Alternating 2'-O-ribose methylation is a universal approach for generating non-stimulatory siRNA by acting as TLR7 antagonist.

Small interfering RNA (siRNA) is widely used to modulate gene expression, but its potential induction of cytokines via Toll-like receptors (TLR) strongly impairs its use. Selective 2'-O-ribose methylation of sense or antisense strand can abolish the immunostimulatory potential, however, no universal approach is available and the mechanism of action is unknown. Here, we demonstrate that alternating 2'-O-ribose methylation of the sense strand within a siRNA duplex specific for eGFP or beta(2)-microglobulin destroyed its immunostimulatory function in primary immune cells, while reduction in target gene expression was functional. Furthermore, addition of siRNA containing a 2'-O-ribose-methylated sense strand to immunostimulatory siRNA abolished its stimulatory activity and binding studies revealed that 2'-O-ribose-methylated RNA bound stronger to TLR7 than unmodified RNA. We conclude that 2'-O-ribose methylation acts as inhibitor for RNA-driven immune stimulation via TLR7 and recommend alternating 2'-O-ribose methylation of the sense strand as a universal approach for the generation of non-immunostimulatory siRNA.