TY - JOUR
AU - Choi, Byong-Seok
AB - Abstract Retinoic acid-inducible gene I (RIG-I) recognizes double-stranded viral RNAs (dsRNAs) containing two or three 5′ phosphates. A few reports of 5′-PPP-independent RIG-I agonists have emerged, but little is known about the molecular principles underlying their recognition. We recently found that the bent duplex RNA from the influenza A panhandle promoter activates RIG-I even in the absence of a 5′-triphosphate moiety. Here, we report that non-canonical synthetic RNA oligonucleotides containing G-U wobble base pairs that form a bent helix can exert RIG-I-mediated antiviral and anti-tumor effects in a sequence- and site-dependent manner. We present synthetic RNAs that have been systematically modified to enhance their efficacy and we outline the basic principles for engineering RIG-I agonists applicable to immunotherapy. INTRODUCTION RIG-I is a cytosolic ATP-dependent RNA sensor in the innate immune system (1–3). When RNA viruses enter the cytosol, RIG-I binds viral RNAs and triggers a signal via mitochondrial antiviral-signaling protein (MAVS) that activates interferon regulatory factor 3 (IRF3) and NF-κB. This induces the type I interferons and suppresses viral propagation (4,5). Aside from its role in host defense against viruses, RIG-I activation leads to immunogenic cell death in carcinomas through the TNF-related apoptosis-inducing ligand (TRAIL) and Noxa (6,7). Immunogenic cell death is a type of programmed cell death that enhances tumor-targeting immunity by stimulating cytokine production and by releasing a number of intracellular molecules (e.g. calreticulin, heat shock proteins and HMGB1) that activate antigen-presenting cells and cytotoxic lymphocytes (8,9). Thus, RIG-I is a promising target not only for antiviral drug development but also for cancer immunotherapy (6,8) and as a vaccine adjuvant (10). The specific molecular structures in viral RNAs that trigger RIG-I activation reside at their 5′ ends. RIG-I recognizes dsRNAs containing uncapped 5′-PPP (11) or 5′-PP (12) which are found in RNA virus genomes and therefore produced during viral replication. Recent publications reported that RIG-I can also be activated by dsRNA (cap-0) containing m7G-capped-5′-PPP without 2′-OH methylation (13,14). Conversely, RIG-I is not activated by the more abundant endogenous RNAs whose 5′ ends have m7G-capped-triphosphates with methylation (cap-1 or cap-2) or monophosphates (5′-P). Thus, the structure of 5′-PPP acts as a molecular switch in the activation of RIG-I-mediated signaling. RIG-I also recognizes polyinosinic:polycytidylic acid (poly (I:C)) (15), poly (A:U) (16), and poly U tracts (17), but the molecular mechanism by which this activation occurs is unknown. We recently reported that the bent structure in the conserved promoter region of the influenza A virus (IAV panhandle promoter) activates RIG-I-mediated immune responses without requiring 5′-triphosphates (18). The RNA helix in this structure contains two G-U wobble base pairs that sandwich an A-U base pair. This configuration bends at an angle of 46°, causing the G–U pairs to pivot around the A–U pair (19). Each G–U base pair causes the RNA helix to twist clockwise by ∼23°, forming an interhelical bend (19) (Supplementary Figure S1). Here, we show that this interhelical bend formed by the two G–U wobble base pairs is a novel structurally-defined molecular pattern recognized by RIG-I. MATERIALS AND METHODS RNA oligonucleotides All 5′-OH RNA oligonucleotides without chemical modifications were purchased from Integrated DNA Technologies or Bioneer. CBS-7, CBS-13-BPS, CBS-21, CBS-22, CBS-23 and CBS-31 were purchased from Dharmacon or Bioneer. 5′-Fluorescein isothiocyanate (FITC)-labeled CBS RNAs were purchased from Bioneer. All RNA oligonucleotides were purified by HPLC. RNA oligonucleotides were dissolved in water and subsequently dialyzed against a storage buffer (10 mM potassium phosphate, pH 6.6, 100 mM sodium chloride, 0.1 mM EDTA). Prior to experiments, all RNA oligonucleotides except CBS-13-BPS were re-annealed by heating at 95°C for 5 min followed by cooling on ice for at least 30 min. CBS-13-BPS was re-annealed by heating at 95°C for 5 min followed by slow cooling to room temperature for an hour. The annealed RNA oligonucleotides were then dialyzed against the desired buffer for each experiment. 5′-PPP RNA (PPP-CBS-4 and CBS-10) was prepared by in vitro transcription using double-stranded DNA encoding the RNA sequence of CBS-4 or CBS-10 and containing the T7 promoter at its 5′-end (Integrated DNA Technologies). T7 RNA polymerase (prepared in house) was used to transcribe the DNA sequence in the presence of nucleotide triphosphates. The RNA was then purified by 20% (w/v) polyacrylamide gel electrophoresis (PAGE) in 7 M urea and 1× Tris–borate–EDTA (TBE). The RNA was electroeluted in 0.5× TBE buffer, precipitated in ethanol, dissolved in water, and desalted using dialysis in water. The desalted RNA was then exchanged into storage buffer. Protein expression and purification The gene encoding human RIG-I C-terminal domain (hRIG-I CTD; 803–925) was amplified by polymerase chain reaction (PCR) and cloned into the 2M-T vector (Addgene). The expression plasmid was transformed into the Escherichia coli BL21 (DE3) strain. The protein was overexpressed in lysogeny broth (LB) medium with 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) until the OD600 reached ∼0.6. After incubation at 17°C for an additional 18 h, the cells were harvested and lysed in Ni-NTA binding buffer (20 mM Tris–HCl buffer, pH 7.9, 500 mM sodium chloride, 5 mM imidazole, 5 mM 2-mercaptoethanol) by sonication. The resulting cellular debris was removed by centrifugation and the supernatant was collected for affinity chromatography purification using Ni-NTA agarose (Qiagen). The protein was then eluted with elution buffer (20 mM Tris–HCl, pH 7.9, 500 mM sodium chloride, 250 mM imidazole). The maltose binding protein (MBP) tag was removed by TEV protease digestion and dialyzed against buffer (20 mM Tris–HCl, 100 mM sodium chloride, 5 mM 2-mercaptoethanol, pH ∼8.0) overnight at 4°C. The protein was further purified using Ni-NTA agarose (Qiagen) and ion exchange chromatography using an SP-HP column (GE Healthcare). The eluted protein was then dialyzed against the final buffer (10 mM potassium phosphate buffer, 50 mM sodium chloride, 5 mM 2-mercaptoethanol, pH 6.87). The human RIG-I gene encoding the helicase and its C-terminal domains (amino acids 200–925 residues) was amplified by PCR using complementary DNA (pEF-BOS-RIG-I, Addgene). The amplified product was cloned into the pET-His6-GST-TEV-LIC vector (Addgene). The protein was then expressed in the BL21(DE3) strain. The bacterial cells were grown in LB medium with 100 