Heterogeneity of Anti-VEGF Aptamer : A Single Molecule Study

Abstract

Age-related macular degeneration (AMD) is a major cause of severe visual loss worldwide. Among the key regulators, inhibition of vascular endothelial growth factor (VEGF) serves as a promising candidate for controlling angiogenesis in AMD. The emergence of aptamers as anti-VEGF (aV) provides a new pathway for targeting VEGF to control the disease. aV exhibits conformational dynamics and heterogeneity, implying a rugged energy landscape. Characterization of the heterogeneity of aptamers by single molecule fluorescence energy transfer (smFRET) has not been extensively elucidated. smFRET efficiency distributions in single-molecule experiments contain both structural and dynamical information. Extraction of this information from these distributions requires a careful analysis of unwanted contributions from dye-photophysics. We used cross-correlation analysis to characterize each time series trajectory and distinguish it from contributions of unwanted dye photophysics. NMR and analytical centrifugation were used to complement the dynamic smFRET conformational analyses. Wavelet denoising and state-finding algorithms were used to process the smFRET data to remove shot-noise and find among the ensembles of DNA aptamer conformational states. Binding of VEGF was found to induce a shift in the equilibrium conformation of the DNA aptamer to two conformations. Increased ionic strength resulted in a decrease of the binding ability of the aV to the VEGF.