phosphoprotein 1  (Cell Signaling Technology Inc)

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting coronaviral inflammation: aptamer-based strategies for emerging threats

doi: 10.1038/s41392-025-02570-8

Figure Lengend Snippet: Identification of a high-affinity DNA aptamer targeting the N protein of coronavirus (NApt8-3). a ELISA analysis used to assess the binding of biotin-labeled candidate DNA aptamers (200 nM) to precoated SARS-CoV-2 N protein (100 ng/well). The optical density (OD) at 450 nm represents the binding signal. RS (random sequence) and the blank (without any treatment) were used as negative controls. b Molecular docking simulation of NApt8 and SARS-CoV-2 N protein binding via Discovery Studio software. The nucleobases of NApt8 (sequence provided in Table ) are labeled in green. Left: docking with the NTD; Right: docking with the CTD; magenta and cyan represent the NTD and CTD, respectively. c ELISA analysis was used to assess the binding of biotin-labeled NApt8 (200 nM) to precoated (100 ng/well) N protein (N), the NTD domain of the N protein (N-NTD) or the CTD domain of the N protein (N-CTD) on microplates. Blank: noncoated microplates. d ELISA analysis of the ability of a biotin-labeled aptamer (200 nM) to bind the mutated N protein (100 ng/well) precoated on microplates. The mutated N protein: arginine (R) was mutated to aspartic acid (D). e ELISAs to assess the binding performance of the biotin-labeled truncated aptamer (200 nM) to N protein (100 ng/well) precoated on microplates. NApt8-3, NApt8-2 and NApt8-1 (sequences provided in Table ) represent truncated variants generated by deleting nonessential sequences at the 5’ end, 3’ end and both ends, respectively. f Molecular docking simulation of NApt8-3 and SARS-CoV-2 (WT) N protein binding via Discovery Studio software. g Biolayer interferometry (BLI) assay to determine the kinetic binding parameters of the aptamer NApt8-3 to immobilized N proteins. h ELISAs were used to assess the binding performance of biotin-labeled NApt8-3 (200 nM) to the N protein of various coronaviruses (SARS-CoV-2 and its variants, SARS, MERS, HCoV-OC43, and HCoV-229E). i ELISA analysis of the kinetics of the binding of the aptamer NApt8-3 to the N protein of various coronaviruses, including SARS-CoV-2 variants (WT, Beta B.1.351, and Omicron BA.2.3), SARS-CoV, MERS-CoV, HCoV-OC43, and HCoV-229E. j Molecular docking simulation of NApt8-3 to the N proteins of SARS-CoV, MERS-CoV, HCoV-OC43, and HCoV-229E via Discovery Studio software. The N protein is shown with residue names. Amino acids shown in magenta represent conserved residues across coronaviruses that interact with NApt8-3, while blue denotes residues uniquely involved in binding in specific viral strains. Green represents the nucleobases of NApt8-3. All the error bars indicate standard deviations ( n = 3). All the P values were calculated via one-way ANOVA. *** P < 0.001

Article Snippet: SARS-CoV-2 Nucleocapsid Phosphoprotein Rabbit Polyclonal antibody was purchased from Proteintech Inc. (28769-1-AP, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Labeling, Sequencing, Protein Binding, Software, Generated, Residue

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting coronaviral inflammation: aptamer-based strategies for emerging threats

doi: 10.1038/s41392-025-02570-8

Figure Lengend Snippet: Broad-spectrum inhibitory effects of NApt8-3 on the inflammatory response induced by the N protein derived from various coronaviruses. a , b qRT‒PCR analysis of the dose-dependent inhibitory effects of NApt8 and NApt8-3 on the expression of cytokines (IL-6 and TNF-α) in Calu-3 cells transfected with a plasmid expressing the N protein. Mock: cells transfected with the N-encoding plasmid but without a random DNA sequence (RS) and aptmer treatment. RS: random sequence. The mRNA level of actin was used for normalization. The methods used to calculate the relative inhibition are described in the Materials and Methods. c qRT‒PCR analysis of the expression of cytokines (IL-6) in Calu-3 cells cotransfected with NApt8-3 together with plasmids expressing the N protein of various coronaviruses and SARS-CoV-2 variants (WT, Beta B.1.351, Omicron BA.2.3), SARS, MERS, HCoV-OC43, and HCoV-229E). d ‒ g qRT‒PCR analysis of the expression of cytokines (IL-6) in Calu-3 cells cotransfected with aptamers (NApt8-3, seq-7, seq-59, seq-333, seq1022, NASO2) and plasmids expressing the N protein of different coronaviruses. h Silver-stained SDS‒PAGE was used for coimmunoprecipitation with anti-Flag antibodies to identify proteins that interact with N-Flag in the lysates of A549 cells transfected with N-Flag-expressing plasmids subjected to the indicated treatments. The white box denotes the differentially expressed protein band (100 kDa). i . Coimmunoprecipitation was performed via the use of anti-FLAG antibodies to confirm the interaction of the N protein and NLRP-3 in the lysates of A549 cells. Cell lysates (40 μg) were used as inputs. j Coimmunoprecipitation using anti-NLRP3 antibodies was used to confirm the interaction between the N protein and NLRP-3 in the lysates of A549 cells subjected to the indicated treatments. Cell lysates (40 μg) were used as inputs. k NApt8-3 inhibits the NLRP3-N protein interaction in a dose-dependent manner. All the error bars indicate standard deviations ( n = 3). P values were calculated via one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: SARS-CoV-2 Nucleocapsid Phosphoprotein Rabbit Polyclonal antibody was purchased from Proteintech Inc. (28769-1-AP, USA).

