We recently showed that 5'-terminal secondary structures in CpG DNA affect activity significantly more than those at the 3'-end [Biochem. Biophys. Res. Commun. 306 (2003) 948]. The need for an accessible 5'-end of CpG DNA for activity suggested that the receptor reads the DNA sequence from this end. In continuation of these studies, we have designed immunomodulatory oligonucleotides (IMOs), consisting of a nine-mer stimulatory domain, containing a CpG motif and a hairpin-loop structure at the 3'-end, referred to as self-stabilized CpG DNAs. We studied the ability of self-stabilized CpG DNAs to stimulate human B-cell proliferation and interferon-alpha (IFN-alpha.) secretion in plasmacytoid dendritic cell (pDC) culture assays. Self-stabilized CpG DNAs activated human B cells and induced plasmacytoid dendritic cells to secrete high levels of IFN-alpha. While both stimulatory and secondary structures in CpG DNAs were required for pDC activation, CpG motifs were sufficient to activate B cells. Interestingly, CpG motifs were not required for activity in the hairpin duplex region. Further modifications of the hairpin duplex region with a mixture of oligodeoxynuclcotides and oliao-2'-O-methylribonucleotides in a heteroduplex formation permitted activation of both human B cells and pDCs. (C) 2003 Elsevier Inc. All rights reserved.