Journal: Theranostics

Article Title: DNA aptamers inhibit SARS-CoV-2 spike-protein binding to hACE2 by an RBD- independent or dependent approach

doi: 10.7150/thno.74428

Figure Lengend Snippet: Selection and characterization of anti-S2 DNA aptamers. (A) SELEX scheme for the selection of aptamers against the S2-domain of the WT spike-protein. (B) Enrichment of the binding capability of the ssDNA library over various selection rounds. The fluorescence emission was measured with a Clariostar microplate reader at λ max = 520 nm. (C) Fluorescence imaging of S2-protein/Ni-NTA resin beads in the presence of the 6-FAM-labeled library was obtained from the 10 th , 11 th , and 15 th rounds (encircled red dots). This shows that the S2-domain-specific pools are satisfactorily enriched after the 15 th round of selection. The complex of the control protein and Ni-NTA resin beads does not produce a fluorescence signal in the presence of the 6-FAM-labeled library obtained from the 15 th round of selection. Both the transmitted light (top panel) and green fluorescence channel (bottom panel) images were collected using a digital inverted fluorescence microscope (Invitrogen EVOS FL). The scale bar represents 200 µm. (D) Binding affinities ( K d ) of the original and truncated anti-S2 aptamers. (E) Secondary structures of anti-S2 aptamers were obtained using NUPACK. (F) Nucleotide sequences of the anti-S2 aptamers. The aptamers S2A1 and S2A2 are originally obtained from the SELEX, while S2A1C1, S2A2C1, and S2A2C3 are the optimized truncated aptamers obtained from the S2A1 and S2A2 aptamers respectively by the deletion of redundant nucleotides. The nucleotides removed in the optimized aptamers are represented by dotted lines.

Article Snippet: For the first round of the selection, the 100 pmol of the S2-protein and 1 μL of nickel nitrilotriacetic acid (Ni-NTA) beads (G-bioscience) were diluted into 100 μL of SELEX buffer (PBST-Mg buffer, PBS with 1 mM MgCl 2 , pH 7.4, 0.01 % tween) and incubated at room temperature (RT), rotating for 1 h. Meanwhile, 3 nmol of DNA library was diluted into 100 μL of SELEX buffer and treated at 95 °C for 5 min, on ice (or 4 °C) for 5 min, RT for 5 min, and placed in ice.

Techniques: Selection, Binding Assay, Fluorescence, Imaging, Labeling, Control, Microscopy

Journal: Theranostics

Article Title: DNA aptamers inhibit SARS-CoV-2 spike-protein binding to hACE2 by an RBD- independent or dependent approach

doi: 10.7150/thno.74428

Figure Lengend Snippet: Determination of the binding affinities and inhibition efficacies of aptamers to the WT SARS-CoV-2 spike-protein. (A) Fluorescence imaging of Ni-NTA resin beads in the presence of the His-tagged WT spike-protein and FAM-labeled mono and fusion aptamers. The protein and 6-FAM-labeled aptamers are used in the same concentration of 100 nM. The rightmost image shows that Ni-NTA resin beads do not give a fluorescence signal in the presence of the unspecific control protein and S1B6C3-A5-S2A2C1, indicating the aptamer does not bind to the control protein. The scale bar represents 200 µm. (B) Binding affinities ( K d ) of mono and fusion aptamers against the His-tagged WT spike-protein. (C) Flow cytometry measurements of the green fluorescence emission from magnetic beads in the presence of the WT spike-protein and various aptamers. The WT spike-protein and 6-FAM-labeled aptamers are used in the same concentration of 500 nM. The library with random sequences showed the minimum, and S1B6C3-A5-S2A1C1 fusion aptamers showed the maximum binding potentials, respectively, to the WT spike-protein. (D) Color of the final product in ELISA tests corresponding to various aptamers: D1 = S1B6C3-A5-S2A2C1, D2 = S2A2C1, D3 = S1B6C3-A10-S2A2C1, D4 = S1B6C3-A15-S2A2C1, D5 = aptamer control, D6 = no aptamer, D7 = S2A2C1-T15-S1B6C3, D8 = S1B6C3, D9 = S1B6C3-T15-S2A2C1, D10 = S1B6C3-PEG-S2A2C1, D11 =TMB only, D12 = SELEX buffer. An intense yellow color was obtained due to HRP-mediated oxidation of the TMB when an unspecific (control) or no aptamer was added. (E) Normalized relative absorbances of the final ELISA products corresponding to neutralization efficacies of the various aptamers. The absorbance is inversely related to the neutralization efficacy of the aptamer.

