Plasmon Rulers

项目摘要 中枢生物学功能，如复制、转录、mRNA剪接、运输和信号传递 都是由分子机器完成的。标准的结构技术无法处理大型的 分子络合物并提供最少的动态信息，而基于荧光的技术是 受限于传统有机染料的光稳定性和复杂的光物理特性。 我们已经发现，成对的纳米颗粒可以用来监测距离- 对它们的等离子体激元耦合的依赖性。这些“血浆尺子”揭示了如下过程的时间动力学 单DNA杂交事件以及单酶与其DNA底物的相互作用。适当地 包被和功能化的等离子激元尺子最近使酶脱氧核糖核酸的单分子研究成为可能 弯曲和劈裂，分辨率为毫秒和纳米。血浆尺子没有聚集或 扰动酶动力学，并允许同时观察约5000个DNA底物。这个 等离激元尺子的可达距离为0-80 nm，其光稳定性使监测成为可能 单个生物分子持续数天。我们提出的研究有三个目标。 首先，我们将优化等离子体激元尺子的光学性质，细化纳米粒子钝化 程序，并开发改进的显微镜来监测血浆尺子。这些技术改进 将允许研究人员在没有等离子体和纳米颗粒专业知识的情况下使用等离子体尺子 他们的研究。第二，我们将使用等离子激元尺子来研究结构动力学，衬底 RNA诱导沉默复合体的中心成分DICER的需求和机械力化学 (RISC)。RNAi是一种广泛的基因调控机制，通过转录后沉默特异性 基因。RISC-组装和功能代表了不易分析的细胞过程 传统的方法，如FRET。第三，作为在体内使用血浆蛋白尺子的第一步，我们将 建立可靠的方法将血浆蛋白尺子输送到细胞内，防止血浆蛋白一旦在细胞内聚集 细胞质，并评估其可能的细胞毒性。总而言之，这项拟议的研究将为生物学家提供一个新的 监控单分子机器的工具，时间和空间分辨率高，几乎不受限制 泰晤士报。项目叙事 公共卫生意义。通过开发具有极高亮度和光稳定性的光学探头， 新的特定和敏感的诊断工具将变得可行。核糖核酸的单分子研究 干扰将有助于建立其基本的机械力化学，并可能使全基因组的发展 扫描microRNA。最后，对RNA干扰的单分子研究也可能有助于设计 有效的短干扰RNA(SiRNA)序列，具有最小的偏离靶标效应，用于治疗用途。

Project Summary Central biological functions such as replication, transcription, mRNA splicing, transport, and signaling are performed by molecular machines. Standard structural techniques fail to deal with the large size of molecular complexes and provide minimal dynamic information, while fluorescence-based techniques are limited by the photo-lability and complex photophysics of conventional organic dyes. We have found that pairs of nanoparticles can be used to monitor distances via the distance- dependence of their plasmon coupling. These `plasmon rulers' reveal the time dynamics of processes such as single DNA hybridization events and the interaction of single enzymes with their DNA substrates. Suitably coated and functionalized plasmon rulers have recently enabled single-molecule studies of enzymatic DNA bending and cleavage with millisecond and nanometer resolution. The plasmon rulers did not aggregate or perturb enzyme kinetics and allowed simultaneous observation of about 5000 individual DNA substrates. The accessible distance range of plasmon rulers is 0-80nm and their photostability makes it possible to monitor single biomolecules for days. Our proposed research has three aims. First, we will optimize the optical properties of the plasmon rulers, refine nanoparticle passivation procedures, and develop improved microscopies for monitoring plasmon rulers. These technical refinements will allow researchers without specialized knowledge of plasmonics and nanoparticles to use plasmon rulers in their research. Second, we will use plasmon rulers to investigate the structural dynamics, substrate requirements, and mechanochemistry of Dicer, a central component of the RNA-induced silencing complex (RISC). RNAi is a widespread mechanism of gene regulation via post-transcriptional silencing of specific genes. RISC-assembly and -function typifies cellular processes not easily amenable to analysis by conventional methods such as FRET. Third, as a first step towards using plasmon rulers in vivo, we will establish reliable methods to deliver plasmon rulers to cells, prevent plasmon ruler aggregation once in the cytoplasm, and evaluate their possible cytotoxicity. Together, the proposed research will give biologists a new tool for monitoring single molecular machines with high temporal and spatial resolution and for nearly unlimited times. Project Narrative Public Health Significance. By developing optical probes with extreme brightness and photostability, new specific and sensitive diagnostic tools will become feasible. The single-molecule studies of RNA interference will help establish its basic mechanochemistry and may enable the development of genome-wide scans for microRNAs. Finally, the single-molecule studies of RNA interference may also facilitate the design of efficient short interfering RNA (siRNA) sequences with minimal off-target effects for therapeutic use.