Discovery and Cryo-EM-Guided Development of a Neuropilin-2-Binding Aptamer for Receptor Antagonism

Receptor antagonists represent a major class of targeted cancer therapeutics, yet SELEX-based aptamer discovery often yields high-affinity binders that display limited functional blockade because epitope positioning is not explicitly selected. Herein, we report a multistage discovery and optimization workflow that integrates cell-SELEX, cryo-electron microscopy (cryo-EM), and a structure-constrained follow-up SELEX to generate ligand-blocking antagonists of Neuropilin-2 (NRP2), a cancer-associated coreceptor for vascular endothelial growth factor (VEGF). We performed initial unbiased cell-SELEX against primary human urine-derived renal progenitor cells and identified an NRP2-binding DNA aptamer (NRP2Apt) with nanomolar affinity and high specificity. Using cryo-EM analysis of the NRP2Apt-NRP2 complex, we defined the spatial relationship between aptamer structural elements and the VEGF-binding pocket, providing direct epitope-level insight that is inaccessible from sequence information alone. Guided by these structural observations, we designed an NRP2Apt-scaffolded library in which a VEGF-proximal loop was extended and randomized, and subjected this library to NRP2 protein SELEX, yielding a lead antagonist with substantially enhanced inhibition of VEGF-NRP2 interactions while preserving strong NRP2 affinity. Finally, inspired by the homodimeric form of VEGF ligands, we engineered a bivalent aptamer that displayed enhanced antagonism and markedly increased serum stability. Collectively, we establish a cryo-EM-guided, iterative SELEX framework in which structural constraints are incorporated into library design to optimize the ligand-blocking activity of receptor-binding aptamers.