Immunostimulatory properties of phosphorothioate CpG DNA containing both 3'-5'- and 2'-5'-internucleotide linkages

Synthetic oligodeoxyribonucleotides containing CpG-dinucleotides (CpG DNA) in specific sequence contexts activate the vertebrate immune system. We have examined the effect of 3'-deoxy-2'-5'-ribonucleoside (3'-deoxynucleoside) incorporation into CpG DNA on the immunostimulatory activity. Incorporation of 3'-deoxynucleosides results in the formation of 2'-5'-internucleotide linkages in an otherwise 3'-5'-linked CpG DNA. In studies, both in vitro and in vivo, CpG DNA containing unnatural 3'-deoxynucleoside either within the CpG-dinucleotide or adjacent to the CpG-dinucleotide failed to induce immunostimulatory activity, suggesting that the modification was not recognized by the receptors. Incorporation of the same modification distal to the CpG-dinucleotide in the 5'-flanking sequence potentiated the immunostimulatory activity of the CpG DNA. The same modification when incorporated in the 3'-flanking sequence had an insignificant effect on immunostimulatory activity of CpG DNA. Interestingly, substitution of a 3'-deoxynucleoside in the 5'-flanking sequence distal to the CpG-dinucleotide resulted in increased IL-6 and IL-10 secretion with similar levels of IL-12 compared with parent CpG DNA. The incorporation of the same modification in the 3'-flanking sequence resulted in lower IL-6 and IL-10 secretion with similar levels of IL-12 compared with parent CpG DNA. These results suggest that site-specific incorporation of 3'-deoxynucleotides in CpG DNA modulates immunostimulatory properties.