μg/ml of ampicillin. Gene expression was induced by the addition of 1 mM IPTG until the OD600 reached 0.4∼0.6. After incubation at 16°C for an additional 20 h, the cells were harvested by centrifugation at 5000 rpm for 15 min, resuspended in 20 mM Tris–HCl, 500 mM sodium chloride, 10 mM imidazole at pH 7.9, and lysed by sonication in the presence of phenylmethanesulfonyl fluoride (PMSF). The resulting cellular debris was removed by centrifugation at 15 000 rpm for 1 h. The proteins were then purified from the supernatant by His-Binding Ni-NTA affinity chromatography (EMD Biosciences). The N-terminal glutathione S-transferase (GST) tag was removed with TEV protease at 4°C. The digested protein was dialyzed against a buffer containing 25 mM HEPES, 100 mM sodium chloride, 5 mM 2-mercaptoethanol, pH 7.4 and purified further in a Q-Sepharose column (GE Healthcare) with a linear gradient from 0 to 1 M NaCl in the same HEPES buffer. The protein-containing fractions were concentrated using an Amicon Ultra-15 (Millipore, MWCO 30,000) and purified with a Superdex 75 (GE Healthcare) gel-filtration column equilibrated with a buffer containing 20 mM Tris–HCl, 100 mM sodium chloride and 1 mM dithiothreitol at pH ∼7.5. Cells and viruses Cell lines of human lung adenocarcinoma (A549), Madin-Darby canine kidney (MDCK) and Calu-3 were purchased from the American Type Culture Collection (ATCC). They were maintained in RPMI 1640 medium (Hyclone), minimum essential medium (MEM; Hyclone), and Eagle's MEM (EMEM; ATCC) respectively, each supplemented with 10% Fetal Bovine Serum (FBS) (Gibco). The influenza A virus, A/Puerto Rico/8/34 (PR8; H1N1), was obtained from ATCC. The mouse-adapted PR8 (maPR8) was kindly provided by Prof. H.-J. Kim (Chung-Ang University, Seoul, Korea). Recombinant PR8-EGFP and PR8(H275Y)-EGFP viruses were constructed as previously reported (20–22). The viruses were amplified in 10-day-old embryonated chicken eggs for 3 days. Allantoic fluid was harvested and infectious virus titers were determined by plaque assays in MDCK cells (20). The stocks were stored at –70°C before use. Human embryonic kidney cell lines (HEK293 and HEK293T) were purchased from ATCC. They were maintained in DMEM medium (Welgene) supplemented with 10% FBS (ThermoFisher Scientific). The murine panc02 cell line was kindly provided by Prof. Christiane Bruns at University Hospital Magdeburg, Germany. The panc02 cells were maintained in DMEM (Welgene) with NEAA (non-essential amino acid solution, Sigma-Aldrich), vitamin (ThermoFisher Scientific) and 10% FBS (ThermoFisher Scientific). The human liver cancer cell line, SNU886 (Korean Cell Line Bank), was maintained in RPMI medium (Welgene) supplemented with 10% FBS. 293-hTLR3 cells (InvivoGen), which are HEK293 cells stably transfected with the human TLR3 gene, were maintained in DMEM supplemented with 10% FBS. Huh 7 and Huh 7.5 cells (kindly provided by Prof. Eui-Cheol Shin, KAIST) were maintained in DMEM supplemented with 10% FBS. Mice Female C57BL/6J mice (6–7 weeks of ages) were purchased from DBL (Seoul, Korea) and used for tumor experiments. All procedures were performed with the approval of the Institutional Animal Care Committee of KAIST. Female BALB/c mice (Orientbio) were maintained until 7 weeks of age before being immunized with the inactivated influenza A virus vaccine. All animal experiments with infectious influenza virus described here were approved by the Institutional Animal Care and Use Committee (IACUC) at KRICT. NMR spectroscopy CBS-4 was exchanged into NMR buffer (10 mM potassium phosphate, pH ∼6.6, 50 mM NaCl, 1 mM dithiothreitol, 10% v/v D2O) by dialysis. CBS-7 was exchanged into the same NMR buffer, except with 1 mM DTT replacing the 0.1 mM EDTA. 1D imino proton spectra of CBS-4 and CBS-4-Min were measured with jump-return pulses for water suppression at 15°C. All experiments were performed in 700 MHz Avance Bruker or Agilent spectrometers equipped with 5 mm triple-resonance cryogenic probes. All NMR spectra were processed using NMRPipe (23) and SPARKY 3 (24). Isothermal titration calorimetry Isothermal titration calorimetry was performed at 25°C using a Microcal VP-ITC instrument (Malvern Instruments Ltd.). The hRIG-I CTD and the RNAs (CBS-4 and CBS-7) were dialyzed against ITC buffer (10 mM potassium phosphate, 50 mM sodium chloride, 5 mM 2-mercaptoethanol, pH ∼6.87). The cell chamber contained 5–10 μM of RNA sample, and the titration syringe was loaded with 50–100 μM of hRIG-I CTD. The concentration of the hRIG-I CTD in the syringe was ∼10 times higher than the concentration of the RNA sample in the chamber (e.g. 10 μM of RNA and 100 μM of hRIG-I CTD). hRIG-I CTD in the syringe was injected at 30 successive points into the chamber containing the RNA sample. A blank experiment for titrating the hRIG-I CTD into a chamber without an RNA sample was also conducted for each RNA sample. These blank data were subtracted from the actual titration data to eliminate miscellaneous heat contributions including the heat of dilution. The resulting titration data were then processed using the Microcal software (Malvern Instruments Ltd.) that was provided with the VP-ITC instrument. The reported thermodynamic parameters including the dissociation constant and the stoichiometry were obtained by fitting the data to a one-site binding model. Electrophoretic mobility shift assay The human RIG-I CTD without its CARD domains (hRIG-I ΔCARD) was incubated with 0.1 mM of ATP and 20 μM of CBS-4, CBS-7, PPP-CBS-4 in a buffer (20 mM Tris–HCl, 100 mM sodium chloride, 1 mM dithiothreitol, pH ∼7.5) at increasing molar ratios of hRIG-I ΔCARD to RNA (i.e. 0:1, 0.5:1, 1:1, 2:1, 3:1). The reaction mixture was incubated on ice for 30 min. The 2× loading buffer (40 mM Tris–HCl, 200 mM sodium chloride, 2 mM dithiothreitol, 80% glycerol by volume, pH ∼7.5) was added to the reaction mixture prior to loading on a non-denaturing gel (9% polyacrylamide, 1× TBE). The reaction mixture was resolved by running the electrophoresis in the cold room (4°C). The gel was then fixed in 1 M acetic acid for 30 min and stained with methylene blue for visualization. RNA interference and quantitative real-time PCR HEK293T cells were seeded at 3 × 106 cells per 100 mm dish in DMEM (Welgene) supplemented with 10% FBS. The cells were incubated at 37°C and 5% CO2 for 24 h. After 1 day, the cells were transfected with 200 pmol hRIG-I siRNA (Bioneer) or control siRNA (Bioneer) using 20 μl of lipofectamine LTX (ThermoFisher Scientific) and 8 μl of plus-reagent (ThermoFisher Scientific) in OPTI-MEM (Thermo Fisher Scientific). The transfection efficiencies of CBS-1, CBS-4 and CBS-7 were measured by using fluorescently labeled RNA constructs and flow cytometry (Supplementary Figure S2). We found that their transfection