Journal: Viruses

Article Title: Structural Characteristics of Heparin Binding to SARS-CoV-2 Spike Protein RBD of Omicron Sub-Lineages BA.2.12.1, BA.4 and BA.5

doi: 10.3390/v14122696

Figure Lengend Snippet: Phylogenetic tree and multiple sequence alignment. ( A ) Phylogenetic relationships of Nextstrain SARS-CoV-2 clades. The phylogenetic tree was adapted from figure provided by Nextstrain and CoVariants . VOCs are represented by colored nodes. ( B ) Mutation profile of S protein RBD of Omicron BA.2.12.1, BA.4/BA.5 compared with WT. Multiple sequence alignment was performed by Clustal Omega (1.2.4). An * (asterisk) indicates positions which have a single, fully conserved residue.

Article Snippet: S protein RBD of Omicron sub-lineages BA.2.12.1 (Cat: 40592-V08H132), BA.4/BA.5 (Cat: 40592-V08H130) were purchased from Sino Biological Inc. (Beijing, China).The proteins were constructed as follows: (1) a DNA sequence encoding the SARS-CoV-2 (BA.2.12.1) Spike RBD (YP_009724390.1, with mutations G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452Q, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H) (Arg319–Phe541) was expressed with a polyhistidine tag at the C-terminus; and (2) a DNA sequence encoding the SARS-CoV-2 (BA.4/BA.5) Spike RBD (YP_009724390.1, with mutations G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H) (Arg319–Phe541) was expressed with a polyhistidine tag at the C-terminus.

Techniques: Sequencing, Mutagenesis

Journal: Viruses

Article Title: Structural Characteristics of Heparin Binding to SARS-CoV-2 Spike Protein RBD of Omicron Sub-Lineages BA.2.12.1, BA.4 and BA.5

doi: 10.3390/v14122696

Figure Lengend Snippet: SPR sensorgrams of S protein RBD of BA.2.12.1 and BA.4/BA.5 binding with heparin. ( A ) SPR sensorgrams of S protein RBD of BA.2.12.1 binding with heparin. Concentrations of RBD (from top to bottom) are 1000, 500, 250, 125, and 63 nM, respectively. ( B ) SPR sensorgrams of S protein RBD of BA.4/BA.5 binding with heparin. Concentrations of RBD (from top to bottom) are 1000, 500, 250, 125, and 63 nM, respectively.

Article Snippet: S protein RBD of Omicron sub-lineages BA.2.12.1 (Cat: 40592-V08H132), BA.4/BA.5 (Cat: 40592-V08H130) were purchased from Sino Biological Inc. (Beijing, China).The proteins were constructed as follows: (1) a DNA sequence encoding the SARS-CoV-2 (BA.2.12.1) Spike RBD (YP_009724390.1, with mutations G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452Q, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H) (Arg319–Phe541) was expressed with a polyhistidine tag at the C-terminus; and (2) a DNA sequence encoding the SARS-CoV-2 (BA.4/BA.5) Spike RBD (YP_009724390.1, with mutations G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H) (Arg319–Phe541) was expressed with a polyhistidine tag at the C-terminus.

Techniques: Binding Assay

Journal: Viruses

Article Title: Structural Characteristics of Heparin Binding to SARS-CoV-2 Spike Protein RBD of Omicron Sub-Lineages BA.2.12.1, BA.4 and BA.5

doi: 10.3390/v14122696

Figure Lengend Snippet: S protein RBD (BA.2.12.1)–heparin interaction inhibited by heparin oligosaccharides/desulfated heparins using solution competition. ( A ) SPR sensorgrams of S protein RBD (BA.2.12.1)–heparin interaction competing with different heparin oligosaccharides. Concentration of S-protein RBD (BA.2.12.1) is 250 nM mixed with 1 µM of different heparin oligosaccharides. ( B ) Bar graphs (based on triplicate experiments with standard deviation) of normalized S-protein RBD (BA.2.12.1) binding preference to surface heparin by competing with different heparin oligosaccharides. ( C ) SPR sensorgrams of S protein RBD (BA.2.12.1)–heparin interaction competing with different desulfated heparins. Concentration of S-protein RBD (BA.2.12.1) is 250 nM mixed with 1 µM of different desulfated heparins. ( D ) Bar graphs (based on triplicate experiments with standard deviation) of normalized S-protein RBD (BA.2.12.1) binding preference to surface heparin by competing with different desulfated heparins. Statistical analysis was performed using unpaired two-tailed t -test (ns: p > 0.05 compared to the control, *: p ≤ 0.05 compared to the control, **: p ≤ 0.01 compared to the control, ***: p ≤ 0.001 compared to the control).

Article Snippet: S protein RBD of Omicron sub-lineages BA.2.12.1 (Cat: 40592-V08H132), BA.4/BA.5 (Cat: 40592-V08H130) were purchased from Sino Biological Inc. (Beijing, China).The proteins were constructed as follows: (1) a DNA sequence encoding the SARS-CoV-2 (BA.2.12.1) Spike RBD (YP_009724390.1, with mutations G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452Q, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H) (Arg319–Phe541) was expressed with a polyhistidine tag at the C-terminus; and (2) a DNA sequence encoding the SARS-CoV-2 (BA.4/BA.5) Spike RBD (YP_009724390.1, with mutations G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H) (Arg319–Phe541) was expressed with a polyhistidine tag at the C-terminus.

