Monofunctional 3 to 10 nm Covalent Gold Labels for CryoEM

用于 CryoEM 的单功能 3 至 10 nm 共价金标签

• 批准号: 8781987
• 负责人: RICHARD DENIS POWELL
• 金额: $ 15.97万
• 依托单位: NANOPROBES, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2016-03-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8781987
• 关键词: Address; Adsorption; Alzheimer' s Disease; Amines; Antibodies; Antigens; Binding; Binding Sites; Biological; Biosensor; Citrates; Collaborations; Complex; Cryoelectron Microscopy; Cysteine; Devices; Diagnosis; Disease; Dose; Early Diagnosis; Electron Microscopy; Electronics; Electrons; Equipment; Event; Generations; Gold; Gold Colloid; Health; Hydrophobicity; Imagery; Immobilization; Label; Ligands; Light; Lysine; Macromolecular Complexes; Malignant Neoplasms; Methods; Microscopic; Modification; Molecular; Multiprotein Complexes; N-terminal; Nanotechnology; National Institute of Arthritis and Musculoskeletal and Skin Diseases; Oceans; Oligonucleotides; Optics; Organelles; Penetration; Pharmaceutical Preparations; Play; Population; Preparation; Procedures; Process; Property; Proteins; Public Health; Radiation; Reaction; Reagent; Research Personnel; Resolution; Role; Series; Site; Solutions; Specimen; Specimen Handling; Sulfhydryl Compounds; Surface; Surface Properties; System; Tannic Acid; Technology; Temperature Sense; Testing; Thick; Thiocyanates; Tissues; United States National Institutes of Health; Variant; Work; base; biomaterial compatibility; chemical reaction; crosslink; developmental disease; effective therapy; electron tomography; functional group; innovation; insight; instrumentation; macromolecule; meetings; milligram; nanoGold; nanodevice; nanomaterials; nanoparticle; nanoprobe; nanoscale; nanoscience; neoplastic cell; novel strategies; particle; programs; public health relevance; research study; stoichiometry; undecagold; water solubility

DESCRIPTION (provided by applicant): Gold nanoparticles are the label of choice for electron microscopy (EM) since they offer a choice of sizes that provide quantitative, high-resolution identification of targets. Recently, high-resolution EM methods including cryoEM and electron tomography have benefitted from significant advances in instrumentation and a series of breakthroughs in specimen processing and preparative equipment that have made these methods widely accessible. However, conventional colloidal gold labels do not allow the full realization of the potential in these methods, because they require extensive stabilization with large macromolecules that limit penetration and antigen access, and also because their mechanism of conjugation, through adsorption to targeting antibodies or proteins, does not allow direct labeling of specific reactive sites within macromolecular complexes at sufficiently high resolution. Nanoprobes has developed 1.4 nm (Nanogold) and 0.8 nm (Undecagold) gold nanoparticle labels with a single reactive group for selective, covalent labeling of thiols (cysteie residues), amines (N-terminal amines or lysine residues), or other functional groups. However, these are too small to be readily visualized in many types of specimens, and do not allow multiple labeling. Although covalent reactivity has been introduced into larger particles, preparing monofunctional reagents capable of 1: 1 labeling has proved highly challenging. To date, no products are available with native monofunctional reactivity. We will synthesize gold nanoparticles that combine a high degree of monodispersity, controlled surface functionalization and selective reactivity with monofunctional reaction stoichiometry. Preparations will be scaled and simplified to deliver nanoparticles with the following properties in an efficient, reproducible and economical manner on any scale: (1) Monodispersity: populations of gold nanoparticles from 3 nm to 10 nm will be prepared with coefficients of variation of 10% or less using thiocyanate reduction, citrate/tannic acid, or homogeneous reduction. (2) Surface properties: A universal base set of organic ligands will be developed that can confer (a) water-solubility and biocompatibility, (b) desired degree of hydrophobicity, (c) specific covalent reactivity, and (d) controlled shell thickness from 0.6 nm to 3 nm. Enable selection of any combination of these properties for any size. (3) Specific reactivity: amine, thiol, and activated carboxyl reactivity ill be introduced by (a) incorporation of a primary amine during synthesis and later modification, and (b) insertion of protected reactivity then deprotection. (4) Monofunctionality: four strategie will be used to generate particles that react with a single conjugate molecule in solution: (a) isolation from statistical mixture; (b) immobilization using a cleavable cross-linker that generate a unique group upon cleavage; or (c) conjugation of one or very few larger protein tags using cleavable cross- linkers, separation of gold particles by numbers of tags and cleavage to yield precisely functionalized gold particles; and (d) polymeric ligands bridging multifunctional gold coordination with a single cross-linking group. Monofunctionality will be tested in collaboration with Alasdair Steven (NIAMS, NIH) using cryoelectron microscopic labeling and electron tomographic experiments of multi-functional proteins in which site-specific labeling without aggregation will be used to confirm truly monofunctional reactivity. Monofunctional gold nanoparticles would also provide innovation to address a critical challenge in nanoscience: enabling the construction of molecular nanodevices in which multiple nanoparticles with unique and different properties and attachment points perform a coordinated set of actions to complete a task. Construction of such a system requires the assembly of particles with different properties into a construct by attachment at different sites. As a key enabling technology for this, monofunctional gold nanoparticles will help afford a new generation of nanotechnology innovations such as biosensors, indicators, and molecular devices that perform targeted, programmed tasks such as oligonucleotide annealing, temperature sensing, or selectively delivering medication or enhanced local radiation dose to tumor cells.

