Materials

His-tagged Nucleocapsid recombinant protein of SARS-CoV-2(WT) was obtained from ACRO Biosystems Inc. (NUN-C5227, USA). His-tagged Spike trimer recombinant protein of SARS-CoV-2 was purchased from Novoprotein Scientific Inc. (DRA49, Shanghai, China). SARS-CoV-2 Spike (WT) Fluc-GFP Pseudovirus was purchased from ACRO Biosystems Inc. (PSSW-HLGB001, USA). SARS-CoV-2 Nucleocapsid Phosphoprotein Rabbit Polyclonal antibody was purchased from Proteintech Inc. (28769-1-AP, USA). SARS-CoV/SARS-CoV-2 Nucleocapsid Mouse Polyclonal antibody (40143-MM05), SARS-CoV-2 Nucleocapsid NTD protein (40588-V07E10), SARS-CoV-2 Nucleocapsid CTD protein (40588-V07E5) were purchased from Sino Biological Inc. (Beijing, China). NLRP3 protein (ab165022) was purchased from abcam (Cambridge, UK). Anti-NLRP3 rabbit polyclonal antibody (D120143), Anti-ASC rabbit polyclonal antibody (D154049) was purchased from Sangon Biotech (China). HRP-conjugated Beta Actin Monoclonal antibody was purchased from Proteintech Inc. (HRP60008, USA). His-tag protein pure Ni-Beads (Ni-Beads) were purchased from BioMag Beads (BMNI-5, Wuxi, China). Polyinosinic-polycytidylic acid (Poly(I:C)) and Resatorvid were obtained from MedChemExpress (HY-107202/HY-11109, Shanghai, China). Dip and Read^TM Biosensors streptavidin (SA) were purchased from ForteBio (California, USA). All media for cell culture were purchased from Gibco (USA). Fetal bovine serum (FBS) was purchased from Excell Bio (Shanghai, China) and penicillin-streptomycin was purchased from Hyclone (USA). All types of DNA sequences with HPLC purification were synthesized by Sangon Biotech (Shanghai, China).

Cell culture

The hACE2-HEK293T cells were gifts from Prof. Zhao Jincun at the Guangzhou Institute of Respiratory Disease. Calu-3 cells (CL-0054) were obtained from Procell Life Science& Technology Co., Ltd (ATCC, HTB-55 BCRJ, 0264).hACE2-HEK293T cells were cultured in DMEM medium (Gibco) supplemented with 100 U/mL Penicillin-Streptomycin, 2 μg/mL Puromycin and 10% (v/v) fetal bovine serum (ExCell Bio), in a humidified atmosphere containing 5% CO₂ incubator at 37 °C. Calu-3 cells were cultured in MEM medium (Gibco) supplemented with 100 U/mL Penicillin-Streptomycin and 10% (v/v) fetal bovine serum (ExCell Bio), in a humidified atmosphere containing 5% CO₂ incubator at 37 °C. Cells were negative for mycoplasma.

Micro-well SELEX procedures

His-tagged full-length SARS-CoV-2 Nucleocapsid protein was used as the bait and His-tag protein was used as the negative control. A synthetic ssDNA library (5′-TGCGTGTGTAGTGTGTCTG-[N]₄₀-CTCTTAGGGATTTGGGCGG-3′ (N = A, T, G, or C)) consisting of 40 nt random core sequence and constant 19 nt arm sequences at both ends were used for target-based SELEX. Briefly, the synthetic ssDNA library was incubated with His-tagged Nucleocapsid proteins conjugated to LOCKWELL C8 MAXISORP (Microplate, 446469, Thermo Fisher Scientific, USA) for SELEX enrichment. This Microplate conjugated His-tagged nucleocapsid proteins was incubated with the library at 25 °C for 30 min in the binding buffer contained 1×PBS buffer with magnesium ions (10 mM Na₂HPO₄, 2 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, 0.55 mM MgCl₂, pH = 7.4), and then separated and washed twice with the washing buffer (PBST: the binding buffer with 0.05% Tween 20). Subsequently, the bound oligonucleotides were eluted by Milli-Q H₂O at 80 °C for 7 min with mild shaking. The selected ssDNA was amplified by PCR. To convert dsDNA to ssDNA, the Lambda exonuclease enzyme was applied to separate and remove the 5′-phosphorylated antisense sequences from the sense sequences. Then, the ssDNA purified using NucleoSpin® Extract II kit was subjected for the next round of SELEX. After 6 rounds, the enriched ssDNA sequences were subjected to counter-selection with the total protein in human lung epithelial cells (A549). After 12 rounds of selection, the enriched aptamer pools were assessed by high-throughput sequencing (Sangon Biotech, Shanghai, China), and the most abundant ssDNA were selected as candidate N protein aptamers.

