Sticky overhangs enhance siRNA-mediated gene silencing

Abstract

siRNA delivery to cells offers a convenient and powerful means of gene silencing that bypasses several barriers met by gene delivery. However, nonviral vectors, and especially polymers, form looser complexes with siRNA than with plasmid DNA. As a consequence, exchange of siRNA for larger polymeric anions such as proteoglycans found outside cells and at their surface may occur and lower delivery. We show here that making siRNAs "gene-like," via short complementary A(5-8)/T(5-8) 3' overhangs, increases complex stability, and hence RNase protection and gene silencing in vitro up to 10-fold. After decomplexation in the cytoplasm, sticky siRNA (ssiRNA) concatemers fall apart. ssiRNAs are therefore not inducing antiviral responses, as shown by the absence of IFN-beta production. Finally, transfection experiments in the mouse lung show that ssiRNA should be particularly suited to silencing with linear polyethylenimine in vivo.

Fig. 1.

Confocal microscopy sections of HeLa cells transfected with a fluorescent siRNA complexed to a cationic lipid or a cationic polymer. Lipid complexes are found in the perinuclear lysosomal compartment (Upper, arrowheads). Polymer complexes are mostly found at the cell surface (Lower, arrowheads) and some times inside the cell (arrows).

Fig. 2.

Gel electrophoresis of siRNA/PEI complexes incubated with increasing amounts of HS. Because of aggregation, siRNA/PEI complexes remain in the well (0 eq HS). Incubation for 30 min with 20–40 (wt/wt) eq HS releases siRNA (Left and Right), but not ssiRNA (Center), from the complexes.

Fig. 3.

Endogeneous luciferase silencing with ssiRNA/PEI complexes requires 10-fold less RNA. A549Luc cells stably expressing GL3 luciferase were incubated with increasing concentrations of ssiRNAs complexed to PEI (N/P = 5). Control RNAs were the corresponding nonsticky siRNA of similar length and the classical GL3 siRNA with 3′(dT)₂ overhangs. Luciferase inhibition was measured after 48 h.

Fig. 4.

ssiRNA-mediated silencing is sequence-specific. Sequences with three mismatches (○ and □) or with a single mismatch (■) in their GL3Luc sequence do not decrease endogeneous GL3 luciferase expression. Experimental conditions are as in Fig. 3.

Fig. 5.

Hybridization of identical (dT)_n overhangs with a complementary oligodeoxyadenylate significantly enhances silencing. Experimental conditions are as in Fig. 3.

Fig. 6.

siRNA oligomerization enhances silencing when delivered by the cationic lipid formulation jetSi-ENDO (N/P = 6). Hybridization conditions are as in Fig. 5, and other experimental conditions are as in Fig. 3.

Fig. 7.

Luciferase gene silencing in the mouse lung. OF1 mice were coinjected retroorbitally with a luciferase-bearing plasmid and siRNA complexed separately with jetPEI. Luciferase gene expression was measured after 24 h. ssiRNA showed significantly higher gene silencing than classical siRNA (∗, P < 0.05; ∗∗, P < 0.01).