Abstract
BackgroundShort interfering RNAs (siRNAs) have been shown to induce immune stimulation through a number of different receptors in a range of cell types. In primary cells, both TLR7 and TLR8 have been shown to recognise siRNAs however, despite the identification of a number of TLR7/8 stimulatory RNA motifs, the complete and definitive sequence determinants of TLR7 and TLR8 are yet to be elucidated.ResultsA total of 207 siRNA sequences were screened for TLR7/8 stimulation in human PBMCs. There was a significant correlation between the U count of the U-rich strand and the immunostimulatory activity of the duplex. Using siRNAs specifically designed to analyse the effect of base substitutions and hybridisation of the two strands, we found that sequence motifs and the thermodynamic properties of the duplexes appeared to be the major determinants of siRNA immunogenicity and that the strength of the hybridisation interaction between the two strands correlated negatively with immunostimulatory activity.ConclusionThe data presented favour a model of TLR7/8 activation by siRNAs, in which the two strands are denatured in the endosome, and single-stranded, U-rich RNA species activate TLR7/8. These findings have relevance to the design of siRNAs, particularly for in vivo or clinical applications.
Highlights
Short interfering ribonucleic acids (RNAs) have been shown to induce immune stimulation through a number of different receptors in a range of cell types
We found that sequence motifs and the thermodynamic properties of the duplexes appeared to be the major determinants of siRNA immunogenicity
Assay development In order to determine the determinants of immunogenicity of siRNAs, we initially sought to develop a sensitive and relatively high-throughput assay for the detection of innate immune stimulation by short duplex RNAs
Summary
Short interfering RNAs (siRNAs) have been shown to induce immune stimulation through a number of different receptors in a range of cell types. The mammalian innate immune system utilises a number of protein receptors for the identification of microbial molecules Amongst these are several receptors specific for foreign ribonucleic acids (RNAs), including Toll-like receptor 3 (TLR3), TLR7, TLR8, RIG-I, MDA5 and PKR (reviewed in [1]). These receptors are activated by different microbial RNA features, including doublestrandedness (TLR3, RIG-I, MDA5, PKR) [2,3,4], extracellular or endosomal localization (TLR3, TLR7, TLR8) [5,6] and/or the presence of 5'-triphosphates (RIG-I, PKR) [79]. A number of groups have demonstrated innate immune stimulation by siRNAs in human peripheral blood mononuclear cells (PBMCs) without defining the cell or receptor specificity [23,24,25,26]
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