efficiencies were comparable to each other. The hRIG-I siRNA sequences are 5′-ACGGAUUAGCGACAAAUUUAA dTdT-3′ (sense) and 5′-UUAAAUUUGUCGCUAAUCCGU dTdT-3′ (antisense). After 4 h of siRNA treatment, the OPTI-MEM was replaced with 7 ml of DMEM with 10% FBS and incubated at 37°C and 5% CO2 for an additional 20 h. The siRNA-treated cells were detached with 0.25% trypsin–EDTA (ThermoFisher Scientific) and then split into six-well plates, with each well containing 1.5 × 105 cells. The cells were then incubated at 37°C and 5% CO2 for 42 h prior to experiments. After 42 h of incubation, the siRNA-treated cells were transfected with 1 μM of each CBS RNA in 4 μl of lipofectamine LTX and 1.6 μl of plus-reagent in OPTI-MEM. After 4 h of transfection, the cells were incubated in DMEM with 10% FBS for another 2 h. The cells were harvested using the TRI-reagent (ThermoFisher Scientific). Their RNA was extracted using chloroform and precipitated with isopropanol. The RNA pellets were washed with 75% ethanol and dissolved in DEPC-treated water. The dissolved RNA was then treated with DNase (Promega) to remove any residual DNA. This RNA was then subjected to reverse transcription using Superior-Script III reverse transcriptase (Enzynomics). Quantitative real-time PCR analysis (Rotorgene-Q, Qiagen) was performed using EvaGreen Dye (Biotium), h-taq DNA polymerase (Solgent), and the following primers: human interferon-stimulated gene 56 (ISG56) forward, 5′-GCCTCCTTGGGTTCGTCTACAA-3′, ISG56 reverse, 5′-GAGGCCATGTGGGCCATGAG-3′, human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward, 5′-GCATTGCCCTCAACGACCAC-3′, GAPDH reverse, 5′-GAGGCCATGTGGGCCATGAG-3′ RIG-I forward, 5′-GCATTGCCCTCAACGACCAC-3′, RIG-I reverse, 5′-GAGGCCATGTGGGCCATGAG-3′. ISG56 and RIG-I expression data were normalized to GAPDH. Huh 7 or Huh 7.5 cells were seeded in six-well plates at 3 × 105 cells per well in DMEM (Welgene) supplemented with 10% FBS. The cells were incubated at 37°C and 5% CO2 overnight prior to transfection of CBS RNA using the same procedure as described above. Western blot analysis A549 cells were seeded at 1 × 106 cells per well in six-well plates. The next day, synthetic CBS RNAs (100 nM) or polyinosine–polycytidylic acid [poly (I:C)] (0.1 μg/ml) were transfected into cells using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. After 24 h, the cell culture supernatants were removed and the cell monolayers were mock-infected or infected with PR8 at a multiplicity of infection (MOI) of 0.1 for 1 h at 37°C. After the unabsorbed virus was removed, the cells were further maintained in culture medium containing 0.1 μg/ml TPCK-trypsin (Sigma-Aldrich). On day 1 or 2 post-infection (p.i.), the cells were lysed with the M-PER reagent (Thermo Scientific). Then, 30 μg of total protein was loaded onto a 10% SDS-PAGE gel for electrophoresis and transferred to a PVDF membrane (Immobilon-P membrane; Millipore). Viral NP protein was detected with an anti-NP antibody (11675-MM03; Sino Biological) and HRP-conjugated goat anti-mouse IgG (Sigma-Aldrich). Viral NS1 protein was detected with an anti-NS1 antibody (sc-17596; Santa Cruz Biotechnology) and HRP-conjugated donkey anti-goat IgG. Beta-actin was detected as a loading control with an anti-β-actin antibody (Sigma-Aldrich) and an HRP-conjugated goat anti-mouse antibody. The protein bands were visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce) or Western ECL Femto Kit (LPS Solution) using an image analyzer (LAS-4000; Fujifilm). For immunoblot of ISG56, HEK 293T cells were seeded at 1 × 106 cells per well in 6-well plates. The next day, synthetic CBS RNAs (1 μM) or polyinosine-polycytidylic acid [poly (I:C)] (2 μg/ml) were transfected into cells using 4 μl of Lipofectamine LTX and 1.6 μl of Plus reagent (Invitrogen). After 6 hours, the cells were harvested by using trypsin–EDTA (Gibco) and lysed with 1× SDS-reducing sample buffer. Cell lysates were analyzed by 10% SDS-PAGE followed by immunoblotting with antibodies against ISG56 (Cell Signaling) and tubulin (Sigma). Live-cell imaging of influenza A virus infected cells Calu-3 cells were seeded onto six-well plates (6 × 105 cells per well). After two days, they were transfected with synthetic RNAs using the Lipofectamine 2000 reagent. The next day, the cells were washed with PBS and infected with PR8-EGFP or PR8(H275Y)-EGFP at an MOI of 0.05 for 1 h at 37°C. The media was exchanged with fresh EMEM with or without 20 μM oseltamivir carboxylate (OSV-C; US Biological). Green fluorescent images were captured every 2 h for 3 days using an IncuCyte FLR (Essen BioScience). Fluorescent objects per well were counted using the built-in software. Vaccination and virus challenge The influenza virus vaccine was prepared by ultracentrifugation of PR8 virus inactivated with 0.02% formalin (25). Seven-week-old female mice (five per group) were immunized intranasally with 0.5 μg vaccine alone or in combination with 5 μg RNA adjuvant, CBS-13-PBS complexed in vivo with jetPEI (Polyplus-transfection) or with poly (I:C). Three weeks after their vaccination, the mice were intranasally challenged with 100-fold excess of 50% minimal lethal dose (MLD50) of maPR8. Then, they were observed for changes in body weight and mortality for an additional 11 days. Measurement of IgG level in mouse serum by ELISA Two weeks after immunization, sera were collected from mice and treated with receptor-destroying enzyme (RDE). The levels of influenza A virus (IAV)-specific IgG were determined by ELISA. 96-well ELISA plates (MaxiSorp; Nunc) were coated with the vaccine (0.5 μg per well). They were incubated with serum samples 10-fold diluted in PBS containing 5% horse serum and 0.05% Tween 20. IgG specifically bound to IAV was detected using HRP-conjugated goat anti-mouse IgG Fab fragment antibody (Southern Biotech). After addition of 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)-diammonium salt (ABTS) and ABTS peroxidase stop solution, optical density was measured at 405 nm using an ELISA microplate reader (SpectraMax M3; Molecular Devices). In vitro apoptotic activity of RIG-I agonists 0.5 × 105 panc02, SNU886, MEF or HEK293 cells were plated per well on 24 well-plates. The cells were then transfected with 1 μg/ml of the appropriate RNA molecule or poly (I:C) (InvivoGen) using lipofectamine LTX and incubated for 24 h at 37°C and 5% CO2. Note that 1 μg/ml of CBS-1, CBS-7, CBS-13-BPS and CBS-31 corresponds to ∼156, ∼149, ∼35 and ∼34 nM, respectively. The cells were then harvested and washed with 1× binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2). The cells were stained with FITC-labeled annexin-V (Biolegend) and 7-aminoactinomycin D (7-AAD; BD Bioscience) in 100 μl of 1× binding buffer for 15 minutes at room temperature. An additional 400 μl of 1× binding