Clonable Nanoparticles

可克隆纳米颗粒

• 批准号: 10608989
• 负责人: Christopher Jeffries Ackerson
• 金额: $ 29.39万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10608989
• 关键词: Address; Animals; Area; Artificial nanoparticles; Binding; Biological; Biological Availability; Biological Models; Biological Process; Biological Testing; Biology; Cell division; Cells; Centrifugation; Chemicals; Collaborations; Color; Colorado; Complement; Contrast Media; Coupled; Cultured Cells; DNA; DNA Shuffling; Data; Development; Diameter; Directed Molecular Evolution; Disease; Disulfides; Drug Metabolic Detoxication; Dyes; Electron Microscopy; Electrons; Elements; Engineering; Environment; Enzymes; Family; Fatty acid glycerol esters; Filament; Fluorescence; Green Fluorescent Proteins; Growth; Homologous Gene; Image; Imaging technology; In Situ; In Vitro; Investigation; Ions; Knowledge; Libraries; Light; Light Microscope; Lighting; Macromolecular Complexes; Magnetic Resonance Imaging; Magnetism; Maintenance; Mercury (II) reductase; Metals; Methods; Minerals; Modality; Modeling; Morphology; Muscle; Mutagenesis; Optics; Organism; Oxidoreductase; Pathologic Processes; Peptide Library; Peptides; Phase; Plasmids; Property; Protein Biosynthesis; Proteins; Public Health; Radiation Accidents; Randomized; Research; Roentgen Rays; Sampling; Selenite; Selenium; Shapes; Site; Skin; Stains; Synaptic Transmission; System; Technology; Testing; Tissues; Variant; Viral; Viral Pathogenesis; Whole Organism; Work; X-Ray Medical Imaging; bioimaging; bone; cell fixation; enzyme activity; experimental study; imaging modality; improved; in vivo; interest; metagenomic sequencing; microscopic imaging; mutant; nanoparticle; nanoparticulate; novel; optical imaging; particle; physical property; portability; pyridine nucleotide; receptor; reconstitution; small molecule; tool

PROJECT SUMMARY The objective of this proposal is to address the contrast problem in images formed by X-Ray, electron, or other scattering based illumination modalities. Briefly, images are made by contrast. In other words, we only see (or acquire information) on things that are distinguishable from their background. In all forms of biological imaging, many things are visible, yet many other things remain camouflaged or indistinguishable from the background. For instance, in an X-ray, it's easy to see bones, but not so easy to see muscles, fat or skin. This is also true in microscopic images, where it's often easy to see the edges of cells, but much harder to see the details inside cells. Green Fluorescent Protein and related fluorescent proteins complement small molecule stains and dyes to essentially solve the contrast problem in optical imaging. For imaging based on X-rays or electrons, however, there are no clonable contrast agents. Clonable contrast (a GFP homolog) in X-Ray or electron-based imaging could be understood as a `clonable nanoparticle.' Such a nanoparticle would scatter incident radiation and would be made by a protein that can be genetically fused to other proteins of interest. Three discrete chemical activities are needed for such a clonable nanoparticle: (1) conversion of bioavailable inorganic ions to insoluble nanoparticulate form; (2) maintenance of the nanoparticle at the protein that synthesizes it; (3) size control of the nanoparticle, where 5nm diameter is suggested as an ideal size. We recently isolated a metalloid reductase that appears to partially fulfill each of these chemical requirements. We propose to build on this finding to create a pipeline that will produce many clonable nanoparticles of distinct size, shape or elemental composition. The most broadly useful clonable nanoparticles will simultaneously incorporate X-ray/electron scattering, magnetism and fluorescence. Such nanoparticles could serve as `universal clonable contrast agents' functioning in optical, MRI, X-Ray and electron imaging. Such a tool would greatly facilitate integrative and/or correlative multiscale bioimaging, integrating information from multiple imaging modalities. The proposed work will proceed in three specific aims. Candidate clonable nanoparticles will be identified and refined in Aims 1a and 1b. Refinement of our existing clonable nanoparticle candidate will proceed through directed evolution methods of saturation mutagenesis, DNA shuffling, and random mutagenesis. Isolation of additional variants and novel enzymes will begin with field samples collected in areas with persistent environmental metal contamination. Candidate clonable nanoparticles will be assessed in in vitro, in situ and in vivo within Aim 2 using 3 model and experimental systems we have identified to assess portability across species and suitability in in vivo, in situ, and in vitro chemical environments.

项目摘要 该提案的目的是解决由X射线、电子或其他成像设备形成的图像中的对比度问题。 基于散射的照明模式。简单地说，图像是通过对比形成的。换句话说，我们只能看到（或 获取信息）的东西是从他们的背景区分。在所有形式的生物成像中， 许多事物是可见的，但许多其他事物仍然被遮蔽或无法从背景中区分出来。 例如，在X光片中，很容易看到骨骼，但不容易看到肌肉、脂肪或皮肤。这也是真实的， 显微图像，通常很容易看到细胞的边缘，但很难看到内部的细节 细胞绿色荧光蛋白和相关荧光蛋白补充小分子染色剂和染料 以基本上解决光学成像中的对比度问题。对于基于X射线或电子的成像， 然而，没有可克隆的造影剂。 基于X射线或电子的成像中的可克隆对比（GFP同系物）可以被理解为“可克隆的 纳米粒子。“这种纳米粒子可以散射入射的辐射，并由一种蛋白质制成， 与其他感兴趣的蛋白质基因融合这种克隆需要三种不同的化学活性 纳米颗粒：（1）将生物可利用的无机离子转化为不溶性纳米颗粒形式;（2）维持 纳米颗粒在合成它的蛋白质上;（3）纳米颗粒的尺寸控制，其中5 nm的直径是 这是一个理想的尺寸。我们最近分离出了一种类金属还原酶，它似乎部分实现了 这些化学要求。我们建议在这一发现的基础上建立一个管道， 具有不同尺寸、形状或元素组成的可克隆纳米颗粒。最广泛使用的克隆 纳米颗粒将同时结合X射线/电子散射、磁性和荧光。等 纳米颗粒可以作为“通用可克隆造影剂”，在光学、MRI、X射线和 电子成像这样的工具将极大地促进综合和/或相关的多尺度生物成像， 整合来自多种成像模式的信息。 拟议的工作将在三个具体目标下进行。将鉴定候选可克隆纳米颗粒， 在目标1a和1b中进行了细化。我们现有的可克隆纳米颗粒候选物的改进将通过 饱和诱变、DNA改组和随机诱变的定向进化方法。分离 其他的变异体和新的酶将开始从在持续存在的地区收集的实地样品开始， 环境金属污染。候选的可克隆纳米颗粒将在体外、原位和体内进行评估。 在Aim 2中使用3个模型和实验系统，我们已经确定了评估跨 物种和在体内、原位和体外化学环境中的适用性。