Synthesis of the antisense strand of the siRNA (27mer) was followed by CpG1668 ODN synthesis 31 , 32 , producing a single stranded oligonucleotide connected through a carbon chain linker (Fig. 1a) . A 25/27mer siRNA was chosen over the conventional 21mer duplex to allow uncoupling of the siRNA from the CpG sequence by the Dicer enzyme once inside the cell. The asymmetric 25/27mer siRNA was optimized for specific processing by Dicer and was more potent in target gene silencing 33 , 34 . Adding either CpG1688 alone or CpG-Stat3 siRNA conjugate to cultured DC2.4 dendritic cells resulted in a similar increase in expression of co-stimulatory CD40 and CD80 molecules, suggesting that CpG-Stat3 siRNA conjugate retains its capacity to activate TLR9 (Fig. 1b) . In addition, the immunostimulatory properties of CpG-siRNA conjugates do not differ from the effect of CpG alone as measured by production of inflammatory cytokines in primary cells and NF-κB/AP1 activation in a stable macrophage cell line ( Supplementary Fig. 1 ). To assess whether linking siRNA with CpG moiety would still allow Dicer processing, we compared in vitro Dicer activity on CpG-Stat3 siRNA substrate versus 25/27mer Stat3 siRNA. Both the CpG-Stat3 siRNA and Stat3 siRNA were processed to 21mer siRNA by recombinant Dicer (Fig. 1c) . Finally, to determine whether the CpG-Stat3 siRNA retains gene silencing function, the chimeric molecule was transfected into cells using Lipofectamine. Results from this experiment indicated that linking CpG ODN to siRNA did not interfere with Stat3 gene silencing (55% and 49% at protein level, respectively, as measured by densitometry) (Fig. 1d ).