描述（由申请人提供）：金纳米颗粒是电子显微镜（EM）的首选标签，因为它们提供了一系列尺寸的选择，可提供靶标的定量高分辨率鉴定。最近，包括冷冻和电子层析成像在内的高分辨率EM方法受益于仪器的重大进展以及标本加工和制备设备的一系列突破，这些设备使这些方法广泛访问。但是，传统的胶体黄金标签不允许在这些方法中充分实现潜力，因为它们需要具有限制渗透和抗原访问的大型大分子的广泛稳定，也需要通过吸附的机制，通过靶向抗体或蛋白质来靶向抗体或蛋白质，不允许在较高的反应型中进行抗反应型的型号。纳米探针已经开发了1.4 nm（纳米元）和0.8 nm（undecagold）的金纳米颗粒标签，具有单个反应性基团，用于选择性，共价标记硫醇（囊肿残基），胺（N-末端胺或赖氨酸残基）或其他功能组，或其他功能组。但是，它们太小，无法在许多类型的标本中容易地可视化，并且不允许多个标签。尽管已将共价反应性引入较大的颗粒中，但事实证明，制备1：1标记的单功能试剂却具有高度挑战性。迄今为止，尚无天然单功能反应性的产品。我们将合成结合高度单分散性，受控的表面功能化和与单功能反应化学计量的选择性反应性的金纳米颗粒。制剂将被缩放和简化，以在有效，可重复的情况下传递具有以下特性的纳米颗粒 在任何规模上的经济方式：（1）单分散性：使用硫氰酸酯减少，柠檬酸/坦氨酸或均质减少，将制备从3 nm到10 nm的金纳米颗粒种群。 （2）表面特性：将开发一组有机配体的通用碱基集，可以赋予（a）水溶性和生物相容性，（b）所需的疏水性程度，（c）特定的共价反应性，以及（d）受控的壳厚度从0.6 nm到3 nm。启用任何尺寸的这些属性组合的选择。 （3）特定的反应性：胺，硫醇和活性羧基反应性通过（a）在合成和随后的修饰过程中掺入原代胺，以及（b）插入受保护的反应性然后剥落。 （4）单功能：将使用四种策略来产生与溶液中单个共轭分子反应的颗粒：（a）与统计混合物分离； （b）使用可裂解的交联链接器固定，该交联时会在切割时产生独特的基团；或（c）使用可裂解的交叉接头将一个或几个较大的蛋白质标签缀合，通过标签数量和裂解将金颗粒分离以产生精确官能化的金颗粒； （d）聚合配体与单个交联组桥接多功能金配位。将使用Cryoelectron显微镜标记和多功能蛋白的电子层析成像实验与Alasdair Steven（NIAMS，NIH）合作测试单功能，其中将使用该蛋白质的电位特异性标记，而无需聚集的位点特异性标记将用于确认真正的单函数反应。单功能的金纳米颗粒还将提供创新，以应对纳米科学中的关键挑战：能够构建分子纳米构造，其中具有独特和不同属性和附件点的多个纳米颗粒执行一组协调的动作以完成任务。这种系统的构建需要通过在不同位置的附件组装具有不同特性的颗粒组装。作为为此的关键促进技术，单功能的金纳米颗粒将有助于新一代纳米技术创新，例如生物传感器，指示器和分子设备，这些创新执行有针对性的，编程的任务，例如寡核苷酸，例如寡核苷酸，温度传感，或选择性地输送药物或增强局部辐射辐射或增强对Tumor dose dumor dose dumor dose dymor dose。