Enzyme linked immunosorbent assay (ELISA)

Microplates were pre-coated with all kinds of Nucleocapsid proteins (8 nM) in 100 μL coating buffer (pH = 9.6, 100 mM NaHCO₃) at 4 °C overnight. After washing two times, 200 μL blocking solution (2% BSA in PBST) was added at room temperature (RT) for 1 h with mild shaking. After blocking, serial dilutions of biotin-labeled aptamers at the 5′-end were added into microplates and incubated at RT for 30 min. Meanwhile, the blocking solution and random sequences were used as baseline control, respectively. Microplates were washed twice to remove free aptamers. Next, streptavidin-horseradish peroxidase (strep-HRP, 1:2000, Solarbio, SE068) was sequentially added into the reactions. After washing three times, the 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate was added to the microplates (100 μL per well) and incubated for 15 min at RT. The reaction was stopped by the addition of 2 M H₂SO₄ (50 μL per well) and the absorbance was measured at 450 nm using a microplate reader.

Flow cytometry analysis

To evaluate the binding performance of selected aptamers, His-tagged SARS-CoV-2 Nucleocapsid proteins (40 nM) coated in Ni-beads were incubated with 200 nM cy3-labeled aptamers in 100 μL binding buffer at RT for 30 min. Meanwhile, Ni-beads incubated with cy3-labeled random sequences were used as the negative control. The beads were washed twice using washing buffer and suspended in 1 mL binding buffer. The fluorescence intensity of beads with counting about 5000 events was measured by flow cytometry2 (FACSVerse, BD). Meanwhile, the data was analyzed using FlowJo (V10, BD). Cells were harvested 48 h after transfection and resuspended in 1 × PBS with 4% FBS and kept on ice before flow cytometry analysis. Flow cytometry data was collected using BD FACScalibur (BD, USA) and analyzed in two channels: green (excitation with 488 nm, emission with 525 ± 25 nm) or red (excitation with 561 nm, excitation with 610 ± 10 nm). The data is processed and analyzed in the FlowJo software.

Fluorescence microscope imaging

Ni-beads with His-tagged SARS-CoV-2 Nucleocapsid protein (5 μL beads and about 2 μg Nucleocapsid protein) or negative Ni-beads with His-tagged protein were incubated with 200 nM FAM-labeled aptamers in 100 μL binding buffer at RT for 30 min. After washing two times using washing buffer, the beads were suspended in 100 μL binding buffer and the fluorescence was monitored by fluorescence microscope (Leica DMi 8, USA). The fluorescence intensity was quantified by Image J software (2021, USA).

Bio-layer interferometry (BLI)

BLI was carried out according to our previous research.⁴¹ Briefly, the sensors were immersed into 96-well added 200 μL PBST/Mg²⁺ (0.55 mM) and shaking (1000 rpm) for 90 s (baseline phase). For loading purposes, streptavidin biosensors were used to capture biotinylated DNA (the thickness signal was 0.6 nm) in PBST/Mg with shaking for 10 min (loading phase). After washing in the PBST/Mg²⁺ for 60 s (second baseline phase), the loaded sensors were immersed into a serial dilution of S proteins with shaking for 3 min (association phase). Then, the sensors were immersed into PBST/Mg²⁺ for an additional 3 min (disassociation phase). Binding affinity kinetic features using ForteBio Octet K2 (Pall, USA).

