Structural basis and binding mechanism of NP14 interaction with the SARS-CoV-2 N protein. (IMAGE)
Caption
(A) Molecular simulation of the binding mode between aptamer NP14 and the SARS-CoV-2 N protein (http://www.rcsb.org, ID:6VYO) via AutoDock. (B) Enlarged view of the presumed binding area. (C) Nucleic acid sequences and corresponding amino acids involved in the docking model. (D) Secondary structure simulation of aptamer NP14 via the Nupack web server at 37 °C. (E) Secondary structure simulation of the truncated chains NP14a via the Nupack web server at 37 °C. (F) Secondary structure simulation of the truncated chains NP14b via the Nupack web server at 37 °C. (G) Binding analysis of NP14 with truncated NP14a, NP14b, and base-mutated 400 nM NP14a1, NP14a2, NP14a3, NP14a4, NP14b1, NP14b2, NP14b3, NP14b4, and NP14b5 to the SARS-CoV-2 N protein by ELONA. Data were presented as mean ± standard deviation of triplicate results (n = 3). The NP14 control: ns, not significant; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (H) Circular dichroism spectroscopy of AS1411 (20 μM) and NP14 (10 μM) was performed in PBS buffer (0.01 M, pH = 7.4) at wavelengths ranging from 220 to 320 nm. (I) Domain organization of the SARS-CoV-2 N protein, with numbers indicating domain boundaries. (J) Immunomagnetic beads (40 μL, 10 mg/mL) labeled with Flag antibodies against the truncated overexpressed protein were reacted with 300 nM biotin-labeled NP14 to assess binding. Data were presented as mean ± standard deviation of triplicate results (n = 3). Compared with the blank control: ∗∗∗∗p < 0.0001. (K) 250 nM biotin-labeled NP14 was mixed with 250 nM unlabeled N1, A58, A61 and competitive binding was analyzed by ELONA. Data were presented as mean ± standard deviation of four replicate results (n = 4). Compared with the NP14: ns, not significant; ∗∗∗p < 0.001. (L) Evaluation of the binding affinity for truncated proteins containing the NTD region at different concentrations of NP14 (0, 2, 5, 10, 20, 50, and 100 nM). Data were presented as mean ± standard deviation of triplicate results (n = 3).
Credit
Shu Zhou, Yuxi Xu, Huan Liao, Hailong Ou, Dan Qi, Yatao Wu, Yunyi Liu, Juan Li, Jiaxuan Li, Bi Shi, Fei Zhu, Siran Zhang, Jason H. Huang, Erxi Wu, Xiaoxiao Hu
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