Structure Determination of Vascular Endothelial Growth Factor Heparin-Binding Domain in Complex with a Dna Aptamer

Hunt, Sabrina Robin

Graduate Thesis Or Dissertation

Structure Determination of Vascular Endothelial Growth Factor Heparin-Binding Domain in Complex with a Dna Aptamer Público Deposited

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Citeable URL: https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/1g05fb71z

Abstract

Vascular endothelial growth factor is a cytokine that is required for the maintenance of healthy vasculature, and it is responsible for a variety of pathogenic conditions, such as age-related macular degeneration and tumor growth in numerous cancers. As an important therapeutic target, vascular endothelial growth factor has been the focus of many in vitro selection strategies to identify aptamers that bind the protein with high affinity and specificity. One of these is a 25-nucleotide DNA aptamer that targets the heparin-binding domain (HBD) of vascular endothelial growth factor. The work presented here focuses on determining the solution structure of the HBD-aptamer complex using NMR. The structure was determined using molecular dynamics simulations restrained with experimental NMR data. The structure of the complex is displayed as an ensemble of the 10 lowest energy structures with a heavy atom RMSD of 2.0 ± 0.8 Å for the well-defined core of the complex. The HBD has two subdomains that retain a similar backbone conformation between the free and bound states, but the orientation of the two subdomains relative to one another changes. The aptamer forms two Watson-Crick base pairs and two tandem base triples that are proximal to a pyrimidine-rich triloop. The 5’ and 3’ ends of the aptamer base pair and form a tetraloop-like structure. The Watson-Crick base pairs and several unpaired nucleotides form the binding interface that spans both subdomains of the HBD. The structure presented here contributes to the larger goal of better understanding the molecular recognition between aptamers and their targets.

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