Enhanced cytokine secretion owing to multiple CpG side chains of DNA duplex

Unmethylated CpG sequences (CpG ODN) stimulate Toll-like receptor 9 (TLR9) to activate innate immunity. We made DNA duplexes from poly(dT)320 and CpG ODN with (dA)40 attached at the 3' end. Circular dichroism and gel electrophoresis indicated that the CpG parts turned outward from the duplex. When we changed the CpG ODN/poly(dT) molar ratio, the amount of IL-12 secreted from J774A.1 cells (murine macrophage-like) reached the maximum at the compositions with two to four CpG portions in one duplex, while the maximum loading was eight CpG ODNs per one poly(dT)320. When the residual free dT parts were hybridized with its control GpC ODN with (dA)40 tail or just (dA)40, the maximum disappeared and the secretion increased with increasing the CpG molar ratio. These results indicated that there is a particular DNA higher-order structure to activate TLR9 more efficiently than single CpG ODN.