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Enhanced immunostimulatory activity of oligodeoxynucleotides by Y‐shape formation

DNA containing unmethylated CpG dinucleotides (CpG DNA) is a potent activator of innate and acquired immune responses. Although the sequence‐specific immunostimulatory activity of CpG DNA has been extensively explored, little information is available about the importance of the stereochemical properties of CpG DNA. In this study, Y‐shaped oligodeoxynucleotides (Y‐ODNs) were prepared using three ODNs with the halves of each ODN being partially complementary to a half of the other two ODNs. Y‐ODN induced greater amounts of tumour necrosis factor‐α and interleukin‐6 from RAW264.7 macrophage‐like cells than did conventional single‐stranded ODN (ssODN) or double‐stranded ODN (dsODN). The Y‐ODN was less stable in serum than dsODN, but greater amounts of Y‐ODN were taken up by macrophage‐like cells compared with dsODN. A newly designed Y‐ODN containing three potent CpG motifs generated significantly higher levels of cytokines compared with dsODN containing the identical sequences. These results indicate that the Y‐shaped form of ODN is a novel, reproducible and reliable approach to enhancing the immunostimulatory activity of ODNs.