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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 血管内皮生长因子（VEGF）是一种分泌的二聚体糖蛋白，其调节从现有血管形成新血管，这是通常称为血管生成的生理过程。 VEGF通过细胞内信号级联增强血管通透性，该信号级联在配体诱导的两种结构相关的受体酪氨酸激酶（RTK）VEGFR-1和VEGFR-2的二聚化后启动。 VEGFR-2依赖性信号传导传递更强的血管生成反应，并且在病理性肿瘤生长期间或当内皮细胞（EC）在缺氧条件下缺氧时在更大程度上观察到。神经纤毛蛋白-1（Nrp-1）增强信号传导，当活化的VEGF/VEGFR复合物组装时，Nrp-1作为细胞表面的共受体发挥作用。 该项目的目标是使用电子显微镜对表达不同VEGF亚型和VEGF受体的肺癌细胞进行成像，这些细胞与其已知的膜结合伴侣复合。 一组预选的SomaLogic dsDNA适体将与2 nm、5 nm或10 nm金颗粒缀合，以使纳米颗粒生物功能化并提供小的电子致密标签，使我们能够在细胞表面定位VEGF/VEGF受体。 我们希望显示共定位已知的蛋白质因子的活性信号复合物的一部分，以更好地了解这个家庭的生长因子/受体在细胞表面的空间显示。 如果被证明是有效的，这种技术可以应用于许多其他生物现象。 由于适体的小尺寸（50-70 bp）和高亲和力（Kds ~1-10 nM），我们认为这种标记方法将优于免疫标记，后者通常需要两个标记步骤，即初级和次级标记！ γ抗体

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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