Multifunctional fluorogenic Ag nanodots for dynamic intracellular single molecule

描述(申请人提供)：真正的单分子敏感探针可以提供实时表征动态和高度异质细胞过程所需的技术进步。到目前为止，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的异质性；然而，细胞内动力学的观察仍然受到光学性质差、生物不相容和不可用、低可持续发射率以及潜在的单分子荧光团的光稳定性差的根本限制。我们组建了一支优秀的团队来创造和优化新型的高度光稳定性、荧光单分子探测器，能够实现非常高的持续单分子发射率，基本上没有实验上的相关闪烁。这些包裹在短链ss-DNA中的少原子银纳米点在近红外区域的低背景中发射，其发射速率独特地实现了捕获自由扩散蛋白质目标的动力学所需的几毫秒帧速率，而不会影响空间分辨率和信号/噪声。它们是唯一具有足够的光稳定性和持续(即基本上不闪烁)高发射率的单价物种，保持较小的总体尺寸(小至3 nm总流体动力学直径)。与可见光激发相比，10倍亮度的近红外纳米点发射器提供的背景减少了10倍，加在一起达到了跟踪和表征真正的细胞内单分子动力学所需的100倍的改进。通过三个具体的目标，我们将把这些坚固的超亮和超小的纳米点发展成特定的、细胞可用的、体内可荧光的生物标记，这些标记在与目标蛋白结合之前是不发射的。在目标I中，我们将创建和表征带有内含素和SNAP标记的荧光近红外发射纳米点探针，用于体内连接，适合通过显微注射进行细胞内单分子研究。在AIM II中，我们将添加膜转运功能，并充分表征胞浆直接或间接摄取和内体逃逸的途径和效率。荧光探针将不会对背景有贡献，因为它们被设计为仅在与目标结合时才发射。这些研究导致了AIM III，其中硫氧还蛋白动力学响应引入的氧化应激优先将Trx1转运到细胞核中。将采用正交双色标记方案，并将在单分子水平上表征应力诱导的Trx1和Trx2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通用成像。一个特定的长期目标是在球形(因此更高的背景)T细胞中研究氧化应激诱导的硫氧还蛋白的动力学，以更全面地了解免疫反应。这种由模块化、小、高发射性和光稳定性纳米点组成的工具箱将普遍适用于各种系统，即使在细胞内快速扩散的情况下也是如此，并将通过该项目向社区提供。公共卫生相关性：生物过程中的异质性和灵活性赋予了对生存至关重要的适应性。这些不同步的动态只能通过极大改进的蛋白质标记的开发来可视化，这种标记能够通过单分子相互作用解开细胞内的通路。虽然普遍适用于其他细胞内动力学研究，但我们开发的多功能、模块化的银纳米点将导致观察与适应性免疫反应和癌症相关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.