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. (C) 2009 Elsevier GmbH. All rights reserved.