Long Double-Stranded RNA-Mediated RNA Interference and Immunostimulation: Long Interfering Double-Stranded RNA as a Potent Anticancer Therapeutics

Abstract

In most applications, small interfering RNAs are designed to execute specific gene silencing via RNA interference (RNAi) without triggering nonspecific responses such as immunostimulation. However, in anticancer therapeutics, immunostimulation combined with specific oncogene silencing could be beneficial, resulting in the synergistic inhibition of cancer cell growth. In this study, we report an immunostimulatory long double-stranded RNA (dsRNA) structure with the ability to trigger RNAi-mediated specific target gene silencing, termed as long interfering dsRNA (liRNA). liRNA targeting Survivin mRNA not only efficiently and specifically triggered target gene silencing via RNAi, but also stimulated the protein kinase R pathway to induce the expression of interferon β. As a result, the ability of Survivin-targeting liRNA to inhibit cancer cell growth was superior over conventional small interfering RNA or nontargeting dsRNA structures. Our results thus provide a simple yet efficient dual function immunostimulatory RNAi-triggering structure, which is potentially applicable for the development of anticancer therapeutics.

Introduction

Long double-stranded (ds) RNA is formed during the replication of most viruses, but is absent in eukaryotic cells. Therefore, eukaryotic organisms recognize the long dsRNA as a virus-associated molecular pattern and trigger potent antiviral innate immune responses (Gantier and Williams, 2007). In mammalian cells, introduction of long dsRNAs activates the protein kinase R (PKR) and 2,5-oligoadenylate synthase (OAS) (Gantier and Williams, 2007). Activated PKR phosphorylates the eukaryotic translation initiation factor eIF-2α, blocking its function and leading to apoptosis. It also phosphorylates IκBα to activate the NF-κB pathway, thereby resulting in the upregulation of type I interferon (IFN) such as IFN-β (Gil et al., 1999). OAS activated by dsRNAs in turn activates RNaseL, resulting in nonspecific mRNA degradation and apoptosis (Iordanov et al., 2001). Therefore, the introduction of long dsRNAs to mammalian cells results in a potent antiproliferative activity along with the induction of various cytokines.

The antiproliferative and immunostimulatory activity of long dsRNAs, such as polyinosinic:polycytidylic acid [poly(I:C)], is currently utilized to develop a novel strategy to kill cancer cells (WEIL, 2006). Levitzki and colleagues delivered poly(I:C) selectively to mouse glioblastoma cells using polyethyleneimine (PEI) decorated with epidermal growth factor (EGF) that induced rapid apoptosis in vitro and in vivo (Shir et al., 2006). Recently, an improved version of PEI-based polymeric carrier, containing EGF as a targeting ligand, was developed for the tumor-specific delivery of poly(I:C) (Schaffert et al., 2011). The systemic administration of this EGF-PEI/poly(I:C) complex showed effective anti-tumor activity in vivo (Schaffert et al., 2011).

Levitzki and colleagues also developed a strategy to selectively activate PKR to kill cancer cells with genes mutated due to deletions or chromosomal translocations (Shir and Levitzki, 2002; Friedrich et al., 2005). A lentiviral vector was introduced to cancer cells that expresses the 39 nt antisense (AS) RNA complementary to the mRNA derived from the mutated genes. The AS RNA formed dsRNA with their target mRNAs in the cancer cells and induced the PKR-mediated selective cell death. Venkataraman et al. (2010) reported an alternative approach to selectively eliminate cancer cells through the formation of long dsRNAs in cells containing mRNA cancer markers. They introduced small conditional RNAs that undergo hybridization chain reactions to generate nicked long dsRNAs only in cells where a cognate mRNA cancer marker is present. Additionally, these dsRNAs caused potent cell death by activating the PKR pathway (Venkataraman et al., 2010).

