Structural Basis of DNA Aptamer A58 Targeting the N-terminal Domain of Sarbecoviruses Nucleocapsid Protein

In the post-pandemic era, the persistent threat of coronaviruses demands broad-spectrum antiviral therapeutic strategies. The SARS-CoV-2 nucleocapsid protein (N protein), an essential factor for genome packaging and immune modulation, poses a promising antiviral target. Here, we determined the crystal structure of the DNA aptamer A58-T10 in complex with the N-terminal domain of the N protein (N-NTD). A58-T10 binds to the N-NTD via a unique three-tiered stem-loop that interacts with the nucleic acid-binding site of N-NTD through extensive hydrogen bonding and stacking. Structural analysis reveals that A58 contains two stem-loops with octanucleotide motifs (5'-11ACCGGATT19-3' and 5'-26ATCGGATT33-3') that specifically recognize N-NTD. Functionally, A58 inhibits N-NTD's binding to viral RNA, disrupting N protein-host cell interactions involved in immune responses. Notably, A58 exhibits broad-spectrum binding activity against N proteins from SARS-CoV-2 variants and related sarbecoviruses. These findings elucidate the specific interaction mechanism between A58 and N-NTD, highlighting its potential as an anti-sarbecovirus agent. RUNNING TITLE: Crystal structure of DNA aptamer in complex with N-NTD.