Controlling activation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification

Abstract

The RNA-dependent protein kinase (PKR) is activated by binding to double-stranded RNA (dsRNA). Activation of PKR by short-interfering RNAs (siRNAs) and stimulation of the innate immune response has been suggested to explain certain off-target effects in some RNA interference experiments. Here we show that PKR's kinase activity is stimulated in vitro 3- to 5-fold by siRNA duplexes with 19 bp and 2 nt 3'-overhangs, whereas the maximum activation observed for poly(I)*poly(C) was 17-fold over background under the same conditions. Directed hydroxyl radical cleavage experiments indicated that siRNA duplexes have at least four different binding sites for PKR's dsRNA binding motifs (dsRBMs). The location of these binding sites suggested specific nucleotide positions in the siRNA sense strand that could be modified with a corresponding loss of PKR binding. Modification at these sites with N2-benzyl-2'-deoxyguanosine (BndG) blocked interaction with PKR's dsRBMs and inhibited activation of PKR by the siRNA. Importantly, modification of an siRNA duplex that greatly reduced PKR activation did not prevent the duplex from lowering mRNA levels of a targeted message by RNA interference in HeLa cells. Thus, these studies demonstrate that specific positions in an siRNA can be rationally modified to prevent interaction with components of cellular dsRNA-regulated pathways.

Figure 1

Domain map for the dsRNA-dependent protein kinase, PKR.

Figure 2

siRNA activates PKR. (Upper panel) PKR autophosphorylation as a function of dsRNA concentration. (Lower panel) Quantification of the stimulation of PKR autophosphorylation as a function of dsRNA concentration. The extent of phosphorylation of PKR was measured by storage phosphor autoradiography and plotted as the ratio to an unstimulated (no RNA) sample. The concentration of each dsRNA tested from left to right is 20 nM, 200 nM and 2 μM except poly(I)•poly(C), which was tested in the absence (−) or presence (+) of 10 μg/ml. This concentration of poly(I)•poly(C) resulted in the highest stimulation of PKR autophosphorylation under these conditions (data not shown).

Figure 3

Directed hydroxyl radical cleavage to probe the binding of an siRNA with the PKR RBD. (A) Storage phosphor autoradiogram of a 19% denaturing polyacrylamide gel separating the RNA cleavage products from the HPV-E7 siRNA duplex with the sense strand 5′-end-labeled. Major cleavage sites are identified with lines and arrows. The lanes had the following reaction conditions as labeled: T, T1 RNAse (G-lane); OH, alkaline hydrolysis; lane 1, RNA with no added reagents (protein, hydrogen peroxide or sodium ascorbate); lane 2, 8 μM PKR RBD E29C-EDTA•Fe (46) in the presence of 0.001% hydrogen peroxide and 5 mM sodium ascorbate (cleavage reagents); lane 3, 8 μM PKR RBD E29C-EDTA•Fe in the absence of 0.001% hydrogen peroxide and 5 mM sodium ascorbate; lane 4, 10 μM PKR RBD Q120C-EDTA•Fe (42) in the presence of 0.001% hydrogen peroxide and 5 mM sodium ascorbate; lane 5, 10 μM PKR RBD Q120C-EDTA•Fe in the absence of 0.001% hydrogen peroxide and 5 mM sodium ascorbate. (B) Conditions are the same as in (A) with antisense strand 5′ end-labeled with the exception that A refers to RNAse A cleavage products (A>C lane) (C) Sites of cleavage on the HPV-E7 siRNA induced by PKR RBD modified with EDTA•Fe at position 29 of dsRBM I. Length of each line is proportional to the extent of cleavage at that nucleotide. (D) Sites of cleavage on the HPV-E7 siRNA induced by PKR RBD modified with EDTA•Fe at position 120 of dsRBM II.

Figure 4

Introduction of N²-benzyl-2′-deoxyguanosine into duplex RNA occludes the minor groove and disrupts dsRBM-RNA binding.