buffer was added, and the cells were analyzed on an LSRFortessa flow cytometer (BD Bioscience) with the FlowJo software (Tree Star, Inc.). Prophylactic anti-tumor effect of apoptotic panc02 cells Before treatment with RNA oligonucleotides, 2 × 106 murine panc02 cells were plated on 100 mm plates. The cells were transfected with 1 μg/ml CBS-13-BPS as described in the in vitro apoptotic activity assay section. After 24 h of incubation with the RNAs, the apoptotic—floating—cells were harvested and quantified by trypan blue staining. C57BL/6J mice, anesthetized by isoflurane inhalation, were injected subcutaneously on the right flank with 5 × 105 apoptotic cells induced by CBS-13-BPS. Seven days later, the mice were injected subcutaneously on the left flank with 5 × 105 viable panc02 cells. The volume of the resulting tumors was monitored beginning on day 5 after tumor cell injection. Tumor volume was calculated by V = (L × W2)/2, where V is tumor volume, L is length in millimeters and W is width in millimeters. In vivo anti-tumor experiment by intratumoral injection of RNA C57BL/6J mice were subcutaneously injected with 2.5 × 105 viable panc02 cells on the left flank. Then, 7 days after cancer cell transplantation, 25 μg of CBS-7 were admixed with in vivo jetPEI® (Polyplus) and 5% glucose, incubated for 15 min at room temperature, and injected directly into the tumor mass. RNA was injected twice a week for 2 weeks for a total of 4 injections. Tumor growth was monitored beginning on day 6 after tumor injection by measuring tumor volume. Tumor volume was calculated using the same equation as above: [V = (L × W2)/2]. RNase I reaction CBS-4 and CBS-7 (1.2 μg each) were incubated with 20 units of RNase I (ThermoFisher Scientific) in a buffer (20 mM Tris–HCl, 100 mM sodium chloride, 1 mM dithiothreitol, pH 7.5) at 37°C for 1, 6, 24 or 48 h. After the incubation, loading dye (0.01% w/v xylene cyanol, 0.01% w/v bromophenol blue, 7 M urea, 0.5× TBE) was added to the reaction mixture. Then, the reaction mixture was heated at ∼95°C for ∼5 min and stored at room temperature. All reaction mixtures stopped at different time points were heated again prior to loading on a denaturing gel (20% polyacrylamide, 7 M urea, 1× TBE). CBS-4 and CBS-7 untreated with RNase I were loaded on the gel in the same manner. After fixation in 1 M acetic acid, the RNA bands were visualized by staining with methylene blue. Dual luciferase reporter gene assay 293-hTLR3 cells (InvivoGen) were seeded in 12-well plates (1 × 105 cells/well). After 24 hours, the cells were transfected with 1 μg of pGL3-IFN-β-FLuc, that was constructed in-house on the basis of the previous report (26), or pNF-κB-Luc (Stratagene) together with 10 ng of phRL-CMV (Promega) using Lipofectamine 2000 (ThermoFisher Scientific). After washing with PBS, the cells were incubated with 1 ml fresh DMEM/10% FBS containing CBS-13-BPS or poly (I:C) in the absence of any transfection reagents for 18 hours. The activities of firefly and Renilla luciferases were then measured using the dual luciferase assay kit (Promega). RESULTS RIG-I-dependent immunogenicity of the Bent RNA duplex To determine the minimal motif in bent RNAs necessary for RIG-I activation, we synthesized CBS-4, a short hairpin RNA of 8 base pairs containing two G-U wobble base pairs flanking an A-U base pair (Figure 1A). We generated CBS-4 by chemical synthesis to ensure that it lacked 5′ phosphates. We also incorporated a UUCG loop (27) to help the RNA form a hairpin structure. By NMR spectroscopy, we observed two characteristic nuclear Overhauser effect (NOE) cross peaks corresponding to the two G–U base pairs and assigned imino protons appropriately (Supplementary Figure S3). Thus, we were able to confirm that CBS-4 folds into the secondary structure depicted in Figure 1A. Figure 1. View largeDownload slide Bent RNAs induce RIG-I-dependent ISG56 expression. (A) Secondary structures of the RNA constructs used in the experiments. The dashed red boxes denote wobble base pairs. (B) RIG-I-dependent induction of ISG56 in HEK293T cells by CBS-4 or CBS-4-Min, as monitored by RT-PCR. CBS-1 and CBS-10 were used as negative and positive controls, respectively. Cells were transfected with 1 μM of the indicated RNA constructs 6 hours prior to measurement. Medium refers to cells transfected with lipofectamine alone. Expression levels of ISG56 and RIG-I were normalized to GAPDH. (C) The induction of ISG56 by 1 μM CBS-4-U3C, CBS-4-U16C, or CBS-4-Mid in HEK293T cells was measured as described in Figure 1B. (B, C) The boxed graphs show the RIG-I siRNA knockdown efficiency. The black and red asterisks indicate significant differences in comparisons to medium and control (Ctrl) siRNA, respectively. Data are representative of two independent experiments (N = 3, mean ± SD). *P < 0.05; **P < 0.01. Figure 1. View largeDownload slide Bent RNAs induce RIG-I-dependent ISG56 expression. (A) Secondary structures of the RNA constructs used in the experiments. The dashed red boxes denote wobble base pairs. (B) RIG-I-dependent induction of ISG56 in HEK293T cells by CBS-4 or CBS-4-Min, as monitored by RT-PCR. CBS-1 and CBS-10 were used as negative and positive controls, respectively. Cells were transfected with 1 μM of the indicated RNA constructs 6 hours prior to measurement. Medium refers to cells transfected with lipofectamine alone. Expression levels of ISG56 and RIG-I were normalized to GAPDH. (C) The induction of ISG56 by 1 μM CBS-4-U3C, CBS-4-U16C, or CBS-4-Mid in HEK293T cells was measured as described in Figure 1B. (B, C) The boxed graphs show the RIG-I siRNA knockdown efficiency. The black and red asterisks indicate significant differences in comparisons to medium and control (Ctrl) siRNA, respectively. Data are representative of two independent experiments (N = 3, mean ± SD). *P < 0.05; **P < 0.01. We then examined the immunogenicity of CBS-4 by monitoring its ability to induce interferon-stimulated gene 56 (ISG56), an IRF-3 target gene that is activated by RIG-I. Indeed, CBS-4 induces ISG56 expression in a RIG-I-dependent manner (Figure 1B) to levels similar to those induced by a 5′-PPP dsRNA (CBS-10, Figure 1A). As expected, CBS-1, a 5′-OH hairpin duplex RNA formed entirely from canonical Watson-Crick base pairs (Figure 1A), fails to induce ISG56 expression (Figure 1B). CBS-1 was therefore used as a negative control throughout this study. We also confirmed that the induction of ISG56 by CBS-4 cannot be attributed to the UUCG loop, since a duplex RNA without the loop (CBS-4-Min) induces ISG56 equally well (Figure 1A and B). We confirmed that CBS-4-Min existed in the double-stranded form, and two single-strands did not confer the immunogenicity (Supplementary Figure S4). We next asked which structural features of CBS-4 