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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 血管内皮生长因子(VEGF)是一种分泌的二聚体糖蛋白，调节现有血管形成新血管，这一生理过程通常被称为血管生成。血管内皮生长因子通过配体诱导两种结构相关的受体酪氨酸激酶(RTK)-1和VEGFR-2二聚化后启动的细胞内信号转导通路增强血管通透性。VEGFR-2依赖的信号传递更强的血管生成反应，在病理性肿瘤生长或缺氧条件下内皮细胞(ECs)缺氧时观察到的程度更高。1神经粘连蛋白-1(NRP-1)可增强信号转导，当激活的血管内皮生长因子/血管内皮生长因子受体复合体组装时，神经粘连蛋白-1作为细胞表面的辅助受体。 该项目的目标是利用电子显微镜对表达不同VEGF异构体和受体的肺癌细胞与其已知的膜结合伙伴形成复合体进行成像。一组预选的SomaLogic dsDNA适配子将被连接到2 nm、5 nm或10 nm的金粒子上，以使纳米粒子具有生物功能，并提供一个小的电子密度标签，使我们能够在细胞表面定位VEGFs/VEGFRs。我们希望展示作为活性信号复合体一部分的已知蛋白质因子的共定位，以便更好地了解这一生长因子/受体家族在细胞表面的空间展示。如果被证明是有效的，这项技术可以应用于许多其他生物现象。由于核酸适体的小尺寸(50-70bp)和高亲和力(Kds~1-10 nM)，我们认为这种标记方法将比免疫标记更好，后者通常需要初级和次级两个标记步骤！ Y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