Monofunctional 3 to 10 nm Covalent Gold Labels for CryoEM

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，因为它们提供了尺寸选择，提供了定量的、高分辨率的靶标识别。近年来，包括低温电子显微镜和电子层析成像在内的高分辨率电磁方法得益于仪器的重大进步以及样品处理和制备设备的一系列突破，使这些方法得到了广泛的应用。然而，传统的胶体金标记物不能充分实现这些方法的潜力，因为它们需要大分子的广泛稳定，限制了穿透和抗原进入，还因为它们通过吸附到靶向抗体或蛋白质的偶联机制不允许以足够高的分辨率直接标记大分子复合体中的特定反应部位。纳米探针已经开发出具有单一反应基团的1.4 nm(纳米金)和0.8 nm(十一金)金纳米颗粒标记，用于选择性、共价标记硫醇(半胱氨酸残基)、胺(N端胺或赖氨酸残基)或其他官能团。然而，这些都太小了，不容易在许多类型的标本中可视化，并且不允许多重标记。虽然共价反应性已经被引入到更大的颗粒中，但事实证明，能够1：1标记的单功能试剂的制备具有很高的挑战性。到目前为止，还没有具有天然单功能反应性的产品。我们将合成金纳米颗粒，它结合了高度的单分散性、可控的表面官能化和单功能反应化学计量比的选择性反应能力。制剂将被规模化和简化，以高效、可重复的方式提供具有以下特性的纳米颗粒 (1)单分散性：采用硫氰酸盐还原、柠檬酸/单宁酸或均相还原的方法可以制备3 nm到10 nm的金纳米粒子，其变异系数小于或等于10%。(2)表面性质：将开发一套通用的有机配体碱基，它可以赋予(A)水溶性和生物相容性，(B)所需的疏水性程度，(C)特定的共价反应活性，以及(D)可控制的壳层厚度从0.6 nm到3 nm。允许选择任意大小的这些属性的任意组合。(3)特殊反应性：胺、硫醇和活性羧基反应性可通过以下方式引入：(A)在合成和后来的修饰过程中加入伯胺，以及(B)加入保护的反应性，然后取消保护。(4)单一功能：四种策略将被用来产生与溶液中的单个共轭分子反应的粒子：(A)从统计混合物中分离；(B)使用可切割的交联剂进行固定化，其在切割时产生独特的基团；或(C)使用可切割的交联剂将一个或极少数较大的蛋白质标签连接，通过标签的数量和切割来分离金粒子，以获得精确功能化的金粒子；以及(D)将多官能团金与单一交联基配位的聚合物配体。将与Alasdair Steven(NIAMS，NIH)合作，使用冷冻电子显微镜标记和多功能蛋白质的电子断层扫描实验进行单功能测试，在这些实验中，将使用无聚集的特定部位标记来确认真正的单功能反应活性。单功能金纳米粒子还将提供创新，以应对纳米科学中的一个关键挑战：使分子纳米设备的构建成为可能，在分子纳米设备中，具有独特和不同性质的多个纳米粒子和连接点执行一系列协调行动，以完成一项任务。这种体系的构建需要具有不同性质的颗粒通过在不同位置附着而组装成构造。作为实现这一目标的关键技术，单功能金纳米粒子将有助于支持新一代纳米技术创新，如生物传感器、指示器和分子设备，这些设备可以执行有针对性的编程任务，如寡核苷酸退火、温度传感，或者选择性地向肿瘤细胞输送药物或增加局部辐射剂量。

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.