Journal: Cell Discovery

Article Title: RNA G-quadruplex formed in SARS-CoV-2 used for COVID-19 treatment in animal models

doi: 10.1038/s41421-022-00450-x

Figure Lengend Snippet: a Top, the sequences of P4 wild-type (P4-WT) and P4 mutant (P4-Mut). Bottom, the schematic representation of the proposed P4-WT and P4-Mut structures. b CD spectroscopy of P4-WT and P4-Mut. CD, circular dichroism. c The formation of P4 G4 detected by nondenaturing polyacrylamide gel electrophoresis experiments. Lane 1, DNA ladder; Lane 2, P4-WT; Lane 3, P4-Mut. d The formation of P4 G4 detected by 1 H NMR. NMR, nuclear magnetic resonance. e The kinetic folding process of P4 G4 detected by the stopped-flow assays. f The thermodynamic parameters of P4 G4 formation estimated from the melting measurements. g The formation of P4 G4 in live cells detected by immunofluorescence assays. Left, the white arrows indicated the colocalized foci of Cy5-labeled RNA (red) with BG4 (green). Scale bars, 4 μm. Right, Cy5/BG4 foci number was quantified. **** P < 0.0001. h , i Chemical structures of PDP ( h ) and TMPyP4 ( i ). j CD thermal melting curves of P4-WT (1.5 μM) with PDP (1.5 μM), TMPyP4 (1.5 μM) or TMPyP2 (1.5 μM). k CD spectroscopy of P4-WT (1.0 μM) without or with PDP (1.0 μM), TMPyP4 (1.0 μM) or TMPyP2 (1.0 μM).

Article Snippet: We first measured the effects of PDP, TMPyP4, TMPyP2 and remdesivir (as a positive control) on the cytotoxicity, viral RNA copies and viral titers in monkey kidney Vero E6 cells.

Techniques: Mutagenesis, Spectroscopy, Polyacrylamide Gel Electrophoresis, Nuclear Magnetic Resonance, Immunofluorescence, Labeling

Journal: Cell Discovery

Article Title: RNA G-quadruplex formed in SARS-CoV-2 used for COVID-19 treatment in animal models

doi: 10.1038/s41421-022-00450-x

Figure Lengend Snippet: a TMPyP4 or PDP treatment inhibits the translation of mRNA with P4 G4 by performing IVT assays. b TMPyP4 or PDP treatment inhibits the protein expression by targeting P4 G4 in HEK293T and Hela cells. c The protein expression of nsp7, nsp8 and nsp12, detected by western blotting assays. d The formation of P4 G4 inhibits the replication of RNA with P4 G4 in HEK293T cells, detected by qRT-PCR assays. e , f TMPyP4 ( e ) or PDP ( f ) treatment inhibits the replication of RNA with P4 G4 in HEK293T cells in a dose-dependent manner, detected by qRT-PCR assays. g HEK293T cells infected with P4-EGFP lentivirus were treated with or without 10 μM TMPyP4 or 2.5 μM PDP. Left, representative confocal images. Right, the relative fluorescent value. Scale bars, 100 μm. Data are shown as means ± SEM of three independent experiments, two-tailed Student’s t -test. n.s. not significant. * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: We first measured the effects of PDP, TMPyP4, TMPyP2 and remdesivir (as a positive control) on the cytotoxicity, viral RNA copies and viral titers in monkey kidney Vero E6 cells.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Infection, Two Tailed Test

Journal: Cell Discovery

Article Title: RNA G-quadruplex formed in SARS-CoV-2 used for COVID-19 treatment in animal models

doi: 10.1038/s41421-022-00450-x

Figure Lengend Snippet: a – c Vero E6 cells infected with SARS-CoV-2 (10 3 TCID 50 virus/mL) were pretreated with different concentrations of TMPyP4, TMPyP2, PDP or remdesivir. At 72 h post infection, viral RNA copies in cell lysates ( a ) and in cell culture supernatants ( b ) were detected by qRT-PCR. Viral titers (log 10 TCID 50 /mL) ( c ) were quantified by TCID 50 . Lower limit of detection for viral titers is indicated with a red dotted line. TCID 50 , median tissue culture infectious dose. d Fitting viral loads (viral RNA copies/mL) and cell viability under increasing concentration of TMPyP4 to determine the EC 50 and CC 50 of TMPyP4, PDP and remdesivir. The drugs cytotoxicity was detected by SRB assays. EC 50 , half-maximal effective doses. EC 90 , 90% maximal effective doses. CC 10 , 10% cytotoxic concentration. CC 50 , half-cytotoxic concentration. Data are shown as means ± SEM of three independent experiments, two-tailed Student’s t -test. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: We first measured the effects of PDP, TMPyP4, TMPyP2 and remdesivir (as a positive control) on the cytotoxicity, viral RNA copies and viral titers in monkey kidney Vero E6 cells.

