Structure-guided optimization of the A58 DNA aptamer targeting the SARS-CoV-2 nucleocapsid protein through integrated computational analysis.

Abstract

The SARS-CoV-2 nucleocapsid protein plays a key role in RNA recognition, genome packaging, and replication. Its RNA-binding domain is an attractive target for the development of nucleic acid-based antiviral and diagnostic strategies. The A58 DNA aptamer is an experimentally derived high-affinity aptamer that targets the nucleocapsid protein via its N-terminal RNA-binding domain to form a defined complex. However, the dynamic stability and binding energetics of this complex remain unexplored. Hence, in this computational study, a structure-guided strategy was employed to investigate the optimization of a 20-nucleotide A58 DNA aptamer. The original nucleotide sequence of the A58 aptamer was mutated to generate a library of 255 non-parental mutated variants at positions 9-12 while preserving the experimentally characterized 5'-TCGGAT-3' loop. The 3D structures were predicted using AlphaFold 3 and docked against the nucleocapsid protein using HADDOCK2.4. The top four best-affinity variants, Apt66, Apt123, Apt17, and Apt94, were selected after docking and subjected to molecular dynamics simulations for 200 ns along with the original PDB structure of the SARS-CoV-2 nucleocapsid protein with the A58 aptamer taken as a reference. After a comprehensive post-simulation analysis, Apt94 was identified as the most favorable computationally prioritized candidate, followed by Apt123, Apt17, and Apt66, indicating that mutations at the targeted nucleotides in the A58 DNA aptamer can remodel its dynamic and energetic behaviors. This study provides a rational framework for identifying potential aptamer variants with distinct structural, dynamic, and energetic signatures for subsequent experimental affinity, specificity, and functional evaluation against the SARS-CoV-2 nucleocapsid protein.

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