unmethylated gpppg  (New England Biolabs)

Journal: medRxiv

Article Title: Human RIG-I Antiviral Deficiency Caused by a Dominant-Negative Variant Locked in a Signaling-Inactive State

doi: 10.64898/2026.03.02.26347088

Figure Lengend Snippet: Novel RIG-I G731R mutation identified in a severe COVID-19 patient. (A) Domain structure of human RIG-I. The arrow indicates the location of the G731R mutation. (B) RNA- and ATP-induced conformational activation of RIG-I. Top view of apo duck RIG-I (PDB: 4A2W) with CARDs (pink/purple) sequestered to Hel2i domain (green) autoinhibitory interface and helicase domains in an open conformation, semi-closed 5’ppp RNA-bound human RIG-I (PDB: 5F9H), and closed RNA-bound duck RIG-I with ADP·AlF 3 with missing CTD (PDB: 4A36). (C) Clustal Omega Multiple Sequence Alignment of helicase motif VI in RIG-I of various species, RLRs, and other SF2 RNA helicases. (D) Zoomed-in view of motif VI (in pink) in the closed state of RIG-I and the associated interactions with ATP and the pincer domain helix (black). (E) Chromatograms from Sanger sequencing of genomic DNA of the patient showing a heterozygous C to G mutation indicated by the arrow.

Article Snippet: Cells were replated in 96-well plates the next day at 250,000 cells/mL density and transfected with 0, 5, or 50 nM 5’ppp ds39 RNA using Lyovec transfection reagent (InvivoGen).

Techniques: Mutagenesis, Activation Assay, Sequencing

Journal: medRxiv

Article Title: Human RIG-I Antiviral Deficiency Caused by a Dominant-Negative Variant Locked in a Signaling-Inactive State

doi: 10.64898/2026.03.02.26347088

Figure Lengend Snippet: RIG-I G731R is a loss-of-function variant with a dominant negative effect. (A) 5’ppp ds39 RNA induced IFN-β reporter activities after transfection with empty vector (EV), WT RIG-I, and G731R plasmids (50 ng) in HEK293T RIG-I -/- cells (Star Methods) (technical replicates, n=3). (B) hPIV-induced IFN-β reporter luciferase activities for G731R. HEK293T cells were transfected with either the WT or G731R construct or EV along with the IFN-β-Luc reporter and a constitutively expressed Renilla reporter for 6 hours. Cells were then infected with hPIV (MOI 3) for an additional 20 hours before measuring luciferase activity. IFN-β-Luc reporter activity in each condition was first normalized to Renilla and then presented relative to WT RIG-I. Data show means ± SD from 3 independent experiments. Statistical analysis was performed using one-sample t-test. ****, P <0.0001. (C) IFN-β promoter response measured by dual luciferase reporter assay in HEK293T RIG-I -/- cells transfected with a constant amount of WT RIG-I plasmid (50 ng), increasing amount of G731R plasmid (0-500 ng), and 5 nM 5’ppp ds39 RNA. The error bars represent the standard error of the mean (technical replicates, n=3), and the curve represents fitting of the mean data to to obtain the half maximal inhibitory concentration IC 50 . (D) hPIV-induced IFN-β reporter luciferase activities in cells co-transfected with WT RIG-I (5 ng) and increasing doses (5 to 20 ng) of G731R or EV plasmids. IFN-β-Luc reporter activity in each condition was first normalized to Renilla and then shown relative to EV. Data show means ± SD from 3 independent experiments. Statistical analysis was performed using one-sample t-test. ***, P <0.001

Article Snippet: Cells were replated in 96-well plates the next day at 250,000 cells/mL density and transfected with 0, 5, or 50 nM 5’ppp ds39 RNA using Lyovec transfection reagent (InvivoGen).

Techniques: Variant Assay, Dominant Negative Mutation, Transfection, Plasmid Preparation, Luciferase, Construct, Infection, Activity Assay, Reporter Assay, Concentration Assay

