anti rig i antibody  (Cell Signaling Technology Inc)

Journal: Molecular Therapy Advances

Article Title: Engineered T7 RNA polymerase to improve mRNA capping efficiency and reduce dsRNA generation during in vitro transcription

doi: 10.1016/j.omta.2026.201722

Figure Lengend Snippet: mRNA generated by T7 RNAP variants had enhanced translation and reduced immunogenicity in vitro mRNA generated by the R632N and Q649L variants at 4 mM and 0.5 mM cap concentration was compared with mRNA generated by WT T7 RNAP for translation and immunogenicity in vitro . mRNAs were transfected to primary human hepatocytes to quantify (A) Cas9 expression and (B) GFP expression. HEK-Lucia RIG-I reporter cells were used to quantity immunogenicity of (C) Cas9 mRNA and (D) GFP mRNA. DC was 142-bp dsRNA as positive control and LC was lipofectamine as negative control. Cell studies were performed with three biological replicates for each condition. Data are presented as mean values, with error bars representing the standard deviation. GraphPad Prism 10.5.0 was used to perform unpaired t test to calculate significance ( p value).

Article Snippet: HEK-Lucia RIG-I reporter cells (InvivoGen) were maintained in DMEM supplemented with 10% heat-inactivated FBS (Thermo Fisher Scientific), normocin (100 μg/mL), penicillin-streptomycin (100 U/mL), blasticidin (30 μg/mL), and zeocin (100 μg/mL).

Techniques: Generated, Immunopeptidomics, In Vitro, Concentration Assay, Transfection, Expressing, Positive Control, Negative Control, Standard Deviation

Journal: Molecular Therapy Advances

Article Title: Engineered T7 RNA polymerase to improve mRNA capping efficiency and reduce dsRNA generation during in vitro transcription

doi: 10.1016/j.omta.2026.201722

Figure Lengend Snippet: mRNA generated by T7 RNAP variants had enhanced translation and reduced immunogenicity in vivo Mice were dosed intravenously with Cas9 mRNA generated by the R632N and Q649L variants at 4 and 0.5 mM cap concentration. These were compared to mRNA produced by WT T7 RNAP to assess (A) Cas9 expression, (B) IFN-α, (C) IFN-β, and (D) IP-10. HEK-Lucia RIG-I reporter cells were used to quantify immunogenicity of (C) Cas9 mRNA and (D) GFP mRNA. Phosphate-buffered saline (PBS) was used as control. Data are presented as mean, with error bars representing the standard deviation ( n = 5 mice). GraphPad Prism 10.5.0 was used to perform unpaired t test to calculate significance ( p value).

Article Snippet: HEK-Lucia RIG-I reporter cells (InvivoGen) were maintained in DMEM supplemented with 10% heat-inactivated FBS (Thermo Fisher Scientific), normocin (100 μg/mL), penicillin-streptomycin (100 U/mL), blasticidin (30 μg/mL), and zeocin (100 μg/mL).

Techniques: Generated, Immunopeptidomics, In Vivo, Concentration Assay, Produced, Expressing, Saline, Control, Standard Deviation

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: FGF8 negatively regulated IFN-β induced by H13N2 infection. (a, B) luciferase reporter assays were used to assess the impact of FGF8 overexpression on IFN-β and ISRE promoter activity in A549 cells infected with H13N2 at an MOI of 1. (C-F) FGF8-overexpressing A549 cells were infected with H13N2 at an MOI of 1. At 12 hours post-infection (hpi), IFN-β levels in the cell supernatant were measured using ELISA (C), and IFN-β mRNA levels were evaluated by RT-qPCR (d). At 24 hpi, the mRNA levels of interferon-stimulated genes MX1 (e) and IFIT1 (f) were assessed by RT-qPCR. (G-J) stable FGF8-knockdown A549 cells were infected with H13N2 at an MOI of 1. At 12 hpi, IFN-β levels in the cell supernatant were quantified by ELISA (G), and IFN-β mRNA levels were evaluated using RT-qPCR (H). At 24 hpi, the mRNA levels of MX1 (i) and IFIT1 (J) were assessed by RT-qPCR. (K and L) Western blot analysis evaluated RIG-I, p-TBK1, and p-IRF3 expression in A549 cells with FGF8 overexpression (L) or knockdown (K) at 12 hours after H13N2 infection (MOI = 1). Band intensities were quantified by densitometric analysis. Statistical analysis was performed using two-tailed unpaired Student’s t-tests, with significance levels of * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, respectively.