Molecular docking and dynamic simulations

The structures of N proteins from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-229E, and NLRP3 were retrieved from the RCSB Protein Data Bank (http://www.rcsb.org). Molecular docking was carried out according to our previous research.^26,41 Briefly, Molecular docking was performed with HDOCK after obtaining the structures of aptamers and their target. The complex structure between the protein and aptamer was predicted using a hybrid protein-DNA docking algorithm, HDOCK. HDOCK used the fast Fourier transform (FFT)-based search strategy to globally sample all possible binding modes between the two molecules. All the sampled binding modes were evaluated by the iterative knowledge-based scoring function ITScorePP. Ultimately the binding modes were ranked according to their binding energy scores, and the top binding modes were provided. During the docking calculation, all the default parameters were used. The binding residues analysis was performed by the HDOCK webserver.

Co-IP

A549 cells were transfected with N-Flag plasmid and NApt8 or NApt8-3 for 48 h, meanwhile, cells were stimulated with LPS. After processing, cells were harvested and resuspended in 1 × PBS with 0.1% NP-40. Cells were centrifuged at 12,000 × g after ultrasonic disruption and harvested the supernatant. Flag-beads were added to the supernatant for mild shake overnight. The Flag-beads were used for SDS-PAGE after washed three times by 1 × PBS with 0.1% NP-40. Then, the gels were for silver staining analysis, which differential bands will be analyzed by Mass Spectrometry.

Western blot analysis

The hACE2-HEK293T cells and Calu-3 cells were washed twice with PBS and dissolved in lysis buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 1% Triton X-100, 5 mM EDTA, 100 mM phenylmethylsulfonyl fluoride (PMSF) and 10% glycerol) after transfection using N with or without aptamer and chimera. Protein concentration was measured by Bicinchoninic Acid assay (BCA). Proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto PVDF membranes (Bio-Rad) and incubated overnight at 4 °C with corresponding antibodies: anti-N (1:2000), anti-NLRP3(1:2000), anti-ASC (1:1000), anti-GAPDH (1:20000), anti-ACTB-HRP (1:10000). A HRP conjugated Goat anti-mouse IgG or Goat anti-rabbit IgG secondary antibody (1:10000) was used for the detection of primary antibody. The protein signals were detected using ECL chemiluminescent substrate (FOREGENE, China).

Confocal microscope of hACE2-HEK293T cells treated with SARS-CoV-2 pseudovirus

Cy3-labeled DNA (200 nM) was incubated with SARS-CoV-2 pseudovirus (low: 1 × 10⁴ TCID₅₀/mL, high: 1 × 10⁵ TCID₅₀/mL, 1 TCID₅₀ ≈ 0.7 PFU) in binding buffer at RT for 10 min. Then, 1 × 10⁵ HEK293T-ACE2 cells were incubated with mixtures of SARS-CoV-2 pseudovirus and DNA at 37 °C for 48 h. After incubating with Hoechst 33342 for 15 min, hACE2-HEK293T cells were observed to image with a Zeiss CELL Observer SD confocal microscope with a 60 × oil objective. The excitation wavelengths were 488 nm (Green Channel) and 561 nm (Red Channel). Exposure times: 200–400 ms. Acquired images were analyzed by the ZEN (blue edition).

Assessment of inflammatory responses of cells

Calu-3 or A549 cells were co-transfected with pan-coronavirus N plasmids and NApt8-3 or NASO2 with Lipofectamine 2000 following the manufacturer’s protocol. After 48 h, the cell was harvested. Subsequently, Trizol was used to extract RNA. qRT-PCR was used to quantitate the mRNA levels of inflammatory cytokines, including IL-6, TNFa, IFN-g.

