Sequence determinants of innate immune activation by short interfering RNAs.
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.


CONCLUSIONS
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.
Goodchild
Amber
A
Johnson and Johnson Research Pty Ltd, Strawberry Hills, NSW, Australia. amber@unswalumni.com
Nopper
Nicole
N
King
Andrew
A
Doan
Tram
T
Tanudji
Marcel
M
Arndt
Greg M
GM
Poidinger
Michael
M
Rivory
Laurent P
LP
Passioura
Toby
T
D045744:Cell Line, Tumor; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology*; D006801:Humans; D007113:Immunity, Innate; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology; D034741:RNA, Small Interfering / Q000737:chemistry / Q000276:immunology*; D051199:Toll-Like Receptor 7 / Q000819:agonists / Q000276:immunology / Q000378:metabolism*; D051200:Toll-Like Receptor 8 / Q000819:agonists / Q000276:immunology / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000276:immunology; D014529:Uridine / Q000737:chemistry / Q000276:immunology
D034741:RNA, Small Interfering; D051199:Toll-Like Receptor 7; D051200:Toll-Like Receptor 8; D014409:Tumor Necrosis Factor-alpha; D014529:Uridine