Another RNA-based anticancer therapeutic modality currently under development is based upon the RNA interference (RNAi) mechanism. RNAi is a post-transcriptional gene silencing mechanism conserved in diverse species (HANNON, 2002). When long dsRNAs are introduced into the cells, they are cleaved into 21–23 bp short dsRNAs by an RNase III enzyme called Dicer. Such short dsRNAs are recognized by the RNA-induced silencing complex (RISC) where the RNA strand with thermodynamically unstable 5′ end is preferentially incorporated into the active RISC complex to execute the specific cleavage of target mRNA. RNAi-based gene silencing has an immense potential for cancer therapeutics because of its ability to specifically inhibit any oncogene, even those that are inaccessible to small molecules or monoclonal antibodies (Pecot et al., 2010).

Originally discovered in Caenorhabditis elegans, long (0.3–1 kb) dsRNA has been successfully used to guide sequence-specific gene silencing in a wide range of organisms (Fire et al., 1998). However, RNAi-mediated specific gene silencing using long dsRNAs was not successful in mammalian cells, as they triggered antiviral responses, resulting in nonspecific mRNA degradation and inhibition in protein synthesis (Stark et al., 1998). Specific gene silencing in mammalian cells was later achieved using 19 bp synthetic RNA duplex with 3′ overhangs as an RNAi trigger, which mimics the structure of the Dicer cleavage products (Elbashir et al., 2001). This small interfering RNA (siRNA) structure caused specific gene silencing without the induction of IFNs and nonspecific mRNA down-regulation. While a recent study demonstrated that synthetic dsRNAs as long as 38 bp can also achieve specific gene silencing in mammalian cells without antiviral responses (Chang et al., 2009a), long RNA duplex was generally avoided as a RNAi trigger for most studies in mammalian cells.

In the RNAi therapeutics development, researchers focus upon executing specific target gene silencing without inducing innate immune responses. However, in the anticancer or antiviral therapeutics development, siRNA-mediated gene silencing along with immune stimulation could be therapeutically beneficial (Schlee et al., 2006). Based on this idea, bifunctional immunostimulatory siRNAs have been designed so that they could provoke both immune responses and gene silencing (Poeck et al., 2008; Gantier et al., 2010). Indeed, a recent report illustrated that a bifunctional immunostimulatory siRNA, consisting of a BCL2-targeting siRNA moiety and 5′-triphosphate modification as an RIG-I activator, exhibited enhanced antitumor activity in the in vivo mouse lung metastasis model (Poeck et al., 2008).

In this study, we present a nicked long dsRNA structure, which we named as long interfering dsRNA (liRNA), as a novel trigger for the immunostimulatory RNAi. The liRNA is composed of a 19 bp siRNA unit, multimerized by base paring between long overhangs. liRNA not only executed specific target gene silencing but also triggered the dsRNA-mediated PKR activation and IFN induction, resulting in cell death. Due to the combined effect of RNAi and immunostimulation, liRNA targeting Survivin mRNA showed enhanced cancer cell growth inhibition compared with the single treatment of siRNA or long dsRNAs, including poly(I:C), suggesting potential application of liRNA for anticancer therapeutics development.

Materials and Methods

Ribonucleic acids

Chemically synthesized RNAs were purchased from Bioneer and annealed according to the manufacture's protocol. The sequences and structures of siRNAs and liRNAs used in the experiments are shown in Supplementary Table S1 (Supplementary Data are available online at www.liebertonline.com/oli) and Fig. 1, respectively. Poly(I:C) was purchased from Sigma-Aldrich.

FIG. 1.

Schematic illustration of liRNA structure. Structure of liRNAs targeting Survivin or GFP. See text for details. liRNA, long interfering double-stranded RNA; AS, antisense; S, sense.

Native gel electrophoresis

Each siRNA or liRNA was resolved on a 3% agarose gel, stained with ethidium bromide, and then observed by ultraviolet transillumination.

Cell culture and RNA transfection

HeLa cells were cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum. One day before transfection, cells were plated in 12-well plates at 70% confluency in complete medium without antibiotics. RNAs were transfected using Lipofectamine2000 (Invitrogen) following the manufacturer's protocol.