The SARS-CoV-2 strains used in this analysis were isolated from COVID-19 patients in Guangdong, China, including wild type (SARS-CoV-2/human/CHN/IQTC-01/2020, NCBI, accession number: MT123290), Beta (B.1.351), Omicron (BA.2.3). Authentic SARS-CoV-2 virus (2 × 10⁶ PFU/mL, MOI = 0.01) were mixed with circular chimera in binding buffer at RT for 10 min. Then, 5 × 10⁵ of Calu-3 cells were incubated for 48 h. After incubating, cells were harvested to detect the inflammatory cytokines. All authentic SARS-CoV-2 infection experiments were performed in Guangzhou Gustoms District Technology Center Biosafety Level 3 (BSL-3) Laboratory.

Agarose gel electrophoresis analysis of circular chimera stability

Three μg chimera/circular chimera was added to the binding buffer containing 10% FBS and incubated at 37 °C for different times (0, 6, 12, 24, 36, 48 and 72 h). Then, the SN/circSN, SASON/circSASON and Random sequences were loaded onto 2% agarose gel in 1×TAE buffer and run at 120 V for 30 min. Gels were incubated in Ethidium bromide (1 μg/ml) for 5 min and bands were imaged using Gel Doc XR + Gel imaging system (Bio-rad, Hercules, California, USA) with ultraviolet excitation (302 nm). The fluorescence intensity was quantified by Image J software (2022, USA).

Immunofluorescence assay (IFA)

A series of circSASON and control oligonucleotides (SApt, RS etc) were incubated with authentic SARS-CoV-2 virus (2 × 10⁶ PFU/mL, MOI = 0.01) at 37 °C for 1 h. Then, 1 × 10⁵ Vero E6 cells were incubated with mixtures of SARS-CoV-2 virus and oligonucleotides at 37 °C for 48 h or 72 h. For immunofluorescence assay, Vero E6 cells were incubated in Paraformaldehyde at 4 °C overnight for permeabilization. The expression of nucleocapsid protein from the SARS-CoV2 virus was detected by using N protein primary antibody for 1 h at 37 °C. After washing with PBST, wells were incubated with secondary antibody for 1 h at room temperature. Meanwhile, the cell nucleus was stained with DAPI. Images were acquired with the fluorescence microscope and analyzed with Image J software.

Animal model

Female BALB/c mice (4–5 weeks old) were purchased from Guangzhou GemPharmatech Co. Ltd or Chengdu GemPharmatech Co. Ltd. All the mice were kept in sterile, autoclaved cages and provided with enough food and water. All animal experiments were undertaken at Sichuan University and Guangzhou Medical University. All protocols were approved by the Institutional Animal Care and Use Committees of the Sichuan University and of the Guangzhou Medical University.

Female BALB/c mice (4–5 weeks old) were purchased from Guangzhou GemPharmatech Co., Ltd. All animal experiments using SARS-Co-2 were undertaken at Guangzhou Medical University and approved by the local regulatory agency. SARS-CoV-2 Beta B.1.351 (2000 FFU) were mixed with saline for 1 h. Then, mice were administrated intranasally with the mixture. At 2 h, 24 h and 72 h after viral infection, circSASON or circSASO (2.5 mg/Kg body weight) were administrated intranasally. Lung and blood samples was obtained at 2 dpi and 4 dpi. Lungs were used for virus quantification using focus forming assay and used for pathological analysis using H.E. staining or Immunohistochemistry staining. Plasma was used for evaluation of inflammation using qRT-PCR and ELISA. All animal experiments were performed with a minimal of 3 mice per experimental condition.

