Figure 1. 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 [29]. 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. Figure 1. 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 [29]. 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.

Figure 2. 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.

Figure 2. 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.

Figure 3. 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).

Figure 3. 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).

Figure 4. S protein RBD (BA.4/BA.5)–heparin interaction inhibited by heparin oligosaccharides/desulfated heparins using solution competition. (A) SPR sensorgrams of S protein RBD (BA.4/BA.5)–heparin interaction competing with different heparin oligosaccharides. Concentration of S-protein RBD (BA.4/BA.5) 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.4/BA.5) binding preference to surface heparin by competing with different heparin oligosaccharides. (C) SPR sensorgrams of S protein RBD (BA.4/BA.5)–heparin interaction competing with different desulfated heparins. Concentration of S-protein RBD (BA.4/BA.5) 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.4/BA.5) 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).

Figure 4. S protein RBD (BA.4/BA.5)–heparin interaction inhibited by heparin oligosaccharides/desulfated heparins using solution competition. (A) SPR sensorgrams of S protein RBD (BA.4/BA.5)–heparin interaction competing with different heparin oligosaccharides. Concentration of S-protein RBD (BA.4/BA.5) 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.4/BA.5) binding preference to surface heparin by competing with different heparin oligosaccharides. (C) SPR sensorgrams of S protein RBD (BA.4/BA.5)–heparin interaction competing with different desulfated heparins. Concentration of S-protein RBD (BA.4/BA.5) 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.4/BA.5) 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).

Figure 5. 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.

Figure 5. 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.

Figure 6. 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).

Figure 6. 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).

Figure 7. IC50 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.

Figure 7. IC50 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.

Figure 8. IC50 measurement of the inhibition of S-protein RBD (BA.4/BA.5) 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.

Figure 8. IC50 measurement of the inhibition of S-protein RBD (BA.4/BA.5) 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.

Table 1. Summary of kinetic data of S protein RBD of BA.2.12.1 and BA.4/BA.5 binding with heparin.

Table 1. Summary of kinetic data of S protein RBD of BA.2.12.1 and BA.4/BA.5 binding with heparin.

ka (M−1s−1)kd (s−1)KD (M) BA.2.12.1 3.4 × 104
( ± 270) * 4.7 × 10−3
( ± 2.1 × 10−5) * 1.4 × 10−7
( ± 5.8 × 10−9) ** BA.4/BA.5 3.4 × 104
( ± 630) 7.9 × 10−3
( ± 8.7 × 10−5) 2.3 × 10−7
( ± 2.6 × 10−8)