Techniques: Infection, Luciferase, Over Expression, Activity Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Knockdown, Western Blot, Expressing, Two Tailed Test

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: FGF8 drives ubiquitin – proteasomal degradation of RIG-I. (a) FGF8 inhibits RIG-I-mediated signaling. A luciferase reporter assay was performed to evaluate the effect of FGF8 overexpression on IFN-β promoter activation induced by RIG-I. (B and C) FGF8 does not affect RIG-I transcription. RIG-I mRNA levels were quantified by RT-qPCR in FGF8-overexpressing A549 cells at 0, 6, and 12 hours post-infection with H13N2 (b) or H1N1 (C) at an MOI of 1. (d) dose-dependent reduction of RIG-I protein. A549 cells were transfected with increasing amounts of Flag-FGF8 plasmid for 24 hours, followed by infection with H13N2 (MOI = 1) for 12 hours. RIG-I protein levels were analyzed by Western blot, and band intensities were quantified by densitometry. (e) FGF8 reduces RIG-I stability. FGF8-overexpressing A549 cells were infected with H13N2 (MOI = 1) and treated with cycloheximide (CHX, 50 µg/mL) for the indicated time periods. Protein levels were analyzed by Western blot, and the relative abundance of HA-RIG-I was quantified to assess protein degradation rates. (F and G) proteasome inhibition restores RIG-I levels. A549 cells infected with H13N2 (f) or H1N1 (G) at an MOI of 1 were treated with DMSO, chloroquine (CQ, 50 µM), or MG132 (10 µM) for 6 hours. RIG-I expression was analyzed by Western blot, with relative protein levels quantified by densitometry. (H and I) FGF8 promotes K48-linked ubiquitination of RIG-I. HEK-293T cells were co-transfected with the indicated plasmids and treated with MG132 for 6 hours. (H) Total ubiquitination of RIG-I was assessed by immunoprecipitation with anti-HA antibody followed by immunoblotting (ib) with anti-Myc. (i) K48- or K63-linked ubiquitination was analyzed using specific ubiquitin mutants. Error bars indicate the mean ± SEM from three independent experiments. Statistical analysis was performed using two-tailed unpaired Student’s t-tests. ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, respectively.

Techniques: Ubiquitin Proteomics, Luciferase, Reporter Assay, Over Expression, Activation Assay, Quantitative RT-PCR, Infection, Transfection, Plasmid Preparation, Western Blot, Inhibition, Expressing, Immunoprecipitation, Two Tailed Test

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: Identification of the ubiquitination site on RIG-I targeted by FGF8. (a) diagram illustrating the truncated constructs of RIG-I. (b) HEK-293T cells were co-transfected with specified plasmids and exposed to MG132 for 6 hours. Western blot analysis was conducted to assess the ubiquitination of various RIG-I truncation constructs. (C) Western blot analysis identified the ubiquitination site on RIG-I targeted by FGF8, and band intensities were quantified by densitometry to assess the degradation of each mutant. (d) a dual-luciferase assay was conducted in HEK293T cells co-transfected with specified RIG-I mutants and FGF8 to evaluate the impact of FGF8 on IFN-β promoter activity. Error bars indicate the mean ± SEM from three independent experiments. Two-tailed unpaired Student’s t-tests were used. ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, respectively.

Techniques: Ubiquitin Proteomics, Construct, Transfection, Western Blot, Mutagenesis, Luciferase, Activity Assay, Two Tailed Test