Focus forming assay (FFA)

FFA was performed as previously described.³² Compared with the traditional plaque assay, FFA provides higher throughput. Vero E6 cells were seeded in 96-well plates one day prior to infection. Serially diluted virus stocks or lung homogenate were added to the Vero E6 cells and inoculated at 37 °C for 1 h. After incubation, the inocula were removed and 125 μL of pre-warmed 1.6% carboxymethylcellulose was added to each well. At the indicated time points post-infection, cells were harvested, and foci were visualized using a peroxidase substrate and counted under a microscope. Viral titers were calculated as focus-forming units (FFU) per milliliter.

Histology and IHC

Lung tissues were harvested and immersion-fixed in 4% paraformaldehyde overnight. Paraffin sections were prepared and stained. Antibodies targeting the SARS-CoV-2 nucleoprotein (N) (Sino Scientific, no. 40143-R019, 1:1000) and NLRP3 (Adipogen, no. AG-20B-0014-C100, 1:200) were used. Pathology evaluation were performed as described in the previous research,^42,43 injuries such as bleeding and edema of the entire left lung tissue can be observed by H&E staining under low microscope magnification (10×), the evaluation method is to observe injury distribution from 6 independent visual fields and score them following criteria (injury area/total area):

0 points: no damage;

• 1 point: mild injury,

• 2 points: moderate injury, increased inflammation with ~25–50% of the total lung involved;

• 3 points: severe inflammation involving 50–75% of the lung;

• 4 points: almost all lung tissue contained inflammatory infiltrates.

Quantitative RT-PCR (qRT-PCR) analysis

Total RNA was isolated from the stimulated cells by RNAiso Plus (108-95-2, TaKaRa, Japan). Then, cDNA was prepared using DNase I (01056834, Thermo Scientific, USA) and RevertAid Reverse Transcriptase (00991337, Thermo Scientific, USA). qRT-PCR was performed using the Applied Biosystems 7500 Real-Time PCR Systems (Thermo Fisher Scientific, USA) with 2×Real PCR EasyTM Mix-SYBR (210520, Foregene, China). ACTB was used as a reference gene. The quantitative analysis of qRT-PCR to evaluate the inhibitory efficiency of aptamer on cytokine gene expression were analyzed by the Livak method (2^-ΔΔCt), described as following steps:

1. 1.

   \(\Delta \mathrm{Ct\; calculation}:\Delta \mathrm{Ct}=\mathrm{Ct}(\mathrm{mRNA})-\mathrm{Ct}(\mathrm{actin})\)

2. 2.

   \(\Delta \Delta \mathrm{Ct\; calculation}:\Delta \Delta \mathrm{Ct}=\Delta \mathrm{Ct}(\mathrm{sample})-\Delta \mathrm{Ct}(\mathrm{blank})\)

3. 3.

   \(\mathrm{Fold\; change}:\mathrm{Fold}={2}^{(-\Delta \Delta \mathrm{Ct})}\)

4. 4.

   \(\mathrm{Relative}\,\mathrm{inhibition}({\rm{ \% }})=100-(\mathrm{Fold}(\mathrm{sample})/\mathrm{Fold}({\rm{N}}))\times 100\)

Bliss independence analysis

To calculate Bliss independence for evaluating drug synergy, you compare the expected combined effect of two drugs (assuming independence) with the observed effect of their combination. Bliss Independence Formula: E_A = effect of drug A alone. E_B = effect of drug B alone. E_AB = observed effect of the combination. The expected effect under Bliss independence is: E_Bliss= E_A + E_B − E_A⋅E_B. If observed E_AB>E_Bliss, it is considered that the combination of A and B exerts a synergistic effect.

Statistical analysis

All analyses were repeated at least three times, and a representative experimental result was presented. Data were analyzed using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA). Continuous variables with normal distribution are expressed as the mean ± standard deviation (SD). Comparisons between groups were all verified for normal distribution by D’Agostino-Pearson omnibus test. Student’s t test (for pairwise comparisons) and one-way ANOVA (for comparisons among three or more groups) were used. The post hoc test with Bonferroni correction was performed for multiple comparisons following ANOVA.