Clonable Nanoparticles

可克隆纳米颗粒

基本信息

批准号：
10378522
负责人：
Christopher Jeffries Ackerson
金额：
$ 29.46万
依托单位：
COLORADO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10378522
关键词：
Address Animals Area Artificial nanoparticles Binding Biological Biological Models Biological Process Biological Testing Biology Caliber Cell division Cells Centrifugation Chemicals Collaborations Color Colorado Complement Contrast Media Coupled Crystallization Cultured Cells DNA DNA Shuffling Data Development 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 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 base 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

项目摘要

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.

项目概要该提案的目的是解决 X 射线、电子或其他图像形成的图像中的对比度问题基于散射的照明方式。简而言之，图像是通过对比形成的。换句话说，我们只看到（或获取与背景不同的事物的信息）。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被伪装或与背景无法区分。例如，在 X 射线中，很容易看到骨头，但不太容易看到肌肉、脂肪或皮肤。这也适用于显微图像，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，没有可克隆的造影剂。 X 射线或电子成像中的可克隆对比（GFP 同系物）可以理解为“可克隆” 纳米粒子。”这种纳米粒子会散射入射辐射，并由一种蛋白质制成，该蛋白质可以与其他感兴趣的蛋白质进行基因融合。这种可克隆的分子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)维护合成蛋白质的纳米颗粒； (3)纳米粒子的尺寸控制，其中5nm直径为建议作为理想尺寸。我们最近分离出一种类金属还原酶，它似乎部分满足以下条件：这些化学要求。我们建议以这一发现为基础，创建一条能够生产许多产品的管道。具有不同尺寸、形状或元素组成的可克隆纳米颗粒。最广泛有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。这样的纳米粒子可以作为“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，整合来自多种成像方式的信息。拟议的工作将朝着三个具体目标进行。将鉴定候选可克隆纳米颗粒并在目标 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完善将通过饱和诱变、DNA改组和随机诱变的定向进化方法。隔离其他变体和新型酶将从在持久性污染地区收集的现场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 Vivo 在 Aim 2 中使用我们确定的 3 个模型和实验系统来评估跨平台的可移植性体内、原位和体外化学环境中的物种和适用性。