allow it to act as a RIG-I agonist despite lacking a 5′-PPP moiety. To determine whether both G-U base pairs are necessary for RIG-I activation, we designed two mutants—CBS-4-U3C and CBS-4-U16C—in which each of the G-U wobble base pairs is replaced with a G–C Watson–Crick base pair (Figure 1A). Neither CBS-4-U3C nor CBS-4-U16C induce ISG56 expression (Figure 1C), indicating that a single G-U base pair is insufficient for RIG-I activation. This is consistent with our previous finding that mutant influenza panhandle promoters containing only one G–U base pair do not form the bent helix (28). To determine whether the location of the G-U pairs affects CBS-4’s ability to activate RIG-I, we moved its G-U base pairs further from the open end of the duplex, generating CBS-4-Mid (Figure 1A). Again, CBS-4-Mid treatment does not induce expression of ISG56 (Figure 1C). Similarly, replacement of its G–U wobble base pairs with U–U or A–G wobble base pairs also prevents CBS-4 from inducing ISG56 expression (CBS-50 and 51; Supplementary Figure S5). Thus, the immunogenicity of CBS-4 depends on the presence of two G–U base pairs located close to the terminus of the RNA duplex. We next asked whether other types of interhelical bends lacking 5′-PPP moieties also activate RIG-I. We synthesized two bent RNA constructs each containing a helix-junction-helix (HJH) motif. One contains a Kt-7 K-turn motif (29) with a GAA bulge (Kinked RNA, Figure 2A); the helices of the K-turn motif bend at an angle of ∼60° around the bulge (Supplementary Figure S1) (29). The other HJH RNA is derived from the transactivation response (TAR) element of human immunodeficiency virus type-1 (HIV-1) (30) and contains a UCU bulge (TAR-Bulge; Figure 2A); the helices of HIV-1 TAR bend at an angle of ∼47° (Supplementary Figure S1) (31,32). Remarkably, both Kinked RNA and TAR-Bulge are capable of inducing RIG-I-dependent ISG56 expression (Figure 2B). Reducing the angle of TAR’s interhelical bend by deleting one or two of its bulge nucleotides (TAR-2 and TAR-1, Supplementary Figure S6A) (33) blocks TAR’s induction of ISG56 (Supplementary Figure S6B). This suggests that interhelical bends are important structural cues in the RIG-I activation. Figure 2. View largeDownload slide RIG-I-dependent immunogenicity of the bulged RNA. (A) Secondary structures of the bulged RNA (green; Kinked RNA and TAR-Bulge). The hairpin RNA without a bulge (CBS-3) used as a control in the experiments. (B) The induction of ISG56 expression by 1 μM Kinked RNA, TAR-Bulge, CBS-3 or CBS-10 was measured as described in Figure 1B. The boxed graphs show the RIG-I siRNA knockdown efficiency. The black and red asterisks indicate significant differences in comparisons to medium and control (Ctrl) siRNA, respectively. Data are representative of two independent experiments (N = 3, mean ± SD). *P < 0.05; **P < 0.01. Figure 2. View largeDownload slide RIG-I-dependent immunogenicity of the bulged RNA. (A) Secondary structures of the bulged RNA (green; Kinked RNA and TAR-Bulge). The hairpin RNA without a bulge (CBS-3) used as a control in the experiments. (B) The induction of ISG56 expression by 1 μM Kinked RNA, TAR-Bulge, CBS-3 or CBS-10 was measured as described in Figure 1B. The boxed graphs show the RIG-I siRNA knockdown efficiency. The black and red asterisks indicate significant differences in comparisons to medium and control (Ctrl) siRNA, respectively. Data are representative of two independent experiments (N = 3, mean ± SD). *P < 0.05; **P < 0.01. Backbone modification of the bent RNA duplex to Gain resistance to degradation by RNase We next explored the potential of synthetic bent RNAs as immunotherapeutic agents. To increase the stability of these RNAs inside the body, we replaced the phosphate backbone of CBS-4 with phosphorothioate linkages to create the nuclease-resistant hairpin CBS-7 (Figure 3A and Supplementary Figure S7). We confirmed by NMR that the secondary structure of CBS-7 is similar to that of CBS-4 (Supplementary Figure S3). Previous structural studies suggest that the binding of a given dsRNA to RIG-I is influenced by electrostatic interactions between RIG-I and the dsRNA phosphate backbone (34–39). In an isothermal titration calorimetry (ITC) analysis, we found that the binding affinity of CBS-7 to the C-terminal domain of human RIG-I (hRIG-I CTD, Supplementary Figure S8A and S8B) is 31-fold lower than that of CBS-4. This may be due to the reduced polarity of phosphorothioate linkages compared to phosphate linkages. Still, we confirmed using an electrophoretic mobility shift assay (EMSA) that CBS-7 does form a complex with a human RIG-I mutant lacking two CARD domains (hRIG-I ΔCARD) (Supplementary Figure S8A and S8C). Furthermore, CBS-7 and CBS-4 induce similar levels of ISG56 expression (Figure 3B). CBS-1-PS, the phosphorothioate-linked version of the CBS-1 control, does not induce ISG56 expression (Figure 3B), excluding the possibility that phosphorothioate backbones in general activate RIG-I. These results suggest the bend structure of our synthetic RNAs permits significant backbone alterations without substantially compromising their ability to activate RIG-I. Figure 3. View largeDownload slide Backbone modification and elongation of the bent RNA do not deteriorate the immunogenicity. (A) Secondary structures of the bent RNA used in this experiment. The red dashed boxes denote G-U wobble motifs. Orange nucleotides denote the nucleotides with phosphorothioate backbone. The boxed A–U base pairs in CBS-13-BPS represent 26 consecutive base-pairs. (B) RIG-I-dependent induction of ISG56 by 1 μM CBS-1, CBS-1-PS, CBS-4 or CBS-7 was monitored by RT-PCR as described in Figure 1B. The boxed graph shows the RIG-I siRNA knockdown efficiency. (C) The induction of ISG56 by 1 μM CBS-1, CBS-4, CBS-7, or CBS-13-BPS in HEK293T cells was measured as described in Figure 1B. (B, C) Data are representative of two independent experiments. The error bars represent the standard deviations of triplicate experiments. The black and red asterisks indicate significant differences in comparisons to medium only and control (Ctrl) siRNA, respectively. *P < 0.05. (D) The induction of ISG56 by 1 μM CBS-1, CBS-13-BPS, or low molecular weight poly (I:C) in Huh 7 and Huh 7.5 cells was measured under the same conditions as described in Figure 1B. Data are representative of two independent experiments. The error bars represent the standard deviations of triplicate experiments. **P < 0.01. Figure 3. View largeDownload slide Backbone modification and elongation of the bent RNA do not deteriorate the immunogenicity. (A) Secondary structures of the bent RNA used in this experiment. The red dashed boxes denote G-U wobble motifs. Orange nucleotides denote the nucleotides with phosphorothioate