Techniques: Concentration Assay, Standard Deviation, Binding Assay, Two Tailed Test

Journal: Viruses

Article Title: Structural Characteristics of Heparin Binding to SARS-CoV-2 Spike Protein RBD of Omicron Sub-Lineages BA.2.12.1, BA.4 and BA.5

doi: 10.3390/v14122696

Figure Lengend Snippet: Molecular Docking and modeling simulation. ( A ) Structure of Omicron S protein (PDB: 7XNS) with the RBD domain in red. ( B ) Model electrostatic potential map for docking binding of BA.2.12.1 and BA.4/BA.5 S protein RBD to heparin dodecasaccharide (PDB:1HPN). ( C ) 2D diagram of the interaction of BA.2.12.1 and BA.4/BA.5 S protein RBDs with heparin dodecasaccharide.

Article Snippet: S protein RBD of Omicron sub-lineages BA.2.12.1 (Cat: 40592-V08H132), BA.4/BA.5 (Cat: 40592-V08H130) were purchased from Sino Biological Inc. (Beijing, China).The proteins were constructed as follows: (1) a DNA sequence encoding the SARS-CoV-2 (BA.2.12.1) Spike RBD (YP_009724390.1, with mutations G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452Q, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H) (Arg319–Phe541) was expressed with a polyhistidine tag at the C-terminus; and (2) a DNA sequence encoding the SARS-CoV-2 (BA.4/BA.5) Spike RBD (YP_009724390.1, with mutations G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H) (Arg319–Phe541) was expressed with a polyhistidine tag at the C-terminus.

Techniques: Binding Assay

Journal: Viruses

Article Title: Structural Characteristics of Heparin Binding to SARS-CoV-2 Spike Protein RBD of Omicron Sub-Lineages BA.2.12.1, BA.4 and BA.5

doi: 10.3390/v14122696

Figure Lengend Snippet: Solution competition between heparin and PPS or MPS. ( A ) Structure of PPS and MPS. ( B ) SPR sensorgrams of S protein RBD (BA.2.12.1)–heparin interaction competing with PPS or MPS. Concentration of S-protein RBD (BA.2.12.1) is 250 nM mixed with 1 µM of PPS or MPS. ( C ) Bar graphs (based on triplicate experiments with standard deviation) of normalized S-protein RBD (BA.2.12.1) binding preference to surface heparin by competing with PPS or MPS. ( D ) SPR sensorgrams of S protein RBD (BA.4/BA.5)–heparin interaction competing with PPS or MPS. Concentration of S-protein RBD (BA.4/BA.5) is 250 nM mixed with 1 µM of PPS or MPS. ( E ) Bar graphs (based on triplicate experiments with standard deviation) of normalized S-protein RBD (BA.4/BA.5) binding preference to surface heparin by competing with PPS or MPS. Statistical analysis was performed using unpaired two-tailed t -test (***: p ≤ 0.001 compared to the control, ###: p < 0.001 compared to the heparin).

Article Snippet: S protein RBD of Omicron sub-lineages BA.2.12.1 (Cat: 40592-V08H132), BA.4/BA.5 (Cat: 40592-V08H130) were purchased from Sino Biological Inc. (Beijing, China).The proteins were constructed as follows: (1) a DNA sequence encoding the SARS-CoV-2 (BA.2.12.1) Spike RBD (YP_009724390.1, with mutations G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452Q, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H) (Arg319–Phe541) was expressed with a polyhistidine tag at the C-terminus; and (2) a DNA sequence encoding the SARS-CoV-2 (BA.4/BA.5) Spike RBD (YP_009724390.1, with mutations G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H) (Arg319–Phe541) was expressed with a polyhistidine tag at the C-terminus.

Techniques: Concentration Assay, Standard Deviation, Binding Assay, Two Tailed Test

Journal: Viruses

Article Title: Structural Characteristics of Heparin Binding to SARS-CoV-2 Spike Protein RBD of Omicron Sub-Lineages BA.2.12.1, BA.4 and BA.5

doi: 10.3390/v14122696

Figure Lengend Snippet: IC 50 measurement of the inhibition of S-protein RBD (BA.2.12.1) binding to heparin using solution competition SPR by sulfated glycans (heparin, PPS, and MPS). S-protein RBD concentration was 250 nM. Error bars represent standard deviations from triplicate tests. ( A , B ) = heparin; ( C , D ) = PPS; ( E , F ) = MPS.

Article Snippet: S protein RBD of Omicron sub-lineages BA.2.12.1 (Cat: 40592-V08H132), BA.4/BA.5 (Cat: 40592-V08H130) were purchased from Sino Biological Inc. (Beijing, China).The proteins were constructed as follows: (1) a DNA sequence encoding the SARS-CoV-2 (BA.2.12.1) Spike RBD (YP_009724390.1, with mutations G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452Q, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H) (Arg319–Phe541) was expressed with a polyhistidine tag at the C-terminus; and (2) a DNA sequence encoding the SARS-CoV-2 (BA.4/BA.5) Spike RBD (YP_009724390.1, with mutations G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H) (Arg319–Phe541) was expressed with a polyhistidine tag at the C-terminus.

Techniques: Inhibition, Binding Assay, Concentration Assay