Journal: medRxiv

Article Title: Human RIG-I Antiviral Deficiency Caused by a Dominant-Negative Variant Locked in a Signaling-Inactive State

doi: 10.64898/2026.03.02.26347088

Figure Lengend Snippet: RIG-I G731R competitively binds 5’ppp dsRNA in an ATPase-deficient manner. (A) Electrophoretic mobility shift assay of fluorescently labeled 5’ppp ds39 RNA upon addition of WT and G731R RIG-I (representative image from n=3). (B) 5’ppp ds39 RNA binding affinity (K D ) was obtained using a competitive fluorescence-based RNA binding assay (Figure S2, Star Methods). Data are mean ± SE from 3 independent experiments. Statistical analysis was performed using unpaired t-test. *, P<0.05. (C) ATPase rates of WT and G731R RIG-I at 1 µM concentration were measured in the presence of 5 nM 5’ppp ds39 RNA. Mean rates of hydrolysis (Moles P i / Moles RNA / sec) are reported (technical replicates, n=3). (D) Mant-ATP binding to WT and G731R RIG-I using fluorescence-based titration. The differences in the fluorescence intensity of Mant-ATP alone (F M ) and Mant-ATP with protein (F MP ) are plotted against Mant-ATP concentration. The error bars represent the standard error of the mean (n=3), and the curve represents hyperbolic fitting of the mean data to (Star Methods) to obtain the indicated Mant-ATP K D values. (E) G731R inhibits the RNA-dependent ATPase activity of WT RIG-I in a dose-dependent manner. ATPase activity was measured in the presence of 5’ppp ds39 RNA at constant WT RIG-I and increasing G731R protein. The error bars represent the standard error of the mean (n=3), and the curve represents fitting of the mean data to (Star Methods) to obtain the indicated IC 50 value.

Article Snippet: Cells were replated in 96-well plates the next day at 250,000 cells/mL density and transfected with 0, 5, or 50 nM 5’ppp ds39 RNA using Lyovec transfection reagent (InvivoGen).

Techniques: Electrophoretic Mobility Shift Assay, Labeling, RNA Binding Assay, Fluorescence, Concentration Assay, Binding Assay, Titration, Activity Assay

Journal: medRxiv

Article Title: Human RIG-I Antiviral Deficiency Caused by a Dominant-Negative Variant Locked in a Signaling-Inactive State

doi: 10.64898/2026.03.02.26347088

Figure Lengend Snippet: HDX-MS analysis of G731R reveals CARDs-CHL protection. (A) Linear mapping of the HDX differential data of WT and G731R with and without 5’ppp HP RNA. In the colorimetric HDX scheme, shifting towards red indicates a higher degree of solvent exchange, while blue indicates less. If there is no significant difference in the solvent exchange of two samples analyzed side-by-side, the HDX differential remains gray. The white/black areas indicate that the peptides were not resolved for mass spectrometry. (B) The differential HDX pattern of G731R apo vs WT RIG-I apo mapped onto a 3D composite model of G731R apo (4A2W, 3TBK, 4AY2, & computationally modeled CH-Linker). (C) The differential HDX pattern of WT RIG-I ± 5’ppp HP RNA mapped onto a 3D composite model of RNA-bound WT RIG-I with computationally modeled CHL and CARDs in the exposed state (PDB: 5E3H, 4P4H, 4AY2). (Ci) Deuterium uptake plots of select RIG-I peptides represented as percent deuterium uptake over time. (D) The differential HDX pattern of G731R ± 5’ppp 10bp HP RNA mapped onto a composite model of RNA-bound G731R with computationally modeled CHL and CARDs sequestered by Hel2i (PDB: 4A2W, 3TBK, 4AY2). (Di) Deuterium uptake plots of select RIG-I peptides represented as percent deuterium uptake over time.

Article Snippet: Cells were replated in 96-well plates the next day at 250,000 cells/mL density and transfected with 0, 5, or 50 nM 5’ppp ds39 RNA using Lyovec transfection reagent (InvivoGen).

Techniques: Solvent, Mass Spectrometry

Journal: medRxiv

Article Title: Human RIG-I Antiviral Deficiency Caused by a Dominant-Negative Variant Locked in a Signaling-Inactive State

doi: 10.64898/2026.03.02.26347088

Figure Lengend Snippet: The CHL regulates IFN-response and ATPase activities of G731R. (A) Amino acid sequence of the intrinsically disordered CHL of RIG-I. Negatively charged residues are colored red and underlined; positive charges are colored blue. The Δ190-200 segment is indicated. (B) IFN-β reporter activities of WT RIG-I, Δ190-200 WT, G731R, and Δ190-200 G731R RIG-I in the absence and presence of 5 nM 5’ppp ds39 RNA (technical replicates, n=3). The fold-change from the 10 aa deletion in WT RIG-I and G731R is shown. (C) Partial rescue of the ATPase function of G731R upon deletion of the CHL-CARDs domains. The ATPase activities of full-length and HelCTD constructs of WT RIG-I and G731R were measured at 1 µM protein in the presence of 5 nM 5’ppp ds39 RNA. Mean rates of hydrolysis (Moles P i / Moles RNA / sec) are reported (technical replicates, n=3).