Techniques: Derivative Assay, Expressing, Transfection, Plasmid Preparation, Sequencing, Inhibition, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting coronaviral inflammation: aptamer-based strategies for emerging threats

doi: 10.1038/s41392-025-02570-8

Figure Lengend Snippet: Design and performance of a circular trivalent chimera for SARS-CoV-2 via NApt8-3. a Schematic illustration of the principle underlying efficient targeted delivery of circSASON to suppress SARS-CoV-2 (WT) replication and inflammation. CorelDraw was used to generate these images. b Flow cytometry analysis of SApt-dependent and SARS-CoV-2 infection-mediated delivery of circSASON into hACE2-HEK293T cells. Blank (without any treatment) and RS were used as negative controls. SApt as a positive control. The upper panel shows the GFP signal from the SARS-CoV-2 Spike (WT) Fluc-GFP pseudovirus. The lower panel displays the Cy3 signal from the Cy3-labeled oligonucleotides (RS, SApt or cirSASON). c Representative fluorescence confocal images of hACE2-HEK293T cells infected with SARS-CoV-2 pseudovirus (5 × 10 4 TCID50 mL −1 ) preincubated with the indicated 200 nM Cy3-labeled oligonucleotides ( n = 3). S-pseudovirus spike-GFP: SARS-CoV-2 spike (WT) Fluc-GFP pseudovirus. Scale bars represent 10 µm. d Representative fluorescence confocal images of HEK293T and hACE2-HEK293T cells incubated with SARS-CoV-2 pseudovirus (5 × 10 4 TCID50 mL −1 ) and Cy3-labeled circSASON (200 nM). Scale bars represent 10 µm

Article Snippet: SARS-CoV-2 Nucleocapsid Phosphoprotein Rabbit Polyclonal antibody was purchased from Proteintech Inc. (28769-1-AP, USA).

Techniques: Flow Cytometry, Infection, Positive Control, Labeling, Fluorescence, Incubation

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting coronaviral inflammation: aptamer-based strategies for emerging threats

doi: 10.1038/s41392-025-02570-8

Figure Lengend Snippet: circSASON inhibits SARS-CoV-2 replication and inflammatory cytokine expression. a The secondary structure of the three circular chimeras (circSASO, circSN and circSASON) predicted via RNAfold. b qRT‒PCR analysis of the expression of IL-6 in Calu-3 cells and A549 cells treated as indicated. mRNA (relative) represents the mRNA expression level normalized to that of actin mRNA. c , d qRT‒PCR analysis of the inhibitory effects of the three circular chimeras on IL-6 expression in cells 48 h after infection with SARS-CoV-2 variants (WT, Beta B.1.351, or Omicron BA.2.3) (2 × 10 6 PFU/mL, MOI = 0.01). e Immunofluorescence staining assay of SARS-CoV-2 replication in Vero E6 cells incubated with SARS-CoV-2 WT or Beta B.1.351 (2 × 10 6 PFU/mL, MOI = 0.01) and the indicated circular chimeras for 48 h. Scale bars represent 1 mm. f Immunofluorescence (IFA) was used to quantify the intracellular nucleocapsids of SARS-CoV-2 WT, Beta B1.351 or Omicron BA.2.3 (2 × 10 6 PFU/mL, MOI = 0.01) in Vero E6 cells after 48 h of the indicated treatment. The IC50 values of circSASO and circSASON are indicated in the curves. All the error bars indicate standard deviations ( n = 3). b , d , f P values were calculated via one-way ANOVA. c , d : P values were calculated via two-way ANOVA. *** P < 0.001

Article Snippet: SARS-CoV-2 Nucleocapsid Phosphoprotein Rabbit Polyclonal antibody was purchased from Proteintech Inc. (28769-1-AP, USA).

Techniques: Expressing, Infection, Immunofluorescence, Staining, Incubation

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting coronaviral inflammation: aptamer-based strategies for emerging threats

doi: 10.1038/s41392-025-02570-8

Figure Lengend Snippet: circSASON protects mice against Beta VOC infection. a Schematic illustration of the mouse infection experiment. The wild-type mice were intranasally inoculated with 10 4 FFU of the SARS-CoV-2 beta variant (B.1.351) and treated intranasally with 50 µg (2.5 mg/kg, body weight = 20 g) circSASON, circSASO or vehicle control (0.9% saline) at 2, 24 and 72 h post-infection. b Representative H&E-stained lung sections ( n = 3 mice per group) collected at 2 days post-infection (dpi) and 4 dpi. Scale bars, 100 μm. c Comparison of the pathology scores of lungs collected from different treatment groups. d Focus formation assay analysis of the viral burden in the lungs of mice ( n = 6 mice per group) using lung homogenates at 2 dpi. LOD: limit of detection. e , f qRT‒PCR and ELISA analysis of cytokines in the lung tissue and plasma of mice at 4 dpi ( n = 6 mice per group), respectively. g Representative images of N protein and NLRP3 immunohistochemical staining at 2 dpi and 4 dpi, respectively. Scale bars, 100 μm. h Quantitative evaluation of N protein and NLRP3 staining. i Representative western blot analysis of N protein and NLRP3 in lung homogenates ( n = 3 mice per group) at 4 dpi. All the error bars indicate standard deviations ( n = 3). All the P values were calculated via one-way ANOVA. * P < 0.05; *** P < 0.001

Article Snippet: SARS-CoV-2 Nucleocapsid Phosphoprotein Rabbit Polyclonal antibody was purchased from Proteintech Inc. (28769-1-AP, USA).

Techniques: Infection, Variant Assay, Control, Saline, Staining, Comparison, Tube Formation Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Immunohistochemical staining, Western Blot