Multifunctional fluorogenic Ag nanodots for dynamic intracellular single molecule

描述(由申请人提供)：对许多生物过程的理解关键取决于在活细胞的背景下跟踪蛋白质的复杂动力学及其与其他生物分子相互作用的能力。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，由于缺乏合适的荧光标记，对细胞内动力学的观察仍然受到根本性的限制。即使是迄今最好的有机荧光团也存在光学性质差、可持续发射速率低和光稳定性差等问题，从而严重限制了它们在单分子研究中的应用。这一问题在存在自发荧光的细胞背景时变得更加严重，并且需要在延长的时间段内跟随在胞浆或细胞隔间内自由扩散的蛋白质。为了开发一类新型的光稳定的单分子探针，我们建议合成和优化封装在特殊设计的保护性有机支架中的少原子尺寸的银纳米团簇。作为对单链DNA封装和实现这一目标的平行途径的补充，这些标记的发射速率应该比最好的有机染料大10-100倍，这是因为它们具有很大的振荡器强度、出色的光稳定性以及几乎完全消除了闪烁路径。与蓝色激发相比，自发荧光的背景减少了10倍，所建议的荧光团应该会超过常规细胞内单分子观察所需的20倍的灵敏度增强。该项目分为两个目标，详细说明了1)所提议的几个原子大小的簇的可伸缩合成和光物理表征，以及2)它们的功能化以用于生物靶向，并并入到母基金建议的体内荧光连接的模块化四功能连接物中。从已发表的多核银簇合物的晶体结构中衍生出来的特殊设计的有机配体不仅可以作为形成簇合物的模板支架，还可以连接可交联的官能团来刚性支架，从而捕获簇合物。标准多肽化学中使用的官能团的附着将允许对支架进行进一步的修饰，以增强水的溶解性和生物偶联。基于单链DNA包裹的纳米点数据，所提出的3个原子大小的银团簇很可能在红色区域强烈发射，但模块化合成很容易扩展到2，4和5，甚至更大的团簇尺寸，以扩大可用的发射体，同时在广泛的生物相关环境中调节化学和光物理性质。建议的小，高发射率和光稳定的基于簇的发射体将通常适用于广泛的细胞内成像任务，即使在存在快速的细胞内扩散的情况下也是如此。作为一条平行的路径，这一竞争性修订将极大地加速执行真正的细胞内单分子研究，详细说明硫氧还蛋白在氧化应激下的核质运输。 公共卫生相关性：许多疾病源于不适当的蛋白质和生物分子相互作用，了解这种相互作用对于改进疾病诊断和治疗至关重要。虽然单分子观察为深入了解蛋白质相互作用的动力学提供了新的机会，但由于缺乏明亮的荧光标记，这些研究目前受到限制。基于少原子银团簇的新发射器有望在光稳定性和可持续发射速率方面提高10倍-这两个问题排除了细胞内单分子动力学的后续研究。这一竞争性修订概述了配体稳定的银纳米点的合成设计，以生产大量高度稳定、极其明亮的下一代生物标记。然后，这些通用的标记应该可供研究团体用于在整体和单分子水平上破译生命系统中的信号动力学。

DESCRIPTION (provided by applicant): The understanding of many biological processes critically depends on the ability to track the complex dynamics of proteins and their interactions with other biomolecules in the context of a living cell. 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 due to the lack of suitable fluorescent labels. Even the best organic fluorophores available to date suffer from poor optical properties, low sustainable emission rates, and poor photostabilities, thus seriously limiting their application in single molecule studies. This problem becomes even more severe in the presence of the autofluorescent cellular background and the need to follow freely diffusing proteins within the cytosol or cellular compartments over extended time periods. To develop a new class of photostable single molecule probes, we propose to synthesize and optimize few-atom sized silver nanoclusters encapsulated into a specifically designed protective organic scaffold. Complementary to ssDNA-encapsulation and a parallel path toward this goal, these labels should exhibit emission rates that are 10-100-fold greater compared to the best organic dyes due to large oscillator strengths, excellent photostability, and nearly complete abolition of blinking pathways. Combined with the 10-fold background reduction in autofluorescence relative to blue excitation, the proposed fluorophores should exceed the 20-fold sensitivity enhancements needed for routine intracellular single molecule observation. The project is structured into two aims that detail 1) the scalable synthesis and photophysical characterization of the proposed few-atom sized clusters, and 2) their functionalization for biological targeting and incorporation into the modular tetrafunctional linker proposed in the parent grant for fluorogenic in vivo conjugation. Specifically designed organic ligands derived from published crystal structures of multinuclear silver clusters will serve not only as the templating scaffold for cluster formation, but also for attaching cross-linkable functional groups to rigidify the scaffold, thereby entrapping the cluster. The attachment of functional groups used in standard peptide chemistry will allow for further modification of the scaffold to enhance water solubility and bioconjugation. Based on ssDNA-encapsulated nanodot data, the proposed 3 atom sized silver clusters are likely to emit strongly in the red region, but the modular syntheses are readily extended to 2, 4, and 5 or possibly even larger cluster sizes to expand the available emitters, while tuning chemical and photophysical properties in a wide range of biologically relevant environments. The proposed small, highly emissive and photostable cluster-based emitters will be generally applicable to a wide range of intracellular imaging tasks, even in the presence of fast intracellular diffusion. Tying into the parent grant as a parallel path this competitive revision should greatly accelerate performing true intracellular single molecule studies detailing the nucleocytoplasmic trafficking of thioredoxins in response to oxidative stress. Public Health Relevance: Many diseases stem from improper protein and biomolecule interactions and the understanding of such interactions is critical to the improvement of disease diagnostics and treatment. While single molecule observations offer new opportunities to gain insights into the dynamics of protein interactions, these studies are currently limited due to the lack of bright fluorescent labels. New emitters based on few-atom silver clusters promise >10-fold improvements in photostability and sustainable emission rate - the two issues precluding intracellular single molecule dynamics from being followed. This competitive revision outlines the synthetic design of ligand-stabilized Ag nanodots to yield large quantities of highly stable, exceedingly bright next-generation biolabels. These generalized labels should then be available to the research community for deciphering signaling dynamics within living systems on bulk and single molecule levels.