RNA:DNA hybrid represents a novel class of high-affinity ligand for Toll Like Receptor-9 (TLR9). TLRs are expressed by various cells of the immune system, such as macrophages and dendritic cells. TLRs are class I receptors, with a single α-helix that spans the cell membrane. They recognize and respond to molecules derived from bacterial, viral and fungal pathogens, such as lipopolysaccharide (LPS) from the outer membrane of Gram negative bacteria, peptidoglycan fragments from bacterial cell walls and single-stranded and double-stranded RNA from viruses. Nucleic acids are of course fundamental components of all pathogens. Several nucleic acid recognizing Pattern Recognition Receptors (PRR) have been identified including TLR3 (dsRNA), TLR7 (ssRNA), TLR8 (ssRNA), TLR9 (bacterial DNA) and TLR13 (23S rRNA) confined to the endosomal compartments, membrane bound C-type lectin receptors, the cytosolic Nod-like receptors and cytosolic RIG-like receptors. Nucleic acid binding to TLRs recruits the adaptor proteins TRIF or MyD88 to trigger NF-κB and/or IRF signaling pathways, inducing pro-inflammatory cytokines and type I interferons.Current understanding of TLR9 is primarily determined from the use of synthetic oligodeoxynucleotides. Most well-known is the interaction of TLR9 with non-methylated cytosineguanosine (CpG) motifs in DNA. Activation of TLR9 is ascribed to the detection of CpG-motifs within pathogen derived DNA. TLR9 signaling not only initiates innate immune cells, but also acquired responses. RNA:RNA hybrids have been described as a new ligand for TLR9. Many pathogenic viruses replicate via a RNA:DNA hybrid intermediate within the cell. Viral genomes are detected as non-self and induce a powerful antiviral response via type I interferon. Indeed, RNA:DNA hybrids are sensed by dendritic cells via TLR9. It was demonstrated that TLR9 mediated cytokine response elicit by the hybrid was MyD88 dependent.The ability to modulate TLR9 activity and subsequent innate responses, through DNA-based immunotherapeutics has been the focus of many research projects. Nucleic acids are attractive as therapeutics for their chemical stability and low costs. The ability to regulate downstream signaling by varying the sequences of the synthetic nucleic acid opens an interesting therapeutic window.HC4072 consists of a RNA:DNA hybrid duplex: 60-mer RNA(GU) annealed to 60-mer DNA (CA).