The critical DNA flanking sequences of a CpG Oligodeoxynucleotide, but not the 6 base CpG motif, can be replaced with RNA without quantitative or qualitative changes in Toll-like receptor 9-mediated activity

Double- and single-stranded oligodeoxynucleotides containing unmethylated cytosine–guanosine (CpG) dinucleotides (CpGODN) activate immune cells via TLR9. In this report we synthesized hybrid DNA–RNA molecules (HDR) in order to further explore the structure–immune function relationship of CpG-ODN in TLR9 signaling and the potential immunomodulatory properties of RNA. We demonstrate that replacement of the deoxyadenosine Xanking sequences, critical for the immune activating properties of CpG-ODN, with a similar number of adenosines, although not guanosines, cytosines, or uracils, maintains complete immunostimulatory activity of the hybrid oligonucleotide in vitro, whereas a similar RNA replacement of even 1 base of the required unmethylated 6 base DNA motif (purine–purine–CpG–pyrimidine–pyrimidine) results in a complete loss of activity. Regardless of whether the critical Xanking sequence was RNA or DNA there was no signiWcant change in the quantitative or qualitative immunestimulating activity, or TLR-speciWcity of the resulting sequences, thus underscoring the relatively permissive functional role of the Xanking sequence, and the more speciWc role of the motif in mediating TLR9 signaling. These data further support a potential role for RNA in immunomodulation. Published by Elsevier Inc.