequences of each base. Among fluoresceinated oligonucleotides, (dG)30 had the highest binding of single base compounds tested while the presence of dG flanks increased binding of compounds with six base motifs, whether active on inactive. In assays of mitogenesis including incorporation of thymidine and uridine as well as expression of cell surface CD69, (dG)30 induced the highest responses among single base compounds. Among compounds with an active six base motif, the extent of proliferation varied with flanking sequence, with dG flanks producing the greatest stimulation in all assays tested. Together, these findings indicate that a variety of base sequences may affect the immunomodulatory properties of DNA, with the activity of dG sequences perhaps resulting from the formation of variant DNA structures.