Figure 5

Directed hydroxyl radical cleavage to probe PKR RBD binding of benzyl-modified siRNAs. (A–D) Directed hydroxyl radical cleavage by PKR RBD E29C-EDTA•Fe to probe PKR dsRBM I binding of benzyl-modified siRNAs. Conditions for A, OH, lane 1, lane 2 and lane 3 for (A–D) were identical to the conditions of A, OH, lane 1, lane 2 and lane 3 for Figure 3B. (A) Cleavage pattern on HPV-E7 siRNA benzylated at position 9 on the sense strand. (B) Cleavage pattern on HPV-E7 siRNA benzylated at positions 6 and 9 on the sense strand. (C) Cleavage pattern on HPV-E7 siRNA benzylated at positions 3, 6 and 9 on the sense strand. (D) Cleavage pattern on HPV-E7 siRNA benzylated at positions 6, 9 and 15 on the sense strand. (E) Directed hydroxyl radical cleavage by PKR RBD Q120C-EDTA•Fe to probe PKR dsRBM II binding of benzyl-modified siRNAs. A and OH were RNAse A and alkaline hydrolysis, respectively. Conditions for lane 1, HPV-E7 siRNA benzylated at position 9 with no added cleavage reagents (protein, hydrogen peroxide or sodium ascorbate); lane 2, same as lane 4 of Figure 3; lane 3, same as lane 5 for Figure 3; lane 4, HPV-E7 siRNA benzylated at position 6 and 9 with no added cleavage reagents; lane 5, same as lane 4 of Figure 3; lane 6, same as lane 5 for Figure 3. Asterisk indicates a hyper-reactive nucleotide showing cleavage independent of directed hydroxyl radical cleavage. The cleavage sites disrupted on the antisense strand are shown in the same color as the benzyl-modified nucleotide on the sense strand that causes the change.

Figure 6

Introduction of BndG in the siRNA sense strand inhibits PKR activation. (A) The positions selected for modification based on the affinity cleavage data are shown on HPV-E7 siRNA. (B) Activation of PKR by (closed circle) unmodified HPV-E7 siRNA, (inverted closed triangle—6,9) (closed diamond—3,6,9) (closed triangle—6,9,15) modified HPV-E7 siRNA and (closed square) HPV-E7 sense single strand. (C) Positions of nucleotides replaced by BndG are shown for GFP siRNA. (D) Activation of PKR by (closed square) unmodified GFP siRNA and (closed diamond—9,11) (closed triangle—9,14) modified GFP siRNA and (closed circle) GFP sense single strand.

Figure 7

siRNAs modified to block PKR activation are capable of inducing RNA interference. (A) Nucleotides of the caspase 2 siRNA substituted by BndG. (B) Activation of PKR by (closed circle) unmodified caspase 2 siRNA, (closed diamond—9,14) (closed triangle—7,9,14) modified caspase 2 siRNA and (closed square) caspase 2 antisense single strand. (C) (Left) Western blot of cell lysate from A172 cells transfected with 50 nM caspase 2 siRNA. (Right) Quantification of western blot analysis of PKR phosphorylation in response to siRNA transfection. PKR T451 phosphorylation is plotted as an increase above background (mock transfection) in arbitrary units (mean ± SD for three independent experiments). (D) Caspase 2 message levels in HeLa cells transfected with varying concentrations of siRNAs. Concentration of each siRNA from left to right were 10, 30 and 100 nM.

Figure 8

(A–C) Model for siRNA binding and activation of PKR (21,22,53). The model was generated using Swiss-PdbViewer (70) based on directed hydroxyl radical cleavage data of the PKR dsRBMs on unmodified HPV-E7 siRNA (Figure 3) and the observed effects of BndG modification on PKR binding and activation (Figures 5–7). Numbers indicate the positions on the siRNA sense strand where BndG modification disrupts PKR interactions.