Quantitative reverse transcription real-time polymerase chain reaction

Total RNAs were extracted from cell lysates using the Isol-RNA Lysis Reagent kit (5 Prime). They were then used as templates for cDNA synthesis, which was performed with the ImProm-II™ Reverse Transcription System (Promega) according to the manufacturer's protocol. Target gene expression levels were analyzed by quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR) using a StepOne^™ real-time PCR system (Applied Biosystems) according to the manufacturer's instructions. The primer sequences for each gene were

GAPDH forward 5′-GAG TCA ACG GAT TTG GTC GT-3′

GAPDH reverse 5′-GAC AAG CTT CCC GTT CTC AG-3′

Survivin forward 5′-GCA CCA CTT CCA GGG TTT AT-3′

Survivin reverse 5′-CTC TGG TGC CAC TTT CAA GA-3′

IFN-ß forward 5′-AGA AGT CTG CAC CTG AAA AGA TAT T-3′

IFN-ß reverse 5′-TGT ACT CCT TGG CCT TCA GGT AA-3′

Cell viability assay

Cells were seeded 24 hours before the transfection with a cell density of 2.5 × 10⁴ in 24-well plates. Immediately before transfection, medium was changed, and 100 μL of the diluted transfection complexes in serum-containing culture medium was added to the cells. All experiments were performed in duplicates. Cell growth inhibition was evaluated 1, 3, and 5 day(s) after the treatment using trypan blue viable cell counting.

PKR inhibition by 2-aminopurine

Cells were transfected with liRNAs or poly(I:C) at the indicated concentrations using Lipofectamine2000 (Invitrogen). Where indicated, 5 mM of 2-aminopurine (Sigma-Aldrich) was added 18 hours before transfection and the medium containing fresh 5 mM of 2-aminopurine was changed every 24 hours. After 5 days, viability was measured by cell counting.

Results

Design of liRNA structure

Figure 1 shows the structure of liRNAs. liRNAs were constructed by annealing of two chemically synthesized 38 nt single-stranded (ss) RNAs. The 5′ end 19 nt of the AS strand was identical to the corresponding 19 bp siRNAs, and the 19 nt extension complementary to the target mRNA was made at the 3′ end to ensure annealing with other siRNA units. The 38 nt sense strand was designed to have both the 5′ end 19 nt and the 3′ end 19 nt that are complementary to their corresponding AS strands, respectively. Thus, annealing of two strands resulted in the formation of multimeric long dsRNAs nicked at every 19 bp.

To develop anticancer liRNA, we designed liRNA targeting Survivin mRNA (liSurvivin). Survivin is an attractive target for cancer therapy, and inhibition of Survivin gene expression with siRNA was shown to efficiently inhibit cell growth (Chang et al., 2009b; Ryan et al., 2009). As negative controls, liSurvivin modified with a seed sequence mutation (second to seventh nucleotide from the 5′ end of the AS strand mutated, termed as liSurvivin-mut), and an liRNA targeting GFP mRNA (liGFP) were used (Fig. 1). The size distribution of liRNAs was analyzed on the agarose gel and compared with that of poly(I:C). The length of liRNAs ranged from 38 bp up to ∼600 bp, which is similar to the size distribution pattern of poly(I:C) (Fig. 2).

FIG. 2.

Size distribution patterns of liRNAs. Each RNA was separated on 3% agarose gel and their size distribution patterns were compared with those of poly(I:C) and siRNAs. poly(I:C), polyinosinic:polycytidylic acid; siRNA, small interfering RNA.

liRNA triggers specific target gene silencing

We first tested whether liRNAs could trigger specific target gene silencing. liSurvivin, liSurvivin-mut, and liGFP were transfected into HeLa cells, and the mRNA levels of Survivin and GAPDH (internal control) were measured by qRT-PCR. liSurvivin showed efficient knock-down of the Survivin mRNA level, which is similar to that of siSurvivin, but not the GAPDH mRNA level (Fig. 3 and Supplementary Fig. S1A). In contrast, liSurvivin-mut and liGFP failed to significantly reduce the Survivin mRNA level (Fig. 3 and Supplementary Fig. S1A). These results reveal that liRNA, a nicked long dsRNA structure, can trigger seed sequence-dependent specific target gene silencing. liSurvivin also triggered strong target gene silencing in another cell line, AGS, demonstrating that the liRNA-mediated gene silencing could be induced in multiple cell lines (Supplementary Fig. S2A–C).

