RNA interference (RNAi) is a natural phenomenon resulting in potent and primarily specific gene silencing, potentially facilitating disease diagnosis and treatment, and improving our understanding of biological processes. RNAi is initiated in cells by the presence of short interfering RNAs (siRNAs). The efficiency of RNAi has been shown to depend not only on the characteristics of the siRNAs but also on the structure of the target mRNA. However, features of the siRNA duplex have been implicated in stimulating an innate immune response, which can complicate their application. Our goal is to define design rules for siRNAs that account for immune stimulation as well as silencing efficiency. Using in vitro binding assays, mRNA secondary structure predictions, cell culture studies, and molecular modeling, we are examining the mRNA and siRNA sequence and structural features that correlate with silencing efficiency and/or immune stimulation. We are currently investigating two known pathways contributing to this response, either via Protein Kinase R (PKR) or Toll-like Receptor 3 (TLR-3), in efforts to describe the importance of target mRNA structure in determining both the gene silencing efficiency of siRNAs and their immunostimulatory capacity. We will describe our findings that siRNAs targeting different predicted structures of the enhanced green fluorescent protein (EGFP) transcript differ in their silencing efficiencies. Furthermore, PKR and TLR-3 expression upregulation is correlated to the degree of EGFP silencing as well as the thermodynamics of the mRNA secondary structure. Immune activation, as characterized by cytokine profiles, is similarly related to local mRNA structural effects. Experiments in cells overexpressing PKR and TLR-3 will also be described. These results have broad implications for experiments employing RNA interference.

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