Techniques: Infection, Cell Culture, Quantitative RT-PCR, Concentration Assay, Two Tailed Test

Journal: Cell Discovery

Article Title: RNA G-quadruplex formed in SARS-CoV-2 used for COVID-19 treatment in animal models

doi: 10.1038/s41421-022-00450-x

Figure Lengend Snippet: a Immunofluorescence of BG4 and fluorescence in situ hybridization (FISH) for viral genomic RNA (gRNA) in Vero E6 cells with or without ARS-CoV-2 infection. Scale bars, 10 μm. In the inset of selected regions, the colocalized foci of gRNA (red) with BG4 (green) are indicated by white arrows. The scale bar of inset, 2 μm. b , c The Vero E6 cells treated with 100 µM TMPyP4 were pre-transfected with the ASOs targeting viral G4s, and then the cells were infected with SARS-CoV-2 (10 3 TCID50 virus/mL). At 72 h post infection, viral RNA copies in cell culture supernatants ( b ) were detected by qRT-PCR. Viral titers (log 10 TCID 50 /mL) ( c ) were quantified by TCID 50 . Lower limit of detection for viral titers is indicated with a red dotted line. d – f Time-of-addition experiment of TMPyP4. The scheme shows the experimental design and the period of cell-drug incubation ( d ). Vero E6 cells were incubated with 100 μM TMPyP4 at the time points indicated. The cells were infected with SARS-CoV-2 (10 3 TCID 50 virus/mL), and the cell culture supernatants viral RNA copies ( e ) and viral titers ( f ) were quantified by qRT-PCR and TCID 50 . Data are shown as means ± SEM of three independent experiments, two-tailed Student’s t -test. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: We first measured the effects of PDP, TMPyP4, TMPyP2 and remdesivir (as a positive control) on the cytotoxicity, viral RNA copies and viral titers in monkey kidney Vero E6 cells.

Techniques: Immunofluorescence, Fluorescence, In Situ Hybridization, Infection, Transfection, Cell Culture, Quantitative RT-PCR, Incubation, Two Tailed Test

Journal: Cell Discovery

Article Title: RNA G-quadruplex formed in SARS-CoV-2 used for COVID-19 treatment in animal models

doi: 10.1038/s41421-022-00450-x

Figure Lengend Snippet: a Schematic of SARS-CoV-2 infected hamster model. Hamsters infected with SARS-CoV-2 (10 5 TCID 50 virus/hamster) were treated with vehicle ( n = 5), 15 mg/kg ( n = 5) or 30 mg/kg ( n = 5) TMPyP4 for consecutive 3 days, with the first dose given at 1 h before infection with SARS-CoV-2. b , c Viral RNA copies ( b ) and viral titers ( c ) in the hamster nasal wash, nasal turbinate and lung tissues of the TMPyP4-treated groups relative to vehicle controls, determined by qRT-PCR and TCID 50 at day 3 after infection. LLOD for viral titers is indicated with a red dotted line. d The representative images of hamster lung histopathological changes at day 3 after infection. The corresponding higher-magnification images were shown. See Supplementary Fig. for whole-lung tissue scan images of all hamsters. e Pathological severity scores in hamsters with SARS-CoV-2 infection. f Representative chemokine and cytokine assessment of the lung tissues ( n = 3) of the indicated groups, as detected in lung tissue homogenate at day 3 after infection. Data are shown as means ± SEM, two-tailed Student’s t -test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: We first measured the effects of PDP, TMPyP4, TMPyP2 and remdesivir (as a positive control) on the cytotoxicity, viral RNA copies and viral titers in monkey kidney Vero E6 cells.

Techniques: Infection, Quantitative RT-PCR, Two Tailed Test

Journal: Cell Discovery

Article Title: RNA G-quadruplex formed in SARS-CoV-2 used for COVID-19 treatment in animal models

doi: 10.1038/s41421-022-00450-x

Figure Lengend Snippet: a Schematic of SARS-CoV-2-infected hamster model. Hamsters infected with SARS-CoV-2 (10 5 TCID 50 virus/hamster) were treated with vehicle ( n = 5), 15 mg/kg TMPyP4 ( n = 5), 15 mg/kg TMPyP2 ( n = 5), 15 mg/kg remdesivir ( n = 5) and 15 mg/kg remdesivir combined with 15 mg/kg TMPyP4 ( n = 5) for consecutive 3 days, with the first dose given at 1 h before infection with SARS-CoV-2. b , c Viral RNA copies ( b ) and viral titers ( c ) in the hamster nasal wash, nasal turbinate and lung tissues of the indicated groups, determined by qRT-PCR and TCID 50 at day 3 after infection. Data are shown as means ± SEM, two-tailed Student’s t -test. n.s. not significant. * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: We first measured the effects of PDP, TMPyP4, TMPyP2 and remdesivir (as a positive control) on the cytotoxicity, viral RNA copies and viral titers in monkey kidney Vero E6 cells.

Techniques: Infection, Quantitative RT-PCR, Two Tailed Test