backbone. The boxed A–U base pairs in CBS-13-BPS represent 26 consecutive base-pairs. (B) RIG-I-dependent induction of ISG56 by 1 μM CBS-1, CBS-1-PS, CBS-4 or CBS-7 was monitored by RT-PCR as described in Figure 1B. The boxed graph shows the RIG-I siRNA knockdown efficiency. (C) The induction of ISG56 by 1 μM CBS-1, CBS-4, CBS-7, or CBS-13-BPS in HEK293T cells was measured as described in Figure 1B. (B, C) Data are representative of two independent experiments. The error bars represent the standard deviations of triplicate experiments. The black and red asterisks indicate significant differences in comparisons to medium only and control (Ctrl) siRNA, respectively. *P < 0.05. (D) The induction of ISG56 by 1 μM CBS-1, CBS-13-BPS, or low molecular weight poly (I:C) in Huh 7 and Huh 7.5 cells was measured under the same conditions as described in Figure 1B. Data are representative of two independent experiments. The error bars represent the standard deviations of triplicate experiments. **P < 0.01. Elongation of the bent RNA duplex to enhance the immunogenicity Poly (A–U) stretches with 5′-PPP moieties have been shown to enhance the immunogenicity of RIG-I (16). Given our finding that the UUCG loop is dispensable (CBS-4-Min, Figure 1A), we synthesized CBS-13-BPS, a modified version of CBS-7 with 35 Watson–Crick base pairs containing 26 consecutive A–U base pairs and a UAA loop in place of the original UUCG loop (Figure 3A). Remarkably, CBS-13-BPS induces 7-fold more ISG56 expression than CBS-7 (Figure 3C) and the immunogenicity was RIG-I-dependent (Figure 3D). CBS-13-BPS also increased the expression level of ISG56 protein (Supplementary Figure S9). This activity of CBS-13-BPS, however, is independent of toll-like-receptor 3 (TLR3; Supplementary Figure S10), whose activation requires at least 40 bp of poly (A:U) dsRNA (40,41). Antiviral activity of the chemically modified bent RNA duplex We next examined the antiviral activity of CBS-7 and CBS-13-BPS. We treated a human lung epithelial cancer cell line, A549, with CBS-7 or CBS-13-BPS for 24 hours before infecting them with an H1N1 strain, PR8, of the influenza A virus for an additional 24 hours. We then monitored viral replication by immunoblotting for the viral nucleoprotein (NP) and non-structural protein 1 (NS1) (Figure 4A). Compared to pre-treatment with CBS-1 and to a lesser extent poly (I:C), pre-treatment with CBS-7 produces a consistent dose-dependent reduction in NP and NS1 (Figure 4A). CBS-13-BPS, in contrast, exhibits a very potent antiviral effect—more so than CBS-7—completely suppressing viral replication at low nanomolar concentrations (Figure 4A). Figure 4. View largeDownload slide Antiviral activities of CBS-7 and CBS-13-BPS in vitro and in vivo. (A) Antiviral activities of CBS-7 and CBS-13-BPS monitored by western blot. Mock refers to cells transfected with lipofectamine alone. CBS-1 (100 nM), CBS-1-PS (100 nM), poly (I:C) (0.1 μg/ml), and oseltamivir carboxylate (OSV-C; 10 μM) were used as controls. (B) Antiviral activity of CBS-13-BPS against oseltamivir-resistant influenza virus. A549 cells were treated with CBS-1, CBS-13-BPS or OSV-C, and infected with EGFP-expressing wild-type (WT) influenza virus [EGFP-PR8 (WT)] or its oseltamivir-resistant variant [EGFP-PR8 (H275Y)] 12 h later. Infected cells were counted at 2-h intervals for 36 h using a live cell imaging system. Mean fluorescence values were normalized to the maximum fluorescence value 24 h post-infection. Images at 24 h post-infection were visualized by fluorescence microscopy. (C) In vivo activity of CBS-13-BPS as an adjuvant for the influenza vaccine. 0.5 μg of inactivated PR8 (A/H1N1; Vaccine) was administered alone, with 5 μg CBS-13-BPS (Vac.CBS13BPS), or with 5 μg poly (I:C) (Vac.Poly(I:C)). Mice were infected with mouse-adapted PR8 (A/H1N1) influenza virus 3 weeks later. The survival rate and weight changes were monitored post-infection for 11 days. Mock refers to mice without any treatment. The mock trace (black) is overlaid with the Vac.CBS13BPS trace (purple). The change of weight: N = 5 (except for Mock, N = 3), mean ± SD. Statistical significance with respect to virus, *P < 0.05, **P < 0.01, ***P < 0.001. (D) IAV-specific antibody production enhanced by CBS-13-BPS. The IAV-specific serum IgG levels were determined by ELISA. Mice were immunized with the inactivated influenza A virus vaccine alone or together with CBS-13-BPS (5 μg per head) or with poly (I:C) (5 μg per head). The asterisks represent statistical significance compared to the vaccine only group. N = 5, mean ± SD. *P < 0.05, ***P < 0.001. Figure 4. View largeDownload slide Antiviral activities of CBS-7 and CBS-13-BPS in vitro and in vivo. (A) Antiviral activities of CBS-7 and CBS-13-BPS monitored by western blot. Mock refers to cells transfected with lipofectamine alone. CBS-1 (100 nM), CBS-1-PS (100 nM), poly (I:C) (0.1 μg/ml), and oseltamivir carboxylate (OSV-C; 10 μM) were used as controls. (B) Antiviral activity of CBS-13-BPS against oseltamivir-resistant influenza virus. A549 cells were treated with CBS-1, CBS-13-BPS or OSV-C, and infected with EGFP-expressing wild-type (WT) influenza virus [EGFP-PR8 (WT)] or its oseltamivir-resistant variant [EGFP-PR8 (H275Y)] 12 h later. Infected cells were counted at 2-h intervals for 36 h using a live cell imaging system. Mean fluorescence values were normalized to the maximum fluorescence value 24 h post-infection. Images at 24 h post-infection were visualized by fluorescence microscopy. (C) In vivo activity of CBS-13-BPS as an adjuvant for the influenza vaccine. 0.5 μg of inactivated PR8 (A/H1N1; Vaccine) was administered alone, with 5 μg CBS-13-BPS (Vac.CBS13BPS), or with 5 μg poly (I:C) (Vac.Poly(I:C)). Mice were infected with mouse-adapted PR8 (A/H1N1) influenza virus 3 weeks later. The survival rate and weight changes were monitored post-infection for 11 days. Mock refers to mice without any treatment. The mock trace (black) is overlaid with the Vac.CBS13BPS trace (purple). The change of weight: N = 5 (except for Mock, N = 3), mean ± SD. Statistical significance with respect to virus, *P < 0.05, **P < 0.01, ***P < 0.001. (D) IAV-specific antibody production enhanced by CBS-13-BPS. The IAV-specific serum IgG levels were determined by ELISA. Mice were immunized with the inactivated influenza A virus vaccine alone or together with CBS-13-BPS (5 μg per head) or with poly (I:C) (5 μg per head). The asterisks represent statistical significance compared to the vaccine only group. N = 5, mean ± SD. *P < 0.05, ***P < 0.001. We then used a PR8 virus expressing enhanced green fluorescent protein (EGFP) [EGFP-PR8 (WT)] to compare the effectiveness of CBS-13-BPS to that of the anti-influenza drug oseltamivir carboxylate in altering the time-course of viral