Two-step target recognition for the competitive inhibition activity of an anti-VEGF aptamer

The anti-vascular endothelial growth factor (VEGF) aptamer, t44.27, is a 27-mer RNA that functions as the active component of pegaptanib, an antiangiogenic medicine for neovascular age-related macular degeneration. The t44.27 aptamer is extensively 2'-modified and tightly binds to the heparin-binding domain (HDB) of VEGF₁₆₅ in a Ca²⁺-dependent manner. However, the molecular mechanism by which the aptamer selectively recognizes VEGF HDB to antagonize its function is poorly understood. We found that t44.27 binds to VEGF HBD in a two-step manner using a different region in the molecule: a transient interaction using a structured region of t44.27 followed by a tight complex formation with a larger interaction surface. Ca²⁺ binding stabilizes t44.27 base-pair formation suitable for the initial transient interaction. Meanwhile, the tight complex formation was essential for t44.27 to exert a competitive inhibition of heparin binding to VEGF HBD. These results provide structural insight into how the RNA aptamer specifically interacts with its target molecule to inhibit its activity.