Plasmon Rulers

项目摘要 核心生物学功能，如复制、转录、mRNA剪接、运输和信号传导 都是由分子机器完成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， 受到常规有机染料的光不稳定性和复杂的光物理学的限制。 我们已经发现纳米粒子对可以用来通过距离来监测距离- 依赖于它们的等离子体耦合。这些“等离子体规则”揭示了诸如 单个DNA杂交事件和单个酶与其DNA底物的相互作用。适当 最近，涂覆和功能化的等离子体激元尺使得能够对酶促DNA进行单分子研究 具有毫秒和纳米分辨率的弯曲和解理。等离子体统治者没有聚集或 干扰酶动力学，并允许同时观察约5000个单独的DNA底物。的 等离激元标尺的可达距离范围为0- 80 nm，其光稳定性使得监测成为可能 一个生物分子数天。我们提出的研究有三个目标。 首先，我们将优化等离子体标尺的光学性质，细化纳米颗粒钝化 程序，并开发改进的显微镜监测等离子统治者。这些技术改进 将允许没有等离子体和纳米粒子专业知识的研究人员使用等离子体统治者， 他们的研究。其次，我们将使用等离子体标尺来研究结构动力学、基底 Dicer是RNA诱导沉默复合物的核心成分， （RISC）。RNAi是一种广泛的基因调控机制，通过转录后沉默特异性转录因子来实现。 基因. RISC的组装和功能是细胞过程的典型，不容易通过以下方法进行分析： 常规方法如FRET。第三，作为在体内使用等离子体规则的第一步，我们将 建立可靠的方法将等离子体规则子传递到细胞，防止等离子体规则子聚集， 细胞质，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个新的 一种监测单分子机器的工具，具有高的时间和空间分辨率， 次项目叙述 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针， 新的特异性和敏感性诊断工具将变得可行。RNA的单分子研究 干扰将有助于建立其基本的机械化学，并可能使全基因组的发展 微RNA扫描最后，RNA干扰的单分子研究也可能有助于设计 具有最小脱靶效应的有效短干扰RNA（siRNA）序列用于治疗用途。

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.