Journal: Nucleic Acids Research

Article Title: A general strategy to enhance aptamer affinity by suppressing dissociation through symmetric assembly

doi: 10.1093/nar/gkag268

Figure Lengend Snippet: Symmetric aptamer trimerization enhances binding affinity. ( A ) Schematic illustration of aptamer assembly. The 3′ end of monomeric aptamer MSA52t was linked to the trident linker (trebler phosphoramidite) with a 15-thymidine spacer (T15) and assembled into dimeric (shDMSA52t) or trimeric (shTMSA52t) constructs. This design provides flexible orientation and optimal target accessibility for each aptamer unit. ( B ) Dot-blot binding assays showing the affinities of MSA52t, shDMSA52t, and shTMSA52t toward the monomeric wild-type S-protein (mS-protein). Apparent dissociation constants ( K d ) were determined by nonlinear curve fitting. Trimeric assembly resulted in a ∼344-fold affinity enhancement over the monomer.

Article Snippet: The wild-type SARS-CoV-2 spike protein subunit S1 (mS-protein, Cat. No. 40591-V08B1) and Troponin I (Cat. No. 501-TNNI0010) were purchased from Sino Biological Inc. VEGF 165 protein (his-tagged, Cat. No. VE5-H5248) was obtained from Acro Biosystems.

Techniques: Binding Assay, Construct, Dot Blot

Journal: Nucleic Acids Research

Article Title: A general strategy to enhance aptamer affinity by suppressing dissociation through symmetric assembly

doi: 10.1093/nar/gkag268

Figure Lengend Snippet: Trimeric assembly enhances affinity primarily by reducing dissociation rate. Binding kinetics of ( A ) MSA52t, ( B ) shDMSA52t, and ( C ) shTMSA52t interacting with the wild-type SARS-CoV-2 S1-protein (mS-protein) were measured by BLI. Concentration-dependent sensorgrams were globally fitted using 1:1 binding model to obtain the kinetic parameters k on , k off , and K d . The experimental curves are shown as solid lines and the fitted curves are shown as black dashed lines. Bar graphs comparing ( D ) k on , ( E ) k off , and ( F ) K d values for each aptamer. While only modest changes in k on were observed, trimerization led to a dramatic reduction in k off , resulting in a 348-fold improvement in K d for shTMSA52t compared to the monomer.

Article Snippet: The wild-type SARS-CoV-2 spike protein subunit S1 (mS-protein, Cat. No. 40591-V08B1) and Troponin I (Cat. No. 501-TNNI0010) were purchased from Sino Biological Inc. VEGF 165 protein (his-tagged, Cat. No. VE5-H5248) was obtained from Acro Biosystems.

Techniques: Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: A macrocyclic peptide-based fusion inhibitor targeting SARS-CoV-2 Spike S2 subunit

doi: 10.64898/2026.03.04.703990

Figure Lengend Snippet: (A) Binding of PA-001 to recombinant SARS-CoV-2 Spike protein of the full length (S1+S2), S1, or S2 regions. Each recombinant Spike protein was immobilized and incubated with HA-tagged PA-001 at various concentrations up to 100 nM, followed by washing out and detection with horseradish peroxidase (HRP)-conjugated anti-HA antibody. (B) Schematic model for the SARS-CoV-2 entry process, in which viral attachment to cells via Spike-ACE2 binding is followed by the fusion of viral envelope with cellular membrane either on the cell surface or in the endosome/lysosome. (C) (a) Virus-cell attachment assay. VeroE6/TMPRSS2 cells were exposed to SARS-CoV-2 at an MOI of 0.01 at 4°C for 15 min in the presence or absence of casirivimab (CAS) + imdevimab (IMD) (50, 500, or 5,000 ng/mL), mefloquine (MFQ) (0.4, 2, or 10 μM), or PA-001 (40, 200, or 1,000 nM), and were then washed out to quantify cell-bound viral RNA by real-time RT-PCR. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons with the DMSO control was performed (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001). (b) Cell-cell fusion assay. Spike-overexpressing HEK293T cells carrying the LgBiT gene were co-cultured with ACE2-expressing VeroE6/TMPRSS2 cells carrying the HiBiT gene upon treatment with or without varying concentrations (up to 200 nM) of PA-001 for 1 h to detect luciferase activity to evaluate fusion activity mediated by Spike and ACE2. IC 50 of PA-001 is also indicated. (D) Induction of PA-001-resistant SARS-CoV-2 variants. (a) Schematic representation of the infection assay for inducing PA-001-resistant SARS-CoV-2 clones. SARS-CoV-2-infected VeroE6/TMPRSS2 cells were continuously cultured with PA-001 at 1,000 nM for 24-48 h (Passage 0). The resultant culture supernatants were collected and diluted to 10-fold, which were then inoculated to naive VeroE6/TMPRSS2 cells in the presence of 1,000 nM PA-001 and cultured for 24 h (Passage 1). The culture supernatants were repeatedly collected and then inoculated to the cells upon 1,000 nM PA-001 for the next passage. At passage 7, the culture supernatants were extracted from eight independent cultures (A8 – H8). (b) PA-001 sensitivity of the collected supernatant clones. VeroE6/TMPRSS2 cells were inoculated with these collected supernatants or the parental virus (WT) upon treatment with varying concentrations of PA-001 (left) or RDV as a reference (right) to determine virus RNA levels in the culture supernatants as shown in . (E) Amino acid sequence of the PA-001-resistant clones. (a) Upper, schematic structure for the SARS-CoV-2 Spike protein. Lower, amino acid sequence of the clones, A8–H8, as well as the parental virus (WT). The substituted amino acids are highlighted by red. The amino acid numbers are indicated. (b) Structure of Spike trimer in the pre-fusion (left, PDB: 6XR8), intermediation (center, PDB: 8Z7P), and post-fusion (right, PDB: 8FDW) forms. Red color indicates the mutated amino acids shown in (a). Blue color indicates FP. (F) Binding capacity of recombinant SARS-CoV-2 Spike carrying the L841R and P862Q substitutions to PA-001. (a) Each recombinant Spike (WT, L841R, and P862Q) was immobilized and incubated with HA-tagged PA-001 (PA-001-HA) at varying concentrations in the presence (gray, competition) or absence (red, S-PA001 binding) of non-tagged PA-001 at 10 μM. After washing out, bound HA-PA-001 was visualized with HRP-conjugated anti-HA antibody. Samples without Spike-immobilization were also prepared to define the background signal (green). (b) Binding signals with Spike WT (left), Spike L841R (center), and Spike P862Q (right) are plotted against the concentrations of treated PA-001-HA. Ordinary two-way ANOVA followed by tukey’s multiple comparisons was performed (*: p<0.05, **: p<0.01, ***: p<0.001). (G) Cell fusion assay performed as described in using the cells overexpressing either Spike WT, L841R, or P862Q co-cultured with ACE2-expressing cells upon PA-001 treatment at various concentrations.

