Clonable Nanoparticles

描述(由申请人提供)：这一探索性(R21)提案的目标是确定最有希望的策略来解决电子显微镜中的对比问题。在所有显微镜中，无论是由光子还是电子照射，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，从根本上解决了光学显微镜的对比度问题。然而，对于电子显微镜而言，并没有类似于GFP的“可克隆的”对比标记。在这里，我们建议探索几种基于多肽或小蛋白的策略来成核、催化或中介电子致密无机纳米颗粒的形成，这可能允许在大分子和全细胞电子显微镜中定位单个蛋白质。拟议的工作对四个调查领域具有重要意义。首先，也是最重要的是，一种强大的可克隆纳米颗粒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，我们将以原核微管蛋白类似物FtsZ为研究对象。我们了解的关于FtsZ的细胞内结构和分布的信息对于开发针对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多肽或小蛋白时，我们将更多地了解金属和生物分子之间重要的、有时鲜为人知的相互作用。第四，就像绿色荧光蛋白可以作为光学读取组织和生物中基因表达的“报告基因”一样，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”，用于通过核磁共振或CT检测基因表达。拟议的工作将以三个具体目标进行。在这些特定目标中确定的所有候选“可克隆纳米颗粒”多肽或蛋白质将在体外、原位和基于FtsZ的结构电子显微镜分析中进行体内评估。目标一和目标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刺激磁性氧化铁纳米颗粒形成的多肽序列。AIM 2在技术上更具挑战性，但更有可能产生一种普遍适用的可克隆纳米颗粒 它可能产生可克隆的纳米颗粒，在原位和体外发挥作用，但不能在体内发挥作用。目标3是检测自然产生的多肽、蛋白质和相关的蛋白质片段，以及其他人分离的多肽，以便与我们在AIMS 1和2中分离的多肽进行比较。该提案确定了现有的已知多肽和蛋白质无法作为EM对比标记的原因，并建议我们可以对这些已知的多肽和蛋白质进行修改，使其成为EM对比标记。拟议的使能技术的发展对公共卫生的意义将来自于关于正常和疾病细胞的更全面的结构信息，也来自对FtsZ生物学的更多了解，这可能使新抗生素的开发成为可能。 公共卫生相关性：显微镜对疾病的诊断和了解至关重要。生物材料在光学显微镜和电子显微镜下基本上都是透明的。正如2008年诺贝尔化学奖所承认的那样，荧光蛋白质从根本上解决了光学显微镜的这一“对比度问题”。受荧光蛋白质的启发，我们建议实施一种可克隆纳米颗粒的策略，以解决高分辨率电子显微镜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.