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. 2018 Feb 21;4(2):e1701854.
doi: 10.1126/sciadv.1701854. eCollection 2018 Feb.

A Minimal RNA Ligand for Potent RIG-I Activation in Living Mice

Free PMC article

A Minimal RNA Ligand for Potent RIG-I Activation in Living Mice

Melissa M Linehan et al. Sci Adv. .
Free PMC article


We have developed highly potent synthetic activators of the vertebrate immune system that specifically target the RIG-I receptor. When introduced into mice, a family of short, triphosphorylated stem-loop RNAs (SLRs) induces a potent interferon response and the activation of specific genes essential for antiviral defense. Using RNA sequencing, we provide the first in vivo genome-wide view of the expression networks that are initiated upon RIG-I activation. We observe that SLRs specifically induce type I interferons, subsets of interferon-stimulated genes (ISGs), and cellular remodeling factors. By contrast, polyinosinic:polycytidylic acid [poly(I:C)], which binds and activates multiple RNA sensors, induces type III interferons and several unique ISGs. The short length (10 to 14 base pairs) and robust function of SLRs in mice demonstrate that RIG-I forms active signaling complexes without oligomerizing on RNA. These findings demonstrate that SLRs are potent therapeutic and investigative tools for targeted modulation of the innate immune system.


Fig. 1
Fig. 1. SLRs are optimally recognized by RIG-I.
(A) SLRs were designed to fold stably into a minimal RIG-I ligand containing 10 or 14 bp and a triphosphorylated 5′ terminus. SLRs were compared against other reported double-stranded RIG-I ligands (dsRNA) 19 to 24 bp in length and control RNAs lacking structure (ppp-NS) or 5′-triphosphates (OH-). Human embryonic kidney (HEK) 293T cells lacking endogenous RIG-I and MDA5 were transfected with plasmids expressing RIG-I (B) or MDA5 (C) and a luciferase reporter under the control of an IFN-β promoter. Cells were then stimulated by various RNAs, and luciferase production was measured as a proxy for IFN-β response. Poly(I:C) stimulates both receptors, whereas SLRs are specific for RIG-I. SLR response is dependent upon the di- or triphosphate moiety and stimulates RIG-I as well or better than other reported RIG-I ligands. RLU, relative luciferase unit.
Fig. 2
Fig. 2. SLR injection induces robust type I IFN responses in vivo.
(A) C57BL/6 mice were injected intravenously with 25 μg of SLR10, SLR14, OH-SLR10, poly(I:C), ppp-NS, or vehicle control complexed with in vivo-jetPEI, and sera were collected 5 hours later. (B and C) C57BL/6 mice were injected intravenously with 25 μg of synthetic SLR10, and sera were collected at the indicated time points. Synthetic RNA was used for all experiments except in (A), where in vitro transcribed SLR (trans SLR) was used for comparison. The concentrations of IFN-α and TNF-α were measured by ELISA. Synthetic SLR10 was superior to transcribed SLR10 in inducing serum IFN-α.
Fig. 3
Fig. 3. Diphosphorylated SLRs induce IFN-α in vivo.
The diphosphorylated counterparts to SLR10 (A) and SLR14 (B) were injected intravenously into C57BL/6 mice, and 5 hours later, RNA was collected from spleens to measure IFN-α by qRT-PCR. Both pp-SLR10 and pp-SLR14 induced comparable IFN-α transcript levels as SLR10 and SLR14.
Fig. 4
Fig. 4. SLRs are specifically recognized by RIG-I.
(A) A549 human lung epithelial cells were transfected with mock or RIG-I–targeting siRNAs, stimulated with SLR10 or SLR14, and assayed by qRT-PCR for knockdown efficiency and induction of IFN-β, viperin, and Mx1. Reduction of RIG-I expression resulted in reduced IFN and ISG production. (B) Mice lacking TLR7, MAVS, and MDA5 were injected intravenously with SLR10, and 5 hours later, sera were collected to measure IFN-α. Asterisks indicate significant difference from WT mouse control (***P < 0.0005).
Fig. 5
Fig. 5. Distinct splenic gene expression between mice treated with SLRs and poly(I:C).
Mice were injected intravenously with SLRs or poly(I:C), and 3 hours later, RNA from spleens was purified for RNA-seq. Heatmaps were generated for genes preferentially regulated by treatment with SLR (A) or poly(I:C) (B). The heatmaps include all genes that are differentially expressed between SLR14 and poly(I:C) [more than twofold change and false discovery rate (FDR) of <0.05]. Highly statistically significant genes (FDR < 1 × 10−5) are labeled. (C) Type I and III IFNs are preferentially expressed following stimulation with SLR and poly(I:C), respectively. (D) Several additional genes, with and without reported immune functions, were differentially induced by SLRs or poly(I:C) (FDR < 1 × 10−5). FPKM, fragments per kilobase of exon per million fragments mapped.

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