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Sequence determinants of innate immune activation by short interfering RNAs.

Goodchild A, Nopper N, King A, Doan T, Tanudji M, Arndt GM, Poidinger M, Rivory LP, Passioura T - BMC Immunol. (2009)

Bottom Line: 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.These findings have relevance to the design of siRNAs, particularly for in vivo or clinical applications.

View Article: PubMed Central - HTML - PubMed

Affiliation: Johnson and Johnson Research Pty Ltd, Strawberry Hills, NSW, Australia. amber@unswalumni.com

ABSTRACT

Background: Short 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.

Results: A 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.

Conclusion: The 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.

Show MeSH
Innate immune stimulation of PBMCs by single-stranded and annealed siRNAs. Supernatant from PBMCs treated with 100 nM DOTAP complexed RNAs for 24 hours was applied to Huh7-Luc cells. Luciferase expression was assayed 24 hours after treatment with supernatant. Each point represents the mean of triplicate samples.
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Figure 3: Innate immune stimulation of PBMCs by single-stranded and annealed siRNAs. Supernatant from PBMCs treated with 100 nM DOTAP complexed RNAs for 24 hours was applied to Huh7-Luc cells. Luciferase expression was assayed 24 hours after treatment with supernatant. Each point represents the mean of triplicate samples.

Mentions: The above analysis made the assumption that the more U rich strand of each duplex was responsible for the majority of the immunostimulatory activity observed for that duplex. To assess the validity of this assumption, we investigated the immunostimulatory activity of the individual strands from 4 of the most highly immunostimulatory duplexes (RS002, RS009, siCyan2 and siRNA9.2). In each case, the U-rich strand elicited greater IFN production from PBMCs (Figure 3), confirming the underlying premise of the analysis and validating the findings of previous studies that TLR 3 is not involved in the sensing of siRNA molecules in isolated human leukocytes (since single-stranded RNA is not a ligand for TLR3).


Sequence determinants of innate immune activation by short interfering RNAs.

Goodchild A, Nopper N, King A, Doan T, Tanudji M, Arndt GM, Poidinger M, Rivory LP, Passioura T - BMC Immunol. (2009)

Innate immune stimulation of PBMCs by single-stranded and annealed siRNAs. Supernatant from PBMCs treated with 100 nM DOTAP complexed RNAs for 24 hours was applied to Huh7-Luc cells. Luciferase expression was assayed 24 hours after treatment with supernatant. Each point represents the mean of triplicate samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724479&req=5

Figure 3: Innate immune stimulation of PBMCs by single-stranded and annealed siRNAs. Supernatant from PBMCs treated with 100 nM DOTAP complexed RNAs for 24 hours was applied to Huh7-Luc cells. Luciferase expression was assayed 24 hours after treatment with supernatant. Each point represents the mean of triplicate samples.
Mentions: The above analysis made the assumption that the more U rich strand of each duplex was responsible for the majority of the immunostimulatory activity observed for that duplex. To assess the validity of this assumption, we investigated the immunostimulatory activity of the individual strands from 4 of the most highly immunostimulatory duplexes (RS002, RS009, siCyan2 and siRNA9.2). In each case, the U-rich strand elicited greater IFN production from PBMCs (Figure 3), confirming the underlying premise of the analysis and validating the findings of previous studies that TLR 3 is not involved in the sensing of siRNA molecules in isolated human leukocytes (since single-stranded RNA is not a ligand for TLR3).

Bottom Line: 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.These findings have relevance to the design of siRNAs, particularly for in vivo or clinical applications.

View Article: PubMed Central - HTML - PubMed

Affiliation: Johnson and Johnson Research Pty Ltd, Strawberry Hills, NSW, Australia. amber@unswalumni.com

ABSTRACT

Background: Short 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.

Results: A 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.

Conclusion: The 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.

Show MeSH