replication. Throughout the course of infection, we found CBS-13-BPS suppresses viral replication at a level comparable to oseltamivir, but at a 200-fold lower molar concentration. Moreover, we also found CBS-13-BPS is active against an oseltamivir-resistant PR8 variant [EGFP-PR8(H275Y)] (Figure 4B), emphasizing the potential of CBS-13-BPS as an antiviral agent. The controls, CBS-1 and CBS-1-PS, which do not induce ISG56 expression, are incapable of suppressing viral replication. CBS-13-BPS’s strong antiviral effect on the influenza virus suggested that it may also be useful as a vaccine adjuvant. We therefore mixed an influenza vaccine containing inactivated PR8 with CBS-13-BPS complexed with the transfection agent jetPEI® or poly (I:C). We then administered these intranasally to mice (Figure 4C). Three weeks later, we challenged the vaccinated mice with a mouse-adapted PR8 virus at its lethal dose and monitored their weight and survival daily for 11 days. The mice given the CBS-13-BPS-admixed vaccine (Vac.CBS13BPS) completely survived this lethal influenza infection, while mice treated with vaccine alone or with the poly (I:C)-admixed vaccine (Vac.Poly(I:C)) suffered 60% or 20% death, respectively (Figure 4C). The influenza A virus (IAV)-specific IgG levels were higher in mice treated with Vac.CBS13BPS than in mice treated with the vaccine alone (Figure 4D). Thus, CBS-13-BPS shows significant potential for use as an adjuvant in vaccine development. Anti-tumor activity of the bent RNA duplex Previous studies have suggested that RIG-I agonists can induce apoptosis of cancer cells (6–8). We therefore asked whether our synthetic RNAs also induce cancer cell death. Twenty-four hours after transfecting the mouse pancreatic cancer cell line panc02 and human liver cancer cell line SNU886 with CBS-1, CBS-7, CBS-13-BPS or poly (I:C), we stained the transfected cells with fluorescently-labeled annexin-V and 7-aminoactinomycin D (7-AAD). Apoptotic cells display phosphatidylserine on the outer leaflet of their plasma membranes. This phosphatidylserine can be detected by annexin-V. Necrotic cells, in contrast, incorporate the membrane impermeable dye 7-AAD. When we detected apoptotic cells (annexin-V+7-AAD−) by flow cytometry, we found that although CBS-7 did induce apoptosis in panc02 and SNU886 cells, it did so less than poly (I:C) (Figure 5A and B). CBS-13-BPS, in contrast, induced apoptosis 4-fold more than CBS-7 and 2-fold more than poly (I:C) in panc02 cells, although it induced less apoptosis than poly (I:C) in SNU886 cells. This suggests that the induction of apoptosis by CBS RNAs may vary depending on the cell types. Importantly, neither CBS-7 nor CBS-13-BPS induced significant apoptosis in the non-cancerous human embryonic kidney 293 (HEK293) (Figure 5C) and mouse embryonic fibroblast (MEF) cell lines (Supplementary Figure S11), suggesting enhanced death of tumor cells triggered by CBS-RNAs. Figure 5. View largeDownload slide Anti-tumor activity of CBS-7 and CBS-13-BPS in vitro and in vivo. (A) Induction of apoptosis in the pancreatic cancer cell line panc02 by CBS RNAs. Panc02 cells were transfected with 1 μg/ml of CBS-1, CBS-7, CBS-13-BPS, and poly (I:C), admixed with lipofectamine. Twenty-four hours later, apoptotic cells were detected by flow cytometry after staining the cells with annexin-V and 7-AAD. Annexin-V+/7-AAD− cells were considered apoptotic and their percentages are shown as a bar graph. Data are representative of two independent experiments (N = 3, mean ± SD). *P < 0.05, ***P < 0.001. (B) Induction of apoptosis in the human liver cancer cell line SNU886 by CBS RNAs. SNU886 cells were treated and analyzed as described in Figure 5A. Data are representative of two independent experiments (N = 3, mean ± SD). ***P < 0.001. (C) No significant induction of apoptosis in the normal human cell line HEK293. HEK293 was treated and analyzed as described in Figure 5A. Data are representative of two independent experiments (N = 3, mean ± SD). (D) Prophylactic anti-tumor effect of apoptotic panc02 cells induced by CBS-13-BPS. 5 × 105 apoptotic panc02 cells, induced in vitro by CBS-13-BPS, were injected subcutaneously to the right flank of mice. A week later, 5 × 105 viable panc02 cells were seeded onto the contralateral side. Injected tumor volumes were then measured for 17 days. Data are representative of three independent experiments (N = 5, mean ± SEM). *P < 0.05. (E) Anti-tumor effect of CBS-7. Five days after tumor seeding, 25 μg of CBS-7 was injected intratumorally twice a week (marked with an asterisk). Changes in tumor volume were then monitored for 16 days. jetPEI (N = 5) and CBS-7 (N = 4). Mean ± SEM. *P < 0.05. Figure 5. View largeDownload slide Anti-tumor activity of CBS-7 and CBS-13-BPS in vitro and in vivo. (A) Induction of apoptosis in the pancreatic cancer cell line panc02 by CBS RNAs. Panc02 cells were transfected with 1 μg/ml of CBS-1, CBS-7, CBS-13-BPS, and poly (I:C), admixed with lipofectamine. Twenty-four hours later, apoptotic cells were detected by flow cytometry after staining the cells with annexin-V and 7-AAD. Annexin-V+/7-AAD− cells were considered apoptotic and their percentages are shown as a bar graph. Data are representative of two independent experiments (N = 3, mean ± SD). *P < 0.05, ***P < 0.001. (B) Induction of apoptosis in the human liver cancer cell line SNU886 by CBS RNAs. SNU886 cells were treated and analyzed as described in Figure 5A. Data are representative of two independent experiments (N = 3, mean ± SD). ***P < 0.001. (C) No significant induction of apoptosis in the normal human cell line HEK293. HEK293 was treated and analyzed as described in Figure 5A. Data are representative of two independent experiments (N = 3, mean ± SD). (D) Prophylactic anti-tumor effect of apoptotic panc02 cells induced by CBS-13-BPS. 5 × 105 apoptotic panc02 cells, induced in vitro by CBS-13-BPS, were injected subcutaneously to the right flank of mice. A week later, 5 × 105 viable panc02 cells were seeded onto the contralateral side. Injected tumor volumes were then measured for 17 days. Data are representative of three independent experiments (N = 5, mean ± SEM). *P < 0.05. (E) Anti-tumor effect of CBS-7. Five days after tumor seeding, 25 μg of CBS-7 was injected intratumorally twice a week (marked with an asterisk). Changes in tumor volume were then monitored for 16 days. jetPEI (N = 5) and CBS-7 (N = 4). Mean ± SEM. *P < 0.05. The tumor cell death induced by RIG-I agonists was recently shown to enhance anti-tumor immunity in vivo (8). To determine whether our synthetic RNAs can also boost anti-tumor immunity, we transfected panc02 cells with CBS-13-BPS in vitro and then injected the resulting apoptotic cells subcutaneously into the right flank of mice (Figure 5D). Seven days