Article Snippet: Binding of HA-tagged PA-001 to SARS-CoV-2 Spike proteins was quantified by ELISA as follows: Recombinant wild type (from Sino biological, 40589-V08B1, 40591-V08B1, and 40590-V08B) or mutated Spike proteins were immobilized on the plates, and then incubated with PA-001-HA at various concentrations together with or without excess amount of non-tagged PA-001, used as a binding competitor.

Techniques: Binding Assay, Recombinant, Incubation, Membrane, Virus, Cell Attachment Assay, Quantitative RT-PCR, Control, Cell-Cell Fusion Assay, Cell Culture, Expressing, Luciferase, Activity Assay, Infection, Clone Assay, Sequencing, Cell Fusion Assay

Journal: Frontiers in Immunology

Article Title: Dual role of ACE2 in regulating inflammation triggered by Omicron S1 and other SARS-CoV-2 Spike variants

doi: 10.3389/fimmu.2025.1667880

Figure Lengend Snippet: Omicron S1 shows reduced immune cells recruitment and expansion compared with the ancestral variant. Recombinant S1WT, S1 Omicron or vehicle (-) were injected in the hindbrain ventricle (HBV) of Tg(lyz:DsRED) (A) , Tg(mfap4:Tomato) (B) , Tg(nfkb:eGFP) (C) of 2-dpf larvae. Neutrophil (A) and macrophage (B) recruitment and number and Nfkb reporter activity (C) were analyzed at 6, 12, and/or 24 hpi by fluorescence microscopy. Representative photos for each treatment are shown from 24 hpi. Scale bar 500 μm. Each dot represents one individual, and the means ± SEM for each group is also shown. P values were calculated using one-way analysis of variance (ANOVA) and Tukey multiple range test. ns, not significant; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** p < 0.0001. auf, arbitrary units of fluorescence.

Article Snippet: Recombinant His-tagged Spike S1 wild-type (#40591-V08B1), S1 Omicron (#40592-V08H121) or Spike S1/S2 TRIMER wild-type (#40589-V08H8), all from Sino Biological at a concentration of 0.25 mg/ml supplemented with phenol red were injected into the hindbrain (1 nl) of 48 hpf zebrafish larvae.

Techniques: Variant Assay, Recombinant, Injection, Activity Assay, Fluorescence, Microscopy

Journal: Frontiers in Immunology

Article Title: Dual role of ACE2 in regulating inflammation triggered by Omicron S1 and other SARS-CoV-2 Spike variants

doi: 10.3389/fimmu.2025.1667880

Figure Lengend Snippet: Omicron is more proinflammatory than the ancestral variant. Recombinant S1WT, S1 Omicron or vehicle (-) were injected in the hindbrain ventricle (HBV) of WT (A–F) 2-dpf larvae. The transcript levels of the indicated genes (A–E) were analyzed at 12 hpi by RT-qPCR in larval head and rest of the body and caspase-1 activity was determined at 24 hpi using a fluorogenic substrate (F) . Graphs shown are representative of three independent experiments; technical replicates are displayed in each graph. The means ± SEM for each group is shown. P values were calculated using one-way analysis of variance (ANOVA) and Tukey multiple range test. n=45 in (A–E) , n=35 in (F) . ns, not significant; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** p < 0.0001. auf, arbitrary units of fluorescence.

