Plasmon Rulers

描述(申请人提供)：复制、转录、mRNA剪接、运输和信号传递等中心生物学功能由分子机器执行。标准的结构技术不能处理大尺寸的分子络合物，提供的动态信息很少，而基于荧光的技术受到传统有机染料的光稳定性和复杂的光物理的限制。我们已经发现，成对的纳米粒子可以通过其等离子体耦合的距离依赖关系来监测距离。这些“血浆尺子”揭示了过程的时间动力学，如单一脱氧核糖核酸杂交事件以及单酶与其脱氧核糖核酸底物的相互作用。经过适当涂层和功能化的等离子激元尺子最近实现了毫秒和纳米级分辨率的单分子酶dna弯曲和切割研究。血浆尺子不聚集或扰乱酶的动力学，并允许同时观察约5,000个单独的dna底物。等离激元尺子的可及距离为0-80 nm，其光稳定性使得对单个生物分子的监测成为可能。我们提出的研究有三个目标。首先，我们将优化等离子激元尺子的光学性质，完善纳米粒子钝化程序，并开发用于监测等离子体激元尺子的改进的显微镜。这些技术改进将允许没有等离子体激元和纳米粒子专业知识的研究人员在他们的研究中使用等离子体激元尺子。其次，我们将使用等离子激元尺子来研究DICER的结构动力学、底物要求和机械力化学，DICER是RNA诱导沉默复合体的中心成分。RNAi是一种广泛存在的通过特定基因转录后沉默进行基因调控的机制。RISC-组装和-功能代表了不易用常规方法如FRET进行分析的细胞过程。第三，作为在体内使用血浆尺子的第一步，我们将建立可靠的方法将血浆尺子输送到细胞，防止血浆尺子一旦聚集在细胞质中，并评估其可能的细胞毒性。总之，这项拟议的研究将为生物学家提供一种新的工具，以高时间和空间分辨率监测单分子机器，并几乎无限次。通过开发具有极高亮度和光稳定性的光学探针，新的特定和灵敏的诊断工具将变得可行。RNA干扰的单分子研究将有助于建立其基本的机械力化学，并可能使全基因组扫描microRNAs的发展成为可能。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，使其脱靶效应最小，用于治疗。

DESCRIPTION (provided by applicant): 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. 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.