Quantitative stepwise analysis of RNA interference

RNA干扰的定量逐步分析

• 批准号: 8352997
• 负责人: Sua Myong
• 金额: $ 96.09万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2015-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8352997
• 关键词: Adopted; Cell Extracts; Cells; Complement; Data; Devices; Double-Stranded RNA; Engineering; Fluorescent in Situ Hybridization; Gene Expression Regulation; Gene Silencing; Genes; Goals; Human; Immunofluorescence Immunologic; Individual; Laboratory Research; MYC gene; Mammalian Cell; Measurement; Measures; Mediating; Melanoma Cell; Messenger RNA; MicroRNAs; Proteins; RNA; RNA Interference; RNA Interference Pathway; RNA Processing; RNA-Induced Silencing Complex; Reporting; Role; Scanning; Series; Slide; Small Interfering RNA; Staging; Techniques; Test Result; Testing; Therapeutic; Time; abstracting; base; biological research; clinical practice; design; human DICER1 protein; novel; public health relevance; single molecule; tool

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.

描述（由申请人提供） 翻译后摘要：RNA干扰（RNAi）的发现已经影响了我们理解基因调控的一个主要方式。由于其独特的下调特定基因的能力，RNAi已被广泛用于生物学研究和应用于治疗目的。尽管它们的来源不同，但小干扰RNA（siRNA）和微小RNA（miRNA）都由人类RNAi途径的相同蛋白质机制加工。RNA沉默蛋白的主要成分包括Dicer、TRBP和Argonaute（AGO），它们组成了RNA诱导沉默复合物（RISC）。虽然个别成分的作用被描绘，RNA和蛋白质成分之间的相互作用和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，最近的研究报告了各种脱靶效应，其导致非预期的基因沉默或极低的沉默效率，即使当RNA底物被优化以达到其最大效应时。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，详细如下。第一阶段是通过TRBP预扫描RNA。这是基于我们最近发现TRBP以ATP非依赖性方式在双链RNA（dsRNA）上滑动。TRBP滑动显示底物依赖性，这意味着它在预扫描RNAi底物中的潜在作用。我们将对各种pre-siRNA和pre-miRNA进行测试，以测试TRBP滑动的选择性。第二个测试是Dicer-TRBP介导的真实的实时切割。使用我们建立的单分子平台，我们期望测量各种pre-siRNA和pre-miRNA底物的切割速率，如我们的初步数据所证明的。这将是切割效率的直接量度。第三阶段涉及Dicer-TRBP-AGO，三链体诱导的解旋和链选择，其中AGO将从哺乳动物细胞提取物中拉下。这项测试的结果将揭示先前隐藏的步骤和负责高精度链分离和选择的参与者。第四步采用单分子荧光原位杂交（smFISH）来直接测量给定RNAi底物的沉默功效。smFISH使人们能够计数单个细胞中单个mRNA的数量，这将作为沉默的直接指标。smFISH数据将通过免疫荧光进行补充，以评估蛋白质水平的沉默效应。我们假设在阶段1-3下待检测的RNAi途径的每个步骤的效率将与RNAi底物沉默靶mRNA的程度相关，使这些测试成为RNAi应用的预测/优化工具。当工程化到装置中时，这种技术可以被研究实验室和临床实践采用，用于设计siRNA以获得高沉默效力。总之，我们的建议涉及开发新的单分子平台，其允许定量和逐步分析RNAi途径，并开辟了评估和预测siRNA功效的新途径。 公共卫生相关性：该提案旨在通过使用一系列单分子平台来剖析RNA干扰途径，目标是开发用于siRNA设计的预测工具。该工具将能够评估和优化siRNA底物以用于治疗目的。我们将在黑色素瘤细胞上测试RNAi治疗是否下调Bcl-2和C-myc癌基因。