FIG. 3.

liRNAs trigger specific gene silencing in mammalian cells. Gene silencing activities of liRNAs targeting Survivin mRNAs. Each siRNA (0.3 nM) or liRNA (0.3 nM) was transfected into HeLa cells using Lipofectamine 2000. Survivin and GAPDH expression levels were analyzed by quantitative reverse transcription real-time polymerase chain reaction 12 hours after transfection. The amount of poly(I:C) used was identical in mass to the siRNA samples used. All data in the graph represent the mean + SD of 3 independent experiments. The concentration of liRNAs is denoted as the concentration of the antisense strand. (A) Survivin mRNA expression levels of liRNA-transfected cells. Y axis represents Survivin mRNA measured with the same amount of total RNA from the liRNA-transfected samples. (B) GAPDH mRNA expression levels of the liRNA-trasnfected cells. Y axis represents the GAPDH mRNA level measured with the same amount of total RNAs from the liRNA-trasnfected samples. (C) Survivin mRNA levels were divided by the GAPDH (control) mRNA level. SD, standard deviation.

IFN induction by liRNAs

Next, the ability of liRNAs to induce IFN response was investigated in the cancer cells. liSurvivin, liSurvivin-mut, and liGFP were transfected into HeLa cells and the IFN-β expression level was measured. We also transfected siSurvivin, siGFP, and poly(I:C) to compare with the IFN-β induction level of liRNAs. siRNAs did not induce any IFN response in HeLa cells as previously reported (Chang et al., 2009a) (Fig. 4). In contrast, poly(I:C) caused strong IFN induction (>100-fold) in both 12 hours and 24 hours post-transfection (Fig. 4). Compared with these RNAs, liRNAs induced the intermediate level of IFN-β induction (∼11 to ∼16-fold) 12 hours after transfection (Fig. 4). Interestingly, the liRNA-triggered IFN-β induction pattern was different from poly(I:C) because the IFN-β mRNA level was reduced close to the basal level 24 hours after transfection, whereas poly(I:C) induced IFN-β level continued to increase (Fig. 4). These results demonstrate that the transfected liRNAs can induce IFN-β expression, with a different magnitude and kinetics compared with poly(I:C). liRNAs also induced IFN-β level in another cell line, AGS (Supplementary Fig. S2D).

FIG. 4.

IFN induction triggered by liRNAs. Each liRNA (0.3 nM) was transfected into HeLa cells and the IFN-β level was measured by quantitative reverse transcription real-time polymerase chain reaction 12 hours and 24 hours post-transfection. The IFN-β mRNA level of mock-treated sample (0 nM) was set at 1. All data in the graph represent the mean + SD of 3 independent experiments. IFN, interferon.

liRNA targeting Survivin inhibits cancer cell growth more efficiently than siRNAs or nontargeting long dsRNAs

We hypothesized that the combination of specific gene silencing and immunostimulation triggered by an oncogene-targeting liRNA might result in enhanced anticancer activity compared with that of siRNA or immunostimulatory dsRNA alone. To test this, liSurvivin, seed-changed liSurvivin, or liGFP was transfected into HeLa cells and their ability to inhibit cell growth was measured. As shown in Fig. 5, siSurvivin-transfected cells showed retarded cell growth, whereas siGFP-transfected cells had the same growth rate as the control (0 nM). liGFP or liSurvivin-mut exhibited efficient cell growth inhibition that is similar to poly(I:C) but slightly less effective than siSurvivin. These results demonstrate that, like poly(I:C), the liRNA structure can induce structure-dependent and sequence-independent cancer cell growth inhibition. Strikingly, liSurvivin caused most potent cell growth inhibition among all the RNAs tested, by almost completely inhibiting the growth of cancer cells up to 5 days (Fig. 5 and Supplementary Fig. S1B). This result suggests that the combination of Survivin gene knock-down and long dsRNA-mediated immunostimulation results in a synergistic inhibition of cancer cell growth.