Article Snippet: Recombinant His-tagged Spike S1 wild-type (#40591-V08B1), S1 Omicron (#40592-V08H121) or Spike S1/S2 TRIMER wild-type (#40589-V08H8), all from Sino Biological at a concentration of 0.25 mg/ml supplemented with phenol red were injected into the hindbrain (1 nl) of 48 hpf zebrafish larvae.

Techniques: Variant Assay, Recombinant, Injection, Quantitative RT-PCR, Activity Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: Dual role of ACE2 in regulating inflammation triggered by Omicron S1 and other SARS-CoV-2 Spike variants

doi: 10.3389/fimmu.2025.1667880

Figure Lengend Snippet: Omicron causes higher neutrophil cell death than the ancestral variant. Recombinant S1WT, S1 Omicron or vehicle (-) were injected in the hindbrain ventricle (HBV) of Tg(lyz:DsRED) (A, B) of 2-dpf larvae. Tunel positive neutrophil number (double positive) was counted at 6 hpi in the head and tail of the larvae (A, B) . Representative photos for each treatment are shown. Scale bar 500 μm. Each dot represents one individual, and the means ± SEM for each group is also shown. P values were calculated using one-way analysis of variance (ANOVA) and Tukey multiple range test. ns, not significant; * P ≤ 0.05, ** P ≤ 0.01 and **** p < 0.0001.

Article Snippet: Recombinant His-tagged Spike S1 wild-type (#40591-V08B1), S1 Omicron (#40592-V08H121) or Spike S1/S2 TRIMER wild-type (#40589-V08H8), all from Sino Biological at a concentration of 0.25 mg/ml supplemented with phenol red were injected into the hindbrain (1 nl) of 48 hpf zebrafish larvae.

Techniques: Variant Assay, Recombinant, Injection, TUNEL Assay

Journal: Frontiers in Immunology

Article Title: Dual role of ACE2 in regulating inflammation triggered by Omicron S1 and other SARS-CoV-2 Spike variants

doi: 10.3389/fimmu.2025.1667880

Figure Lengend Snippet: Trimeric ancestral variant induces a weaker immune response than its monomeric form. Recombinant S1WT (monomeric), S1/S2WT-T (trimeric) or vehicle (-) were injected in the hindbrain ventricle (HBV) of Tg(lyz:DsRED) (A) , Tg(mfap4:Tomato) (B) , Tg( nfkb :eGFP) (C) 2-day postfertilization larvae (dpf). Neutrophil (A) and macrophage (B) recruitment and number and Nfkb reporter activity (C) were analyzed at 6, 12, and/or 24 hpi by fluorescence microscopy. Representative photos for each treatment are shown from 24 hpi. Scale bar 500 μm. Each dot represents one individual, and the means ± SEM for each group is also shown. P values were calculated using one-way analysis of variance (ANOVA) and Tukey multiple range test. ns, not significant; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** p < 0.0001. auf, arbitrary units of fluorescence.

Article Snippet: Recombinant His-tagged Spike S1 wild-type (#40591-V08B1), S1 Omicron (#40592-V08H121) or Spike S1/S2 TRIMER wild-type (#40589-V08H8), all from Sino Biological at a concentration of 0.25 mg/ml supplemented with phenol red were injected into the hindbrain (1 nl) of 48 hpf zebrafish larvae.

Techniques: Variant Assay, Recombinant, Injection, Activity Assay, Fluorescence, Microscopy

Journal: Frontiers in Immunology

Article Title: Dual role of ACE2 in regulating inflammation triggered by Omicron S1 and other SARS-CoV-2 Spike variants

doi: 10.3389/fimmu.2025.1667880

Figure Lengend Snippet: Trimeric ancestral variant induces a weaker proinflammatory response than its monomeric form. Recombinant S1WT, S1/S2WT-T or vehicle (-) were injected in the hindbrain ventricle (HBV) of WT (A–E) 2-day postfertilization larvae (dpf). The transcript levels of the indicated genes (A–D) were analyzed at 12 hpi by RT-qPCR in larval head and rest of the body and caspase-1 activity was determined at 24 hpi using a fluorogenic substrate (E) . Graphs shown are representative of three independent experiments; technical replicates are displayed in each graph. The means ± SEM for each group is shown. P values were calculated using one-way analysis of variance (ANOVA) and Tukey multiple range test. n=40 in A-D, n=35 in E. ns, not significant; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** p < 0.0001. auf, arbitrary units of fluorescence.

Article Snippet: Recombinant His-tagged Spike S1 wild-type (#40591-V08B1), S1 Omicron (#40592-V08H121) or Spike S1/S2 TRIMER wild-type (#40589-V08H8), all from Sino Biological at a concentration of 0.25 mg/ml supplemented with phenol red were injected into the hindbrain (1 nl) of 48 hpf zebrafish larvae.

Techniques: Variant Assay, Recombinant, Injection, Quantitative RT-PCR, Activity Assay, Fluorescence