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: TRIM16 mediated RIG-I degradation and promoted influenza virus replication. (a) Co-immunoprecipitation analysis was performed in cells transfected with Flag-TRIM16 and HA-RIG-I, with or without H13N2 infection (MOI = 1), to verify the interaction. (b) immunofluorescence microscopy showing the localization of TRIM16 (green) and RIG-I (red) in cells infected with H13N2 or mock-infected (NC). Nuclei were stained with DAPI (blue). Note that TRIM16 and RIG-I show diffuse distribution in the NC group but form co-localized puncta (yellow) upon H13N2 infection. Scale bar: 5 μm. (C) in vitro ubiquitination assay to verify the direct E3 ligase activity of TRIM16 using wt and ΔB-Box mutant proteins. (d) in vitro ubiquitination assay to determine the linkage specificity of TRIM16-mediated RIG-I ubiquitination using K48-only and K63-only ubiquitin mutants. (e) bioinformatic analysis using PONDR revealed the presence of intrinsically disordered regions (IDRs) in the FGF8 protein sequence. (f) fluorescence microscopy of A549 cells transfected with EGFP-FGF8 (green). Nuclei were stained with DAPI. Scale bar represents 10 μm. (G) TurboID-based proximity labeling assay was performed in cells expressing FGF8-TurboID. Biotinylated proteins were captured using streptavidin beads, and the pulled-down proteins were analyzed by Western blot to detect the presence of RIG-I and TRIM16. (H and I) validation of TRIM16 knockdown. RT-qPCR (H) and Western blot (i) confirmed the silencing efficiency in A549 cells. (J) control and TRIM16-silenced A549 cells were infected with H1N1 or H13N2 (MOI = 0.5) for 24 hours. Viral protein levels (NP, PB1, PB2) were analyzed by Western blot, and band intensities were quantified by densitometry. (K) RT-qPCR analysis of IFN-β mRNA levels in TRIM16-silenced A549 cells 12 hours post-infection with H13N2 (MOI = 1). (L) Western blot confirmation of TRIM16 overexpression (OE-TRIM16). (M) A549 cells overexpressing TRIM16 were infected with H1N1 or H13N2 (MOI = 0.5) for 24 hours. Viral protein expression was analyzed by Western blot and quantified by densitometry. Error bars indicate the mean ± SEM from three independent experiments. Statistical analysis was performed using two-tailed unpaired Student’s t-tests. ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, respectively.

Techniques: Virus, Immunoprecipitation, Transfection, Infection, Immunofluorescence, Microscopy, Staining, In Vitro, Ubiquitin Proteomics, Activity Assay, Mutagenesis, Sequencing, Fluorescence, Labeling, Expressing, Western Blot, Biomarker Discovery, Knockdown, Quantitative RT-PCR, Control, Over Expression, Two Tailed Test

Journal: bioRxiv

Article Title: Enhanced lung delivery of an immunostimulatory duplex RNA augments the antitumor activity by reshaping systemic cytokine pharmacodynamics

doi: 10.64898/2026.05.03.722518

Figure Lengend Snippet: (a) Schematic illustrating how BD shapes the in vivo immunostimulatory activity of self-dimerizing RNA-1 delivered by LungLNPs or LiverLNPs. LungLNP enhances delivery of RNA-1 to the lungs (1, pink), whereas conventional LiverLNP delivery directs RNA-1 to the liver (1, blue). In each case, organ-specific accumulation leads to uptake of RNA-1 into tissue resident immune or non-immune cell populations expressing pattern recognition receptors (PRRs) (2, pink/blue), thereby influencing pharmacodynamic responses, cytokine release, immune activation, and tumor suppression. (b) IFN-luciferase reporter assay in A549 IRF3 dual reporter cells showing induction by RNA-1 formulated in LungLNPs vs LiverLNPs, compared with free RNA-1 and empty controls. Data presented as average ± SD, n = 3. (c) Schematic of the in vivo pharmacodynamic (PD) model used to assess plasma cytokines following systemic administration of LungLNP/RNA-1, LiverLNP/RNA-1 formulations and corresponding empty LNPs. Mice were dosed with 2.2 mg/kg of RNA-1. (d-h) Quantification of peak plasma cytokine levels (2h for IFNα, IFNβ, TNFα and 6h for IFNγ, IFNλ), (i–m) Temporal kinetics of plasma cytokines (IFNα, IFNβ, IFNλ, IFNγ, and TNFα) following treatment at 2, 6 and 24 h. Data are represented as mean ± SEM from a representative experiment of three independent experiments with n = 6–7 (d–h) and n = 5–7 (i–m) biologically independent samples. (n) Schematic presentation depicting the knockout models used to study the innate immune pathway activated by LungLNPs/RNA-1 (2.2 mg/kg) in mice. (o) Quantification of IFNα plasma levels in RIG I KO mice (cytoplasmic sensing) compared with wildtype (WT) control. (p) Quantification of IFNα plasma levels in TLR3 and TLR7 KO mice compared with wildtype (WT) control. Data are represented as mean ± SD from a representative experiment of two independent experiments with n = 3-6 (o–p) biologically independent samples. (q) Molecular illustration depicting an Alphafold3 modeling of mouse RIG I and mouse TLR7 engaged with dsRNA-1 or ssRNA-1 respectively. Panels a, c and n were created with BioRender.com. The data were analyzed by ordinary one-way ANOVA with Tukey’s multiple-comparisons test; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: Human NF-κB-SEAP & IRF-Luc Reporter lung carcinoma (A549) cells (A549 RIG I) and human RIG-I-KO Dual Reporter A549 cells (A549 RIG I KO) (InvivoGen) were used to study the in vitro innate immune activity of RNA-1.