FIG. 5.

Cancer cell growth inhibition by liRNAs. Each liRNA, siRNA, or poly(I:C) was transfected into HeLa cells. (a) Cell growth was measured at indicated time points by counting cell numbers. (b) Cell growth at 5 days after transfection. All data in the graph represent the mean + SD of 3 independent experiments.

The anticancer activity of liRNAs is PKR-dependent

It is well known that long dsRNAs induce the antiproliferative activity by activating the PKR pathway (Friedrich et al., 2004). We wondered whether the cancer cell growth inhibition triggered by liRNAs is also PKR-dependent. To test this, the cells were pre-treated with a PKR inhibitor 2-AP before RNA transfection. As expected, 2-AP treatment did not affect the cell growth inhibition by siSurvivin (Fig. 6). In contrast, like poly(I:C), the cell growth inhibition by liGFP and seed-changed liSurvivin was dramatically reduced when HeLa cells were pre-treated. In addition, 2-AP treatment also reduced the liSurvivin-mediated cell growth inhibition. Interestingly, liSurvivin showed a cell growth inhibitory activity similar to siSurvivin, when the cells were treated with 2-AP. These results are consistent with the mechanism that the sequence-independent antitumor activity by liRNA structure is PKR-dependent, and the enhanced antiproliferative activity of liSurvivin is due to the combination of the PKR-dependent immunostimulation and PKR-independent target gene silencing.

FIG. 6.

liRNA-mediated cell death is protein kinase R dependent. HeLa cells were pre-treated with the protein kinase R inhibitor 2-AP or buffer before liRNA transfection. Cell growth was measured as described in Fig. 5. All data in the graph represent the mean + SD of 3 independent experiments.

Discussion

In this study, we demonstrated that liRNA designed to inhibit Survivin gene expression has a potent antiproliferative activity. Our results are consistent with the hypothesis that the enhanced antiproliferative activity of liRNAs operates by the dual function of the structure: PKR activation by the structural feature of liRNA mimicking long dsRNAs, and sequence-specific silencing of oncogene by siRNA units within the liRNA structure. We believe that the specific gene silencing by liRNAs is via RNAi mechanism, based on the observation that seed-changed liRNA (liSurvivin-mut) showed compromised target gene silencing (Fig. 3).

It was believed that specific gene silencing was not possible using dsRNAs longer than 30 bp (Elbashir et al., 2001). Recently, however, our group has demonstrated that dsRNAs as long as 38 bp can trigger specific gene silencing via RNAi (Chang et al., 2009a). Nonetheless, the liRNA structure used in this experiment is as long as ∼600 bp. How could this long dsRNA guide specific target gene silencing? One hypothesis is that the presence of multiple nicks in the liRNA duplex might either help break down liRNAs into smaller siRNA units or facilitate unwinding and guide strand incorporation into RISC. Future mechanistic and structure–function relationship studies on liRNAs should reveal its precise mechanism of action.

Long dsRNAs such as poly(I:C) have been used to eliminate cancer cells by inducing potent antiviral responses (Friedrich et al., 2004). However, poly(I:C) triggers strong and persistent induction of cytokine expression, which could potentially lead to toxicity when remains uncontrolled. In contrast to poly(I:C), liRNAs induce intermediate level of IFN-β induction, and the induction pattern was temporal, rather than persistent. More importantly, when combined with Survivin gene silencing, this mild level of immunostimulation induced a more potent antiproliferative effect than poly(I:C), suggesting that the liRNA structure could replace poly(I:C) for future dsRNA-based anticancer therapeutics development. We believe that, along with the specific tumor-targeting delivery system (CHOWDHURY, 2011), the liRNA structure has a great potential to be developed into a highly potent anticancer therapeutic modality.

Footnotes

Acknowledgment

This work was supported by Global Research Laboratory grant from Ministry of Education, Science and Technology of Korea (No. 2008-00582).

Author Disclosure Statement

No competing financial interests exist.

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