later, we injected viable panc02 cells subcutaneously into the left flank of mice and then monitored tumor growth for 17 days. We observed a clear suppression of tumor growth in mice vaccinated with CBS-13-BPS-induced apoptotic cells, but not in un-vaccinated mice (Figure 5D). We also found that two weeks of CBS-7 injections into panc02-derived mouse tumors induces substantial tumor regression with one mouse cleared of tumors (Figure 5E). Together, these results indicate that our RNA-based RIG-I agonists have tremendous potential as cancer immunotherapeutics. DISCUSSION Here, we present our results in systematically editing a group of RIG-I agonists to alter their ligand recognition, stability, and potency. We found the incorporation of a terminal interhelical bend facilitates recognition by RIG-I even in the absence of a 5′-PPP moiety. The RNA backbone can be replaced with phosphorothioate linkages to enhance cytosolic delivery and stability (42,43). The hairpin structure of these agonists facilitates duplex formation and helps maintain their stability. We also found additional poly (A:U) base pairs increase the potency with which these RNAs act as RIG-I agonists. We were able to demonstrate the immunogenicity of our synthetic RNA oligonucleotides against viruses and tumor cells both in vitro and in vivo, verifying the rationality of our design process. The fact that the 5′-end of our RNA agonists can be modified gives them a distinct advantage over classical RIG-l agonists, which must harbor 5′-PPP or 5′-PP. To further confirm this, we coupled biotin to the 5′-end of CBS-4 and CBS-13-BPS to synthesize CBS-21 and CBS-31, respectively (Figure 6A). We also coupled cholesterol to the 5′-end of CBS-7 to synthesize CBS-23 (Figure 6A). Then, we measured their immunogenicity. We found CBS-21 and CBS-23 induce ISG56 expression at levels comparable to CBS-4 (Figure 6B and C, respectively) and CBS-31 induces apoptosis in the hepatic cancer cell line (SNU886) at levels comparable to CBS-13-BPS (Figure 6D). This confirms the flexibility of our RIG-I agonists because the chemical modification of their 5′-ends does not seem to hinder their activity. In contrast to the situation with their 5′-ends and consistent with a previous report that showed 5′-PPP dsRNAs containing 3′-overhangs induce reduced expression of interferon-β (44), we found 3′ conjugation of cholesterol (CBS-22) prevents the induction of ISG56 expression (Figure 6B). From these observations, we infer that the 5′-end of our RNA agonists tolerates chemical conjugations that may improve their pharmacokinetic properties. Figure 6. View largeDownload slide Chemical modifications of CBS RNAs at their 5′-end do not affect their activity. (A) Secondary structures of CBS-21, CBS-22, CBS-23 and CBS-31. The dashed red boxes denote the bent duplex motif. Orange nucleotides denote phosphorothioate modifications. The boxed A–U base pairs in CBS-31 represent 26 consecutive base pairs. Biotin and cholesterol are abbreviated as bi and chol, respectively. Chemical structures of 5′-biotin, 3′-cholesterol, 5′-cholesterol are shown. (B) The induction of ISG56 by 1 μM CBS-1, CBS-1-PS, CBS-4, CBS-21 or CBS-22 in HEK293T cells was measured as described in Figure 1B. Mean ± SD. *P < 0.05, **P < 0.01. (C) The induction of ISG56 by 1 μM CBS-1, CBS-4, or CBS-23 in HEK293T cells was measured as described in Figure 1B. Mean ± SD. *P < 0.05. (D) Induction of apoptosis in the human liver cancer cell SNU886 by treatment with 1 μg/ml of CBS-1, CBS-7, CBS-13-BPS or CBS-31 analyzed as described in Figure 5A. Data are representative of two independent experiments (N = 3, mean ± SD). ***P < 0.001. Figure 6. View largeDownload slide Chemical modifications of CBS RNAs at their 5′-end do not affect their activity. (A) Secondary structures of CBS-21, CBS-22, CBS-23 and CBS-31. The dashed red boxes denote the bent duplex motif. Orange nucleotides denote phosphorothioate modifications. The boxed A–U base pairs in CBS-31 represent 26 consecutive base pairs. Biotin and cholesterol are abbreviated as bi and chol, respectively. Chemical structures of 5′-biotin, 3′-cholesterol, 5′-cholesterol are shown. (B) The induction of ISG56 by 1 μM CBS-1, CBS-1-PS, CBS-4, CBS-21 or CBS-22 in HEK293T cells was measured as described in Figure 1B. Mean ± SD. *P < 0.05, **P < 0.01. (C) The induction of ISG56 by 1 μM CBS-1, CBS-4, or CBS-23 in HEK293T cells was measured as described in Figure 1B. Mean ± SD. *P < 0.05. (D) Induction of apoptosis in the human liver cancer cell SNU886 by treatment with 1 μg/ml of CBS-1, CBS-7, CBS-13-BPS or CBS-31 analyzed as described in Figure 5A. Data are representative of two independent experiments (N = 3, mean ± SD). ***P < 0.001. The quintessential feature of 5′-PPP-independent RIG-I agonists is a non-canonical RNA structure that forms an interhelical bend near the terminal end of the RNA duplex. Here, we provide evidence that dsRNAs with a terminal interhelical bend are recognized by RIG-I in eukaryotic cells. Most cytosolic RNAs—including ribosomal RNAs (rRNA), transfer RNAs (tRNA) and precursor microRNAs (pre-miRNA)—do not conform to the structural qualifications we have discovered for 5′-PPP-independent RIG-I agonists. For example, pre-miRNAs and tRNAs have two- and four-nucleotide 3′-overhangs, respectively. These should block RIG-I activation even in the presence of a terminal interhelical bend. The 3D structures of rRNAs are finely-tuned to interact with ribosomal proteins for the hierarchical assembly of the ribosome (45), which makes rRNAs unlikely targets for recognition by RIG-I. Our findings thus highlight the unique ability of RIG-I to discern the structural features of pathogen-derived RNAs from the variety of endogenous host RNA structures. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We would like to thank the Korea Basic Science Institute for access to its NMR facility. We would like to thank Prof. Christiane Bruns at University Hospital Magdeburg in Germany for generously providing the panc02 cell line. We would like to thank Prof. Eui-Cheol Shin at KAIST for kindly providing the Huh 7 and Huh 7.5 cell lines. FUNDING National Research Foundation of Korea [2015R1A2A2A01005688, 2015R1A2A1A15055329 to B.-S.C., 2017R1A2B4007817 to S.-J.K.]; Korea Drug Development Fund funded by MSIT, MOTIE, and MOHW [KDDF-201706-06 to B.-S. 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TI - Systematic editing of synthetic RIG-I ligands to produce effective antiviral and anti-tumor RNA immunotherapies
JF - Nucleic Acids Research
DO - 10.1093/nar/gky039
DA - 2018-01-24
UR - https://www.deepdyve.com/lp/oxford-university-press/systematic-editing-of-synthetic-rig-i-ligands-to-produce-effective-xHGipXIEPm
SP - 1635
EP - 1647
VL - 46
IS - 4
DP - DeepDyve
ER -