Secondary structures in CpG oligonucleotides affect immunostimulatory activity

Oligodeoxynucleotides containing CpG dinucleotides in specific sequence contexts activate the vertebrate immune system. Our previous studies showed that the 5(')-end of a CpG oligonucleotide should be accessible for receptor recognition and subsequent immune stimulation. Activity is abrogated if this end is blocked by joining two CpG oligos through 5(')-5(') linkage. It was not known whether a similar effect would arise from secondary structures at either end of a CpG oligo, such as hairpin loops or terminal dimers. In the present study we found that 5(')-terminal secondary structures affect activity significantly more than those at the 3(')-end. The need for an open 5(')-end suggests that the receptor responsible for immune stimulation reads the DNA sequence from this end. These results may also provide insights to place CpG motifs appropriately in DNA vaccines to induce additional Th1 type responses.