Single-stranded RNAs act as ligands of Toll-like receptors (TLRs) 7 and 8 and induce immune responses. In the present study, we have designed and synthesized phosphorothioate oligoribonucleotides (ORNs) with self-complementary Sequences that form duplex structures with either 3'- or 5'-overhanging sequences. We studied the new ORNs for their duplex formation, nuclease stability, and ability to Induce immune-stimulatory activate through TLR7 and TLR8 in TLR-transfected cell lines, PBMCs, human pDCs, and in vivo in mice. Thermal melting and gel electrophoresis studies showed that all ORNs formed secondary structures and that the thermal stability of the duplex is depended oil the length and GC composition of the duplex. Nuclease stability of ORNs increased with increasing thermal stability of the duplex formed. All ORN showed TLR8 activity in HEK293 cells, and induced cytokine and chemokine production in human PBMC cultures. In addition to TLR8 activity, two ORNs containing a 'CUGAAUU' motif in the duplex-forming region induced immune stimulation through TLR7 in HEK293 cells, human PBMC and pDC cultures, and in vivo in mice. These results suggest that secondary structures in ORN provide nuclease stability and lead to stimulation Of immune responses through TLR8 as well as TLR7 depending oil the presence of specific nucleotide motifs. (C) 2009 Elsevier Inc. All rights reserved.