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背景下跟踪蛋白质复杂动力学及其与其他生物分子相互作用的能力。迄今为止，单分子方法在揭示体外生物系统的环境和机制异质性方面是有效的；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限制。即使是迄今为止最好的有机荧光团也存在光学性能差、可持续排放率低和光稳定性差的问题，因此严重限制了它们在单分子研究中的应用。在自身荧光细胞背景的存在下，这个问题变得更加严重，并且需要在很长一段时间内跟踪胞浆或细胞室内自由扩散的蛋白质。为了开发一类新的光稳定单分子探针，我们提出了合成和优化包裹在特殊设计的保护性有机支架中的小原子大小的银纳米团簇。作为ssdna封装的补充和实现这一目标的平行路径，这些标签的发射速率应该比最好的有机染料高10-100倍，因为它们具有大的振荡器强度、优异的光稳定性和几乎完全消除闪烁路径。结合自身荧光相对于蓝色激发的10倍背景降低，所提出的荧光团应超过常规细胞内单分子观察所需的20倍灵敏度增强。该项目分为两个目标，详细说明1)可扩展的合成和光物理表征所提出的少原子大小的团簇，以及2)其功能化的生物靶向和整合到模块化的四功能连接在父母的资助提出的荧光体内偶联。根据已发表的多核银团簇晶体结构，专门设计的有机配体不仅可以作为团簇形成的模板支架，还可以连接交联官能团，使支架刚性，从而包裹团簇。标准肽化学中使用的官能团的连接将允许对支架进行进一步修饰，以提高水溶性和生物偶联性。基于ssdna封装的纳米点数据，所提出的3原子大小的银簇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甚至更大的簇尺寸，以扩大可用的发射器，同时在广泛的生物相关环境中调整化学和光物理性质。所提出的小型，高发射和光稳定的簇基发射器将普遍适用于广泛的细胞内成像任务，即使存在快速细胞内扩散。作为一个平行的途径，这种竞争性的修订将极大地加速进行真正的细胞内单分子研究，详细说明核胞质中硫氧还毒素在氧化应激下的运输。

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.