Molecular and functional characterization of a novel Toll-like receptor 9-specific DNA aptamer with therapeutic...

Toll-like receptor 9 (TLR9) plays a pivotal role in immune activation through recognition of DNA motifs, making it a compelling target for immunomodulatory therapies. We employed an integrative approach combining computational docking, biophysical characterization, and functional assays to identify and characterize DNA aptamers targeting TLR9. From an initial pool of candidates, ATA2 (agonistic TLR9 aptamer) emerged as a potent TLR9 agonist, demonstrating dose-dependent induction of TNF-α production in macrophages with specificity confirmed by TLR9 antagonist inhibition. ATA2 also enhanced macrophage migration and activated dendritic cells but showed limited effects on B cells. Surface plasmon resonance revealed a moderate affinity interaction between ATA2 and TLR9 (KD = 2.94 μM). Structural analyses via HPLC indicated the presence of discrete conformational states and high thermal and chemical stability of ATA2. Molecular dynamics simulations further highlighted ATA2's superior conformational stability and flexibility in binding TLR9 compared to another aptamer, ATA1, suggesting a versatile mode of interaction. Collectively, these findings elucidate the molecular basis of ATA2's TLR9 agonistic activity and underscore its therapeutic potential as a stable and specific immunomodulatory agent.