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The binding of a DNA aptamer (5'-CCGTCTTCCAGACAAGAGTGCAGGG-3') to recombinant human vascular endothelial growth factor (VEGF(165)) was characterized using surface plasmon resonance (SPR), fluorescence anisotropy and isothermal titration calorimetry (ITC). Results from both fluorescence anisotropy and ITC indicated that a single aptamer molecule binds to each VEGF homodimer, unlike other VEGF inhibitors that exhibit 2(ligand):1(VEGF homodimer) stoichiometry. In addition, ITC revealed that the association of the aptamer to VEGF at 20 degrees C is enthalpically driven, with an unfavorable entropy contribution. SPR kinetic studies, with careful control of possible mass transfer effects, demonstrated that the aptamer binds to VEGF with an association rate constant k(on) = 4.79 +/- 0.03 x 10(4) M(-1) s(-1) and a dissociation rate constant k(off) = 5.21 +/- 0.02 x 10(-4) s(-1) at 25 degrees C. Key recognition hot-spots were determined by a combination of aptamer sequence substitutions, truncations, and extensions. Most single-nucleotide substitutions, particularly within an mfold-predicted stem, suppress binding, whereas those within a predicted loop have a minimal effect. The 5'-end of the aptamer plays a key role in VEGF recognition, as a single-nucleotide truncation abolished VEGF binding. Conversely, an 11-fold increase in the association rate (and affinity) is observed with a single cytosine nucleotide extension, due to pairing of the 3'-GGG with 5'-CCC in the extended aptamer. Our approach effectively maps the secondary structural elements in the free aptamer, which present the unpaired interface for high affinity VEGF recognition. These data demonstrate that a directed binding analysis can be used in concert with library screening to characterize and improve aptamer/ligand recognition.