Article Snippet: Cells were replated in 96-well plates the next day at 250,000 cells/mL density and transfected with 0, 5, or 50 nM 5’ppp ds39 RNA using Lyovec transfection reagent (InvivoGen).

Techniques: Sequencing, Construct

Journal: medRxiv

Article Title: Human RIG-I Antiviral Deficiency Caused by a Dominant-Negative Variant Locked in a Signaling-Inactive State

doi: 10.64898/2026.03.02.26347088

Figure Lengend Snippet: G731R RIG-I mutant is trapped in the “CTD-mode” RNA-bound conformation. (A) RIG-I forms kinetically distinguishable “CTD-mode” and “Helicase-mode” conformations on 5’ppp RNA. (B) Experimental setup to measure the off-rate of RIG-I from 5’ppp RNA using a rapid stopped-flow instrument. (C) Biphasic kinetics of WT RIG-I and G731R dissociation from 5’ppp ds27 RNA with and without ATP. The time axis is shown on a log scale to clearly show the two phases. The dissociation kinetics (average of n=3) best fit to the sum of two exponentials , providing slow and fast off-rates and the corresponding amplitudes plotted in panel D. (D) The “CTD-mode” and “Helicase-mode” phase amplitudes from data in panel C in the absence and presence of ATP are shown as percent population from three biological replicates with SEM. (E) The lifetimes (1/off-rate) in seconds of the “CTD-mode” and “Helicase-mode” phases are shown in the absence and presence of ATP from three biological replicates with SEM.

Article Snippet: Cells were replated in 96-well plates the next day at 250,000 cells/mL density and transfected with 0, 5, or 50 nM 5’ppp ds39 RNA using Lyovec transfection reagent (InvivoGen).

Techniques: Mutagenesis

Journal: medRxiv

Article Title: Human RIG-I Antiviral Deficiency Caused by a Dominant-Negative Variant Locked in a Signaling-Inactive State

doi: 10.64898/2026.03.02.26347088

Figure Lengend Snippet: Bulky and charged substitutions at G731 correlate with loss-of-function. (A) IFN-β responses of various G731 X mutants with and without RNA were measured by dual luciferase reporter assay (technical replicates, n=3). (B) PyMOL Mutagenesis Wizard analysis predicts clashes with bulky substitutions of G731 in the closed state of RIG-I (4A36). The visible red disks indicate pairwise overlap of atomic van der Waals radii. (C) Mechanistic model of G731R mutation locking RIG-I in an inactive CTD-mode RNA-bound conformation, where the CARDs-CHL are not exposed. The high affinity of G731R for the 5’ppp RNA competitively interferes with the WT RIG-I binding to the RNA.

Article Snippet: Cells were replated in 96-well plates the next day at 250,000 cells/mL density and transfected with 0, 5, or 50 nM 5’ppp ds39 RNA using Lyovec transfection reagent (InvivoGen).

Techniques: Luciferase, Reporter Assay, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: Evaluating the reliability of tools for mRNA annotation and IRES studies

doi: 10.64898/2026.03.29.707813

Figure Lengend Snippet: (A) A-cap / stemloop IRES reporter RNA carrying the negative control “scramble 1” sequence was mixed into a polysome lysate made from HEK293T cells for five minutes on ice and loaded on a sucrose gradient. (B) The sucrose gradient was fractionated and cDNA was prepared from groups of fractions as shown (numbered 1 -6). qRT-PCR was performed to quantify the exogenous “scramble 1” RNA across the fractions, as compared to endogenous Gapdh mRNA. The “scramble 1” transcript migrated into polysomal fractions 4-6, even though it is not translated .

Article Snippet: The ARCA m 7 G-cap analog (New England Biolabs; S1411) was used at a 4:1 ratio to GFP for transcription of T7-nluc constructs, while an A-cap analog (New England Biolabs S1406), was used to generate T7HP-nluc IRES reporter RNAs.

Techniques: Negative Control, Sequencing, Quantitative RT-PCR

Journal: Immunity

Article Title: A type I interferon-mitochondrial axis regulates efferocytosis and interferon-stimulated gene induction in macrophages

doi: 10.1016/j.immuni.2025.12.010

Figure Lengend Snippet:

Article Snippet: 5’ triphosphate double stranded RNA , Invivogen , Cat#tlrl-3prna.

Techniques: Virus, Western Blot, Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Detection Assay, Lactate Assay, Bicinchoninic Acid Protein Assay, Reverse Transcription, Software, Real-time Polymerase Chain Reaction