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.

项目总结 这项建议的目的是解决由X射线、电子或其他方式形成的图像中的对比度问题 基于散射的照明模式。简而言之，图像是通过对比来制作的。换句话说，我们只看到(或 获取信息)可与其背景相区别的事物。在所有形式的生物成像中， 许多东西是可见的，但许多其他东西仍然伪装着或与背景难以区分。 例如，在X光中，很容易看到骨骼，但不太容易看到肌肉、脂肪或皮肤。这一点也适用于 显微图像，通常很容易看到细胞的边缘，但很难看到里面的细节 细胞。绿色荧光蛋白及相关荧光蛋白对小分子染料的补充 从根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，目前还没有可克隆的造影剂。 X射线或电子成像中的可克隆对比度(GFP同系物)可被理解为可克隆 纳米颗粒。这种纳米粒子将散射入射辐射，并将由一种蛋白质制成，这种蛋白质可以 在基因上与其他感兴趣的蛋白质融合。这种可克隆的化合物需要三种不同的化学活动 纳米颗粒：(1)将生物可利用的无机离子转化为不溶的纳米颗粒形式；(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直径是 建议为理想的尺码。我们最近分离出一种金属还原酶，它似乎部分地满足了每一个 这些化学要求。我们建议在这一发现的基础上建立一条管道，以生产许多 具有不同大小、形状或元素组成的可克隆纳米颗粒。最广泛使用的可克隆 纳米粒子将同时结合X射线/电子散射、磁性和荧光。是这样的 纳米粒子可作为光学、核磁共振、X射线和光学等领域的“通用可克隆造影剂” 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， 集成来自多个成像模式的信息。 拟议的工作将以三个具体目标进行。候选的可克隆纳米颗粒将被识别并 目标1a和1b中的精炼。我们现有的可克隆纳米候选粒子的精炼将通过 饱和突变、DNA改组和随机突变的定向进化方法。隔离 更多的变种和新的酶将从在持续存在的地区收集的田间样本开始 环境金属污染。候选可克隆纳米颗粒将在体外、原位和体内进行评估 在Aim 2中使用我们确定的3个模型和实验系统评估可移植性 在体内、原位和体外化学环境中的种类和适宜性。