Quantitative stepwise analysis of RNA interference

描述(由申请人提供) 摘要：RNA干扰(RNAi)的发现在很大程度上影响了我们理解基因调控的方式。由于其独特的下调特定基因的能力，RNAi已被广泛应用于生物学研究和治疗目的。尽管小干扰RNA(SiRNA)和微小RNA(MiRNA)的来源不同，但在人类中，它们都是由RNAi途径的相同蛋白质机制加工的。RNA沉默蛋白的主要成分包括DICER、TRBP和ArgAerte(AGO)，它们组成了RNA诱导沉默复合体(RISC)。虽然单个组分的作用已被描述，但RNA和蛋白质组分之间的相互作用以及链选择和分离所涉及的机制尚未阐明，主要是由于缺乏可用的测量工具。此外，最近的研究报道了各种非靶点效应，这些效应会导致意外的基因沉默或极低的沉默效率，即使在RNA底物为其最大作用进行优化的情况下也是如此。我们建议发展一系列的单分子和单细胞测量来逐步剖析RNAi途径。我们的建议需要四个阶段的测试，具体如下。第一阶段是用TRBP对RNA进行预扫描。这是基于我们最近的发现，TRBP以不依赖于ATP的方式在双链RNA(DsRNA)上滑动。TRBP滑动显示依赖于底物，这意味着它在预扫描RNAi底物中的潜在作用。我们将测试各种前siRNA和前miRNA来测试TRBP滑动的选择性。第二个测试是Dester-TRBP介导的实时切割。使用我们建立的单分子平台，我们希望测量各种前siRNA和前miRNA底物的切割速度，如我们的初步数据所示。这将是对骰子效率的直接衡量。第三阶段包括DICER-TRBP-AGO，三链诱导解离和链选择，AGO将从哺乳动物细胞提取液中提取下来。这次测试的结果将以高精度揭开之前隐藏的步骤和负责链分离和选择的玩家。第四步采用单分子荧光原位杂交技术(SmFISH)直接检测给定RNAi底物的沉默效果。SmFISH使人们能够计算单个细胞中单个mRNAs的数量，这将作为沉默的直接指标。SmFISH数据将得到免疫荧光的补充，以在蛋白质水平上评估沉默效应。我们假设，在阶段1-3下检查的RNAi途径的每一步的效率将与RNAi底物沉默靶mRNA的程度相关，从而使这些测试成为RNAi应用的预测/优化工具。当这种技术被设计成一种设备时，可以被研究实验室和临床实践所采用，以设计具有高沉默效力的siRNA。综上所述，我们的建议包括开发新的单分子平台，允许定量和逐步分析RNAi途径，并开辟了一种评估和预测siRNA有效性的新方法。 公共卫生相关性：该提案寻求通过使用一系列单分子平台来剖析RNA干扰途径，目的是开发一种用于siRNA设计的预测工具。这一工具将能够评估和优化用于治疗目的的siRNA底物。我们将在黑色素瘤细胞上测试RNAi治疗下调Bcl-2和C-myc癌基因的作用。

DESCRIPTION (Provided by the applicant) Abstract: The discovery of RNA interference (RNAi) has impacted the way we understand gene regulation in a major way. Owing to its unique ability to down regulate a specific gene, RNAi has been widely used for biological research and applied for therapeutic purposes. Despite the differences in their origin, both small interfering RNA (siRNA) and micro RNA (miRNA) are processed by the same protein machineries of the RNAi pathway in human. The main components of the RNA silencing proteins include Dicer, TRBP and Argonaute (AGO), which make up the RNA induced silencing complex (RISC). Although the roles of individual components are delineated, the interplay between RNA and protein components and the mechanism involved in strand selection and separation has not been elucidated, mainly due to the lack of measurement tools available. Moreover, recent studies report various off-target effects which result in unintended gene silencing or extremely low efficiency of silencing even when the RNA substrates are optimized for its maximal effect. We propose to develop series of single molecule and single cell measurement to dissect the RNAi pathway step by step. Our proposal entails four stages of tests as detailed below. First stage is pre-scanning of RNA by TRBP. It is based on our recent discovery that TRBP slides on double stranded RNA (dsRNA) in ATP independent manner. TRBP sliding displays substrate dependens which implies its potential role in pre-scanning RNAi substrates. We will subject various pre-siRNA and pre-miRNA to test the selectivity of TRBP sliding. Second test is Dicer-TRBP mediated cleavage in real time. Using our established single molecule platform, we expect to measure the cleavage rate of various pre-siRNA and pre- miRNA substrates as demonstrated by our preliminary data. This will be a direct measure of the dicing efficiency. Third stage involves Dicer-TRBP-AGO, triplex induced unwinding and strand selection, where AGO will be pulled down from mammalian cell extract. The result from this test will unfold the previously hidden steps and players responsible for strand separation and selection with high precision. Fourth step employs single molecule fluorescence in situ hybridization (smFISH) to directly measure the silencing efficacy of a given RNAi substrate. The smFISH enables one to count the number of individual mRNAs in a single cell and this will be serve as a direct indicator of silencing. The smFISH data will be complemented by immunofluorescence to assess the silencing effect at the protein level. We hypothesize that the efficiency at each step of RNAi pathway to be examined under stage 1-3 will be correlated with how well the RNAi substrate will silence the target mRNA, making these tests a prediction/optimization tool for RNAi application. When engineered into a device, such technique can be adopted by research laboratories and clinical practices for designing siRNA for high silencing potency. In summary, our proposal involves developing novel single molecule platforms which allow quantitative and stepwise analysis of RNAi pathway and opens a new way of assessing and predicting siRNA efficacy. Public Health Relevance: This proposal seeks to dissect the RNA interference pathway by using series of single molecule platforms with the goal of developing a prediction tool for siRNA design. This tool will enable assessment and optimization of siRNA substrate to be applied for therapeutic purposes. We will test the RNAi treatment on melanoma cells for down regulating Bcl-2 and C-myc oncogenes.