Techniques: In Vivo, Activity Assay, Expressing, Activation Assay, Luciferase, Reporter Assay, Clinical Proteomics, Knock-Out, Control

Journal: bioRxiv

Article Title: Enhanced lung delivery of an immunostimulatory duplex RNA augments the antitumor activity by reshaping systemic cytokine pharmacodynamics

doi: 10.64898/2026.05.03.722518

Figure Lengend Snippet: (a) Schematic illustration depicting a cross-section of the human lung cancer chip model, which recapitulates key physiological and pathophysiological features of human lung cancer. The microfluidic chip top channel containing human lung epithelial cells and human A549 adenocarcinoma alveolar basal epithelial cells stably expressing GFP, bottom channel containing human lung microvascular endothelial cells cultured on all four walls of the lower channel. (b) Treatment regimen for the human lung cancer-chip using LungLNPs/RNA-1 (100 and 200 nM), and empty LungLNP control (LungLNPs/Empty, 200 nM) and untreated chips. The first treatment was administered 4 days post-seeding, followed by establishment of the air–liquid interface on the same day. A second dose was administered on day 8. LNPs were delivered by vascular perfusion for 6 h per treatment. (c) A549 tumor growth curves during the treatment regimen, quantified by longitudinal GFP fluorescence imaging and measurement of fluorescence intensity. Data were analyzed using a two-way mixed effects model with time and treatment as fixed effects, followed by Tukey’s multiple-comparisons test. (d) Representative fluorescence images showing A549 tumor cells (green) on day 11 (scale bar = 1000 µm). (e) Quantification of cytokines and chemokines measured 2 h following the second dose. Data were analyzed by one way ANOVA with Tukey’s multiple comparisons test. (f) LNP uptake in the lung cancer-chip following perfusion of fluorescently labeled LungLNPs/RNA-1 Cy (yellow) at 100 and 200 nM. Endothelial cells were stained for VE-cadherin (purple), A549 tumor cells expressing GFP are shown in blue, and nuclei are shown in white. Chips were imaged 4 days post-treatment using confocal microscopy (scale bar = 20 µm). (g) Schematics depicting the mechanistic insight into RIG I-mediated lung cancer immunotherapy in human lung cancer chip demonstrating internalization into endothelial cells and RIG I activation and secretion of cytokines. Uptake into epithelial cells via direct exposure or via transport through gaps in the endothelial barrier. Panels a, b and d were created with BioRender.com. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: Human NF-κB-SEAP & IRF-Luc Reporter lung carcinoma (A549) cells (A549 RIG I) and human RIG-I-KO Dual Reporter A549 cells (A549 RIG I KO) (InvivoGen) were used to study the in vitro innate immune activity of RNA-1.

Techniques: Stable Transfection, Expressing, Cell Culture, Control, Fluorescence, Imaging, Labeling, Staining, Confocal Microscopy, Activation Assay

Journal: Nucleic Acids Research

Article Title: Engineering polymeric RNA scaffolds as programmable combinatorial innate immune agonists

doi: 10.1093/nar/gkag328

Figure Lengend Snippet: PolyRNAs of varying structures and sequences activate multiple PRRs. ( A ) Proposed structural features of polyRNA for PRR-specific recognition and activation. A dumbbell-shaped DNA template is processively transcribed by T7 RNA polymerase to generate 5′ triphosphate-containing polyRNA structures comprising repeat units of dsRNA and ssRNA regions. Panel of polyRNAs screened for activation of specific PRRs. All polyRNAs in the panel have a 25 bp dsRNA stem in each repeat, with varying ssRNA loop and connecting region lengths and sequences as indicated in the schematics. GUU labels indicate GU-rich ssRNA sequences. ( C, D ) Co-transcriptional structure prediction of monomeric units ( C ) and oligomeric RNAs ( D ) by KineFold . Pseudoknots are visualized as coloured single-stranded regions connected by two straight lines as predicted by KineFold. Structure prediction images were created with KineFold and polished using Adobe Photoshop. ( E ) In vitro activation of PRRs by polyRNAs transfected with Mirus TransIT-X2 (Mirus) in HEK-Blue hTLR3, hTLR7, and Null1 cells at 2 μg/ml. HEK-Blue Null1 is the parental cell line of HEK-Blue TLR cell lines, with baseline PRR expression levels. ( F ) In vitro RIG-I activation by polyRNAs transfected by Lipofectamine 3000 (Lipo) in HEK-Lucia RIG-I cells at 0.5 μg/ml. ( G ) In vitro IRF activation of IRF in RAW-Dual cells by polyRNAs transfected by Lipofectamine at 0.5 μg/ml. For panels (E)–(G), established agonist benchmarks were included for each PRR reporter cell line: high molecular weight poly(I:C) for TLR3, Null1, and RAW-Dual, R848 for TLR7, and 3p-hpRNA for RIG-I. The data represent the mean ± standard deviation of n = 3 technical replicates. Data were analysed by one-way Analysis of Variance (ANOVA) with Šidak’s multiple comparisons test. Ns, no significant difference between bracketed groups. **/***/**** denotes significance between bracketed groups ( P <.01/.001/.0001). Figure and were created in BioRender. Yang, Y. (2026) https://BioRender.com/gd4yhbl .

