We present a comprehensive investigation of the molecular interactions between DNA aptamers aV and aH and their target, vascular endothelial growth factor (VEGF165). Fluorescence anisotropy, isothermal titration calorimetry and sedimentation studies with aV aptamer indicate that, at equilibrium, a single aptamer binds to each VEGF homodimer, suggesting an involvement of the subunit interface in aptamer recognition. We further present kinetics of association and dissociation for VEGF with various aptamer mutant forms using surface plasmon resonance. Using single-molecule intramolecular fluorescence resonance energy transfer, we investigate the Mg2+-dependent conformational dynamics of aV-VEGF. Without its protein target, aV aptamer appears to favor the closed conformation, shifting to a more open conformation upon interaction with VEGF. The association of the aH aptamer to VEGF,  by contrast, is not sequence specific and is mediated by electrostatic interactions, sensitive to ionic strength, and entropically-driven, compared to enthalpically-driven aV-VEGF interaction.

Dynamics and interactions of VEGF-DNA aptamers: Ensemble and single molecule studies

243rd National Meeting (2012)

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