LOCALIZATION OF VEGF AND VEGF-RECEPTORS USING GOLD-CONJUGATED DNA APTAMERS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Vascular Endothelial Growth Factor (VEGF) is a secreted dimeric glycoprotein that regulates the formation of new blood vessels from existing vessels, a physiological process often referred to as angiogenesis. VEGF enhances vascular permeability through an intracellular signaling cascade that is initiated after ligand induced dimerization of two structurally related receptor tyrosine kinases (RTKs), VEGFR-1 and VEGFR-2. VEGFR-2 dependent signaling conveys a stronger angiogenic response and is observed to a greater degree during pathological tumor growth or when endothelial cells (ECs) are starved of oxygen under hypoxic conditions. 1 Signaling is enhanced by Neuropilin-1 (Nrp-1), which acts as a co-receptor at the cell surface when the activated VEGF/VEGFR complex assembles. The goal of this project is to image lung cancer cells which express different VEGF isoforms and VEGF receptors in complex with their known membrane binding partners using electron microscopy. A panel of pre-selected SomaLogic dsDNA aptamers will be conjugated to 2 nm, 5 nm or 10 nm gold particles in order to biofunctionalize the nanoparticle and provide a small electron dense tag that will enable us to locate the VEGFs/VEGFRs on the cell surface. We hope to show colocalization of known protein factors that are part of an active signaling complex in order to better understand the spatial display of this family of growth factors/receptors at the cell surface. If proven to be effective, this technique could be applied to a host of other biological phenomena. Because of the small size (50-70 bp) and high affinity (Kds ~1-10 nM) of the aptamers, we think this labeling approach will be better than immunolabeling, which often requires two labeling steps with primary and secondar! y antibodie