Article Snippet: 3p-hpRNA - RIG-I Agonist (3p-hpRNA), R848 (Resiquimod), and Poly (I:C) High Molecular Weight [poly(I:C)] were purchased from InvivoGen.

Techniques: Activation Assay, In Vitro, Transfection, Expressing, High Molecular Weight, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Engineering polymeric RNA scaffolds as programmable combinatorial innate immune agonists

doi: 10.1093/nar/gkag328

Figure Lengend Snippet: Assembly of CpG-DNA motifs onto polyRNA scaffolds induces activation of multiple PRRs in vitro . ( A ) Structure of a CpG-DNA comb, comprising a region complementary to a polyRNA repeat sequence, a linker, and a CpG-DNA sequence. Agarose gel electrophoresis of T52 polyRNA and DNA comb-patterned polyRNA indicates successful hybridization. T52 polyRNA comprises hairpin repeat motifs and averages 1000 bases in length. Complementary DNA comb: D52. The D52 DNA comb was hybridized to T52 polyRNA at a DNA comb/polyRNA repeat molar ratio of 1:3. ( C, D ) In vitro activation of the IRF ( C ) and NF-κB ( D ) pathways by T19 polyRNA and its CpG-DNA hybrid, transfected by Lipofectamine 3000 (Lipo) in RAW-Dual reporter cells, compared to the PRR agonist benchmarks poly(I:C) and 5′ triphosphate hairpin RNA (3p-hpRNA). A CpG-DNA comb only quantitative control was dosed at the equivalent amount used for hybridizing to polyRNA. T19 polyRNA comprises nonhairpin repeat motifs and averages 7000 bases in length. The D19-CpG comb is a DNA oligo mTLR9 agonist with a complementary region to T19 repeats and was hybridized to T19 polyRNA at a DNA comb/polyRNA repeat molar ratio of 1:4. ( E, F ) In vitro activation of PRRs by T52 polyRNA and its CpG-DNA hybrid, transfected with D-Lin-MC3-DMA (MC3)-based LNPs in RAW-Dual ( E ) and HEK-Blue mTLR9 reporter cells ( F ). D52-CpG is a DNA oligo mTLR9 agonist with a complementary region to T52 repeats. D52.mismatch-CpG is a mismatch comb control that does not contain any region complementary to T52 repeats. CpG-DNA combs were hybridized to T52 polyRNA at a DNA comb/polyRNA repeat molar ratio of 1:3. All dosages of polyRNAs, polyRNA/CpG-DNA (dosing based on polyRNA mass), and benchmark PRR agonists were 0.5 µg/ml. All CpG DNA comb-only samples were dosed at levels equivalent to the amounts hybridized to their corresponding base polyRNA scaffolds. The data represent the mean ± standard deviation of n = 3 technical replicates ( C–F ). Data were analysed by one-way ANOVA with Šidak’s multiple comparisons test. ns, no significant difference between bracketed groups. **/***/**** denotes significance between bracketed groups ( P <.01/.001/.0001). Figure and were created in BioRender. Yang, Y. (2026) https://BioRender.com/99l41mi .

Article Snippet: 3p-hpRNA - RIG-I Agonist (3p-hpRNA), R848 (Resiquimod), and Poly (I:C) High Molecular Weight [poly(I:C)] were purchased from InvivoGen.

Techniques: Activation Assay, In Vitro, Sequencing, Agarose Gel Electrophoresis, Hybridization, Transfection, Control, Standard Deviation