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跟踪蛋白质的复杂动力学及其在活细胞背景下与其他生物分子相互作用的能力。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，由于缺乏合适的荧光标记，细胞内动力学的观察仍然从根本上受到限制。即使是迄今为止最好的有机荧光团也存在光学性质差、可持续发射速率低和光稳定性差的问题，从而严重限制了它们在单分子研究中的应用。这个问题变得更加严重的存在下，自发荧光的细胞背景和需要遵循自由扩散的蛋白质在胞质溶胶或细胞隔室在延长的时间段。为了开发一类新型的光稳定单分子探针，我们建议合成和优化几个原子大小的银纳米团簇封装到一个专门设计的保护性有机支架。作为对ssDNA包封的补充和实现这一目标的平行路径，这些标记应该表现出比最好的有机染料大10-100倍的发射速率，这是由于大的振荡器强度、优异的光稳定性和几乎完全消除闪烁途径。结合自发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，详细说明了1）所提出的几个原子大小的簇的可扩展合成和生物物理表征，以及2）它们的生物靶向功能化，并纳入母基金中提出的用于荧光体内缀合的模块化四功能接头中。衍生自多核银簇的公开晶体结构的专门设计的有机配体将不仅用作簇形成的模板支架，而且用于连接交叉交联官能团以使支架刚性化，从而包埋簇。在标准肽化学中使用的官能团的连接将允许支架的进一步修饰以增强水溶性和生物缀合。基于ssDNA封装的纳米点数据，所提出的3个原子大小的银簇可能在红色区域中强烈发射，但是模块化合成容易扩展到2、4和5或甚至可能更大的簇大小以扩展可用的发射体，同时在广泛的生物相关环境中调节化学和生物物理性质。所提出的小的，高发射和光稳定的基于簇的发射器将通常适用于广泛的细胞内成像任务，即使在存在快速细胞内扩散的情况下。作为一个平行的路径，这种竞争性的修订将大大加快执行真正的细胞内单分子研究，详细说明了氧化应激反应中硫氧还蛋白的核质运输。 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的相互作用和理解这种相互作用是至关重要的疾病诊断和治疗的改进。虽然单分子观测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。基于少原子银簇的新发射体承诺在光稳定性和可持续发射速率方面有>10倍的改进-这两个问题排除了细胞内单分子动力学。这一竞争性修订概述了配体稳定的银纳米点的合成设计，以产生大量高度稳定，非常明亮的下一代生物标记。然后，这些通用标签应该可用于研究社区，用于在批量和单分子水平上破译生命系统内的信号动力学。

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.