Plasmon Rulers

描述（由申请人提供）：中心生物学功能，如复制，转录，mRNA剪接，运输和信号传导是由分子机器执行的。标准的结构技术无法处理大尺寸的分子复合物并提供最小的动态信息，而基于荧光的技术则受到传统有机染料的光不稳定性和复杂的光物理特性的限制。我们发现纳米粒子对可以通过等离子体激元耦合的距离依赖来监测距离。这些“等离子体标子”揭示了过程的时间动力学，如单DNA杂交事件和单酶与其DNA底物的相互作用。合适的涂覆和功能化等离子体标尺最近使单分子DNA酶促弯曲和切割的研究具有毫秒和纳米分辨率。等离子体标尺不会聚集或扰乱酶动力学，并允许同时观察约5000个单独的DNA底物。等离子体标尺的可达距离范围为0-80nm，其光稳定性使得对单个生物分子进行数天的监测成为可能。我们提出的研究有三个目的。首先，我们将优化等离子体标尺的光学特性，完善纳米粒子钝化程序，并开发用于监测等离子体标尺的改进显微镜。这些技术的改进将允许没有等离子体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尺。其次，我们将使用等离子体尺子来研究Dicer的结构动力学、底物要求和机械化学，Dicer是rna诱导沉默复合体（RISC）的核心成分。RNAi是一种广泛的基因调控机制，通过特定基因的转录后沉默。risc组装和功能典型的细胞过程不容易适应分析的传统方法，如FRET。第三，作为在体内使用等离子体调控子的第一步，我们将建立可靠的方法将等离子体调控子输送到细胞中，防止等离子体调控子在细胞质中聚集，并评估其可能的细胞毒性。总之，拟议的研究将为生物学家提供一种新的工具，用于监测单分子机器，具有高时间和空间分辨率，并且几乎无限次。通过开发具有极高亮度和光稳定性的光学探针，新的特异和敏感的诊断工具将成为可能。RNA干扰的单分子研究将有助于建立其基本的机械化学，并可能使全基因组扫描的发展成为可能。最后，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，以最小的脱靶效应用于治疗。

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.