Multifunctional fluorogenic Ag nanodots for dynamic intracellular single molecule

描述（由申请人提供）：真正的单分子敏感探针可以提供动态和高度异质性细胞过程实时表征所需的技术进步。迄今为止，单分子方法已经有效地揭示了体外生物系统的环境和机制异质性;然而，细胞内动力学的观察仍然从根本上受到光学性质差、生物不相容性和不可用性、低可持续发射速率和潜在单分子荧光团的光稳定性差的限制。我们组建了一支优秀的团队，以创建和优化一类新型的高度光稳定的荧光单分子探针，能够实现非常高的持续单分子发射率，基本上没有实验相关的闪烁。这些封装在短ss-DNA链中的少原子Ag纳米点在低背景近IR区域中发射，其发射速率独特地使得能够实现捕获自由扩散蛋白质靶标的动力学所需的几毫秒帧速率，而不会使空间分辨率和信号/噪声中毒。这些是唯一具有足够光稳定性和持续（即基本上不闪烁）高发射速率的单价物质，保持小的总体尺寸（小至3 nm的总流体动力学直径）。相对于由10倍更亮的近IR纳米点发射体提供的可见光激发的10倍背景降低，一起达到了要跟踪和表征的真实细胞内单分子动力学所需的100倍改进。通过三个特定的目标，我们将这些强大的超亮和超小的纳米点开发成特定的，胞质内可用的，在体内荧光生物标记物，这些标记物在与靶蛋白结合之前是不发射的。在目的I中，我们将创建和表征荧光近红外发射纳米点探针与内含肽和SNAP标签在体内共轭，通过显微注射适合于细胞内单分子研究。在目标II中，我们将附加膜转运功能，并充分表征直接或间接胞质摄取和内体逃逸的途径和效率。荧光探针不会对背景产生影响，因为它们被设计为仅在与靶标缀合时发射。这些研究导致目标III，其中硫氧还蛋白动力学响应于引入的氧化应激优先运输Trx 1进入细胞核。将采用正交双色标记方案，并在单分子水平上表征Trx 1和Trx 2的应激诱导运输的微观速率。我们的长期目标是生产和传播足够灵敏的探针，用于细胞内单分子动力学的广义成像。一个具体的长期目标是球形（因此更高的背景）T细胞中硫氧还蛋白的氧化应激诱导动力学，以更充分地了解免疫反应。这种模块化的，小的，高发射和光稳定的纳米点工具箱将普遍适用于广泛的系统，即使在存在快速细胞内扩散的情况下，并将通过该项目提供给社区。公共卫生相关性：生物过程的异质性和灵活性赋予了对生存至关重要的适应性。这些不同步的动力学只能通过开发大大改进的蛋白质标签来可视化，这些标签能够通过单分子相互作用解开细胞内途径。虽然通常适用于其他细胞内动力学研究，但我们开发的多功能模块化Ag纳米点将导致观察与适应性免疫反应和癌症中涉及的氧化还原调节相关的同步多蛋白质动力学。

DESCRIPTION (provided by applicant): True single-molecule-sensitive probes can provide the technological advances needed for real-time characterization of dynamic and highly heterogeneous cellular processes. To date, single molecule methods have been effective in revealing the environmental and mechanistic heterogeneity of biological systems in vitro; however observation of intracellular dynamics remains fundamentally limited by poor optical properties, biological incompatibility and unavailability, low sustainable emission rates, and poor photostabilities of potential single molecule fluorophores. We have assembled an outstanding team to create and optimize a new class of highly photostable, fluorogenic single molecule probes capable of very high sustained single molecule emission rates with essentially no experimentally relevant blinking. These few-atom Ag nanodots encapsulated in short ss-DNA strands emit in the low background near IR region with emission rates that uniquely enable the few msec frame rates necessary to capture dynamics of freely diffusing protein targets without from poisoning spatial resolution and signal/noise. These are the only monovalent species with sufficient photostability and sustained (i.e. essentially non-blinking) high emission rates, maintaining small overall size (as small as 3nm total hydrodynamic diameter). The 10-fold background reduction relative to visible excitation afforded by the 10-fold brighter near IR nanodot emitters, together reaches the 100-fold improvements necessary for true intracellular single molecule dynamics to be followed and characterized. Through three specific Aims, we will develop these robust ultrabright and ultrasmall nanodots into specific, cytosolically available, in vivo fluorogenic biological labels that are non-emissive until bound to target protein. In Aim I we will create and characterize fluorogenic near IR-emitting nanodot probes with intein and SNAP tags for in vivo conjugation, suitable for intracellular single molecule studies through microinjection. In Aim II we will attach membrane transport functionality and fully characterize the pathways and efficiencies of direct or indirect cytosolic uptake and endosomal escape. The fluorogenic probes will not contribute to background as they are designed to be emissive only upon conjugation to the target. These studies lead to Aim III in which thioredoxin dynamics in response to introduced oxidative stress preferentially transports Trx1 into the nucleus. An orthogonal two-color labeling scheme will be employed and microscopic rates characterizing stress-induced trafficking of Trx1 and Trx2 will be characterized on the single molecule level. Our long-term goal is the production and dissemination of sufficiently sensitive probes for generalized imaging of intracellular single molecule dynamics. A specific long-term goal is the oxidative stress-induced dynamics of thioredoxin in spherical (and therefore higher background) T-cells to more fully understand immune response. This toolbox of modular, small, highly emissive and photostable nanodots will be generally applicable to a wide range of systems, even in the presence of fast intracellular diffusion, and will be made available to the community through this project. Public Health Relevance: Heterogeneity and flexibility in biological processes confer the adaptability that is crucial to survival. These unsynchronized dynamics can only be visualized through the development of greatly improved protein labels that enable the unraveling of intracellular pathways through single molecule interactions. While being generally applicable to other intracellular dynamics studies, our development of multifunctional, modular Ag nanodots will lead to observation of synchronous multi-protein dynamics associated with redox regulation implicated in adaptive immune response and cancer.