Article Snippet: For the first round of the selection, the 100 pmol of the S2-protein and 1 μL of nickel nitrilotriacetic acid (Ni-NTA) beads (G-bioscience) were diluted into 100 μL of SELEX buffer (PBST-Mg buffer, PBS with 1 mM MgCl 2 , pH 7.4, 0.01 % tween) and incubated at room temperature (RT), rotating for 1 h. Meanwhile, 3 nmol of DNA library was diluted into 100 μL of SELEX buffer and treated at 95 °C for 5 min, on ice (or 4 °C) for 5 min, RT for 5 min, and placed in ice.

Techniques: Binding Assay, Inhibition, Fluorescence, Imaging, Labeling, Concentration Assay, Control, Flow Cytometry, Magnetic Beads, Enzyme-linked Immunosorbent Assay, Neutralization

Journal: Theranostics

Article Title: DNA aptamers inhibit SARS-CoV-2 spike-protein binding to hACE2 by an RBD- independent or dependent approach

doi: 10.7150/thno.74428

Figure Lengend Snippet: Binding affinities, inhibition efficacies, and stability of the mono and fusion aptamers. (A) Binding affinity ( K d ) of the aptamers with the Delta variant spike-protein. (B) Inhibition efficacies of aptamers or the control aptamer against Delta spike-protein by employing the competitive ELISA experiment. Relative normalized absorbances obtained from the colorful product in the presence of aptamer is inversely proportion to its inhibition efficacies. (C) Inhibition efficacies of the aptamers against the Omicron spike-protein. (D) The comparison of the binding affinities of aptamers toward the WT and Delta spike-protein reveals that the aptamers bind to both spike-proteins with comparable affinities. (E) The stability assessment of the S2A2C1 and S1B6C3-A5-S2A2C1 in 25 % FBS. The 4 ng/μL aptamer samples were prepared in nuclease free water and incubated at 37 °C for various time in the absence (-) or presence (+) of the 25 % FBS solution. the aptamer samples showed the noticeable digestion in 30 min and complete digestion within 3 h by the action of 25 % FBS. The stability of the aptamers in the biological environment maybe improved by the construction of nucleic acid mimics (NAM) aptamers, which comprise the chemical modifications on the heterocyclic base or sugar-phosphate backbone of native nucleic acids. (F-N) The determination of K d of the 6-FAM-labeled S1B6C3-A5-S2A2C1 aptamer by using the flow cytometry approach. The flow cytometry measurement of the aptamer bound to (F-H) WT spike-protein, (I-K) Delta spike-protein, and (L-N) Omicron spike-protein. Figures F, I, and L are the histograms obtained from the flow cytometry measurement to determine the mean green fluorescence emission intensity ( X C ) of 6-FAM-labeled S1B6C3-A5-S2A2C1 aptamer bound to the complex of Ni-NTA beads and the His-tagged spike-protein. Each histogram was constructed by the integration of 3 trials of experiments that contain a total of 15,000 flow events (5000 events from a trial). Figures G, J, and M show the increment of X C value as a function of 6-FAM aptamer concentration that bound to virtually the same quantity of spike-protein and Ni-NTA bead complex. The shorter error bars indicate the high reproducibility of X C values for various trials. Figures H, K, and N represent the K d of the 6-FAM labeled S1B6C3-A5-S2A2C1 aptamer against various spike-proteins. The K d values were determined by intensity vs concentration plot using the Origin software.

Article Snippet: For the first round of the selection, the 100 pmol of the S2-protein and 1 μL of nickel nitrilotriacetic acid (Ni-NTA) beads (G-bioscience) were diluted into 100 μL of SELEX buffer (PBST-Mg buffer, PBS with 1 mM MgCl 2 , pH 7.4, 0.01 % tween) and incubated at room temperature (RT), rotating for 1 h. Meanwhile, 3 nmol of DNA library was diluted into 100 μL of SELEX buffer and treated at 95 °C for 5 min, on ice (or 4 °C) for 5 min, RT for 5 min, and placed in ice.

Techniques: Binding Assay, Inhibition, Variant Assay, Control, Competitive ELISA, Comparison, Incubation, Labeling, Flow Cytometry, Fluorescence, Construct, Concentration Assay, Software