(260b) VEGF - DNA Aptamer Interactions: Ensemble and Single Molecule Studies and Implications for Aptamer Design

Here we present a comprehensive investigation of the association of a DNA aptamer and its binding partner, recombinant human Vascular Endothelial Growth Factor (VEGF165). We employed surface plasmon resonance (SPR), fluorescence anisotropy (FA), isothermal titration calorimetry (ITC), sedimentation velocity, and single-molecule fluorescence resonance energy transfer (SM-FRET) to characterize the dynamics of this molecular pairing. FA, ITC and sedimentation velocity all indicate that, at equilibrium, a single aptamer molecule binds to each VEGF homodimer, suggesting an involvement of the subunit interface in aptamer recognition. Kinetic studies with mass-transfer-compensated Biacore SPR were used to determine rates of association and dissociation of VEGF with various aptamer derivatives. These studies helped to identify those nucleotides in the aptamer particularly important for binding of the protein. Specifically, the terminal nucleotide at the 5'-end appeared to be essential for binding, and a one nucleotide extension at the 3'-end was found to increase affinity 11-fold. The aptamer contains an mfold predicted weak stem-loop structure; base substitutions in the loop region do not strongly affect VEGF binding, but alterations in the stem can nearly abolish VEGF affinity.

Most recently, single-molecule intramolecular FRET across the stem-loop structure was used to investigate more extensively the Mg²⁺-dependent conformational dynamics within the VEGF-DNA aptamer pair. Without its protein target, the aptamer favors a closed conformation, but in a manner which is highly dependent on Mg²⁺ concentration. The same dynamics occur in the presence of VEGF, but interaction with VEGF shifts the aptamer equilibrium toward a more open conformation. The observed kinetics of fluctuation suggest that two processes mediate the aptamer's return to the closed conformation in the presence of VEGF; a fast transition characteristic of unbound aptamer changing its conformation, and a slow transition representing aptamer/protein dissociation and return of the aptamer to the closed conformation.