Clonable Nanoparticles

描述（由申请人提供）：本探索性（R21）提案的目的是确定解决电子显微镜对比度问题的最有前途的策略。在所有的显微镜中，无论是用光子还是电子照明，生物组织基本上都是透明的。绿色荧光蛋白（GFP）和相关的荧光蛋白补充了小分子染色剂，以基本上解决光学显微镜中的对比度问题。然而，对于电子显微镜，没有类似于GFP的“可克隆的”对比标记。在这里，我们建议探索几个肽或小蛋白质为基础的战略成核，催化或介导的电子致密的无机纳米粒子的形成，这可能会允许在大分子和全细胞电子显微镜的个别蛋白质的本地化。 拟议的工作对四个调查领域具有重要意义。首先，也是最重要的，一种强大的可克隆纳米粒子将彻底改变电子显微镜，使其对所有形式的生物EM都具有重要意义。其次，我们将研究FtsZ，原核微管蛋白类似物作为初始目标。我们了解FtsZ的细胞内结构和分布的信息对于开发靶向FtsZ的抗生素具有重要意义。第三，在寻找与金属离子或配位络合物相互作用的新型肽或小蛋白质时，我们将更多地了解金属和生物分子之间重要的、有时知之甚少的相互作用。第四，正如GFP可以作为“报告基因”用于光学阅读组织和生物体中的基因表达，可遗传编码的磁性纳米颗粒也可以作为广泛使用的“报告基因”用于通过MRI或CT检测基因表达。 拟议的工作将在三个具体目标下进行。在这些特定目的中鉴定的所有候选“可克隆纳米颗粒”肽或蛋白质将在基于FtsZ的结构电子显微镜测定中在体外、原位和体内进行评估。目的一和二分别使用噬菌体展示和核糖体展示的定向进化技术来分离能够引起磁性氧化铁纳米颗粒形成的肽序列。AIM2在技术上更具挑战性，但更有可能产生普遍有用的可克隆纳米颗粒 目的1，这可能会产生可克隆的纳米颗粒，在原位和体外发挥作用，但在体内没有。目的3是测定天然存在的肽、蛋白质和相关蛋白质片段，以及由他人分离的肽，以与我们在目的1和2中分离的肽进行比较。该提案确定了为什么现有的“已知”肽和蛋白质不能作为EM对比标记物，并建议我们可以对这些已知肽和蛋白质进行修改，以使其适应EM对比标记物。 开发拟议的使能技术的公共卫生意义将来自正常和患病细胞的更全面的结构信息，以及对FtsZ生物学的更深入了解，这可能有助于开发新的抗生素。 公共卫生相关性：显微镜对疾病的诊断和理解至关重要。生物材料在光学显微镜和电子显微镜下基本上是透明的。这种“对比度问题”在光学显微镜中基本上被荧光蛋白解决了，这一点得到了2008年诺贝尔化学奖的认可。受荧光蛋白的启发，我们提出了一种可克隆纳米粒子的策略，用于解决电子显微镜更高分辨率技术中的对比度问题。

DESCRIPTION (provided by applicant): The objective of this exploratory (R21) proposal is to identify the most promising strategy for solving the contrast problem in electron microscopy. In all microscopes, whether illuminated by photons or electrons, biological tissues are essentially transparent. Green Fluorescent Protein (GFP) and related fluorescent proteins have complemented small molecule stains to essentially solve the contrast problem in optical microscopy. For electron microscopy, however, there are no 'clonable' contrast markers analogous to GFP. Herein we propose to explore several peptide or small protein based strategies for nucleating, catalyzing or mediating the formation of electron dense inorganic nanoparticles which may allow the localization of individual proteins in macromolecular and whole cell electron microscopy. The proposed work is significant for four areas of inquiry. First and most significantly, a robust clonable nanoparticle will revolutionize electron microscopy, making it significant for all forms of biological EM. Second, we will study FtsZ, the prokaryotic tubulin analog as an initial target. Information we learn about the intrcellular structure and distribution of FtsZ is significant for the development of antibiotics that target FtsZ. Third, we will, in finding novel peptides or small proteins that interact with metal ions or coordination complexes learn more about the important and sometimes poorly understood interactions of metals and biomolecules. Fourth just as GFP can act as a 'reporter gene' for reading optically for gene expression in tissues and organisms, a genetically encodable magnetic nanoparticle may also serve as a widely used 'reporter gene' for detecting gene expression by MRI or CT. The proposed work will proceed in three specific aims. All candidate 'clonable nanoparticle' peptides or proteins identified in these specific aims will be evaluated in vitro, in situ and in vvo in an FtsZ based structural electron microscopy assay. Aims one and two use the directed evolution techniques of phage display and ribosome display, respectively, to isolate peptide sequences capable of provoking the formation of magnetic iron oxide nanoparticles. Aim 2 is technically more challenging but is more likely to yield a universally useful clonable nanoparticle than Aim 1, which may yield clonable nanoparticles that function in situ and in vitro, but not in vivo. Aim 3 is to assay naturally occurring peptides, proteins and relevant protein fragments, as well as peptides isolated by others, for comparison to the peptides we isolate in aims 1 and 2. The proposal identifies why existing 'known' peptides and proteins fail to function as EM contrast markers, and suggests modifications that we may make to these known peptides and proteins to adapt them as EM contrast markers. The public health significance of the development of the proposed enabling technologies will derive from both more comprehensive structural information on normal and diseased cells, and also from greater understanding of FtsZ biology, which may enable development of new antibiotics. PUBLIC HEALTH RELEVANCE: Microscopes are critical to the diagnosis and understanding of disease. Biological material is essentially transparent in both light and electron microscopes. This 'contrast problem' was essentially solved for light microscopy by fluorescent proteins, as recognized by the 2008 Nobel prize in chemistry. Inspired by fluorescent proteins, we propose to implement a strategy of clonable nanoparticles for solving the contrast problem in the much higher resolution technique of electron microscopy.