Thermodynamic Evaluation of Optimized Heparin–DNA Origami Nanostructures Targeting the SARS-CoV-2 Spike RBD Using MM/PBSA–IE

Although DNA origami scaffolds functionalized with antiviral ligands have been explored as potential platforms against SARS-CoV-2, the equilibrium thermodynamic behavior of heparin-bearing DNA origami constructs in interaction with the spike receptor-binding domain (RBD) remains incompletely understood. Building upon a previous steered molecular dynamics (SMD) study, we performed an equilibrium MD-based analysis of U-shaped DNA origami nanocages functionalized with heparin oligosaccharides of different lengths, including tetra-, hexa-, and decasaccharide chains, using all-atom molecular dynamics simulations and MM/PBSA–Interaction Entropy (MM/PBSA-IE) free-energy calculations. Molecular docking was first used to generate plausible initial binding poses, and the docking results showed a qualitative trend consistent with the subsequent MD-based free-energy estimates. Each RBD-containing complex was simulated in three independent 100 ns replicas to improve statistical reliability. Among the modeled systems, the decasaccharide-functionalized complex showed the most favorable predicted tethered heparin–RBD binding free energy (ΔG bind = −110.1 ± 14.6 kJ/mol), together with the greatest total heparin–RBD contact formation. This relative ranking was qualitatively consistent with the trend observed in the earlier SMD study. However, because the three ligands differ simultaneously in oligosaccharide length, molecular size, total negative charge, and number of available interaction sites, the observed ranking cannot be attributed uniquely to chain length or interpreted as evidence of greater size-normalized interaction efficiency. Overall, these results provide a relative computational ranking of the three modeled tethered-heparin systems under the selected binding configurations. Additional validation using free-heparin controls, more complete spike models, alternative docking poses, and experimental assays is required to confirm biological relevance and antiviral activity.