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Double-Stranded RNA and Antisense Regulation in Vivo

双链 RNA 和体内反义调控

基本信息

批准号：
7067911
负责人：
STEPHEN H MUNROE
金额：
$ 0.59万
依托单位：
MARQUETTE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-02-01 至 2007-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7067911
关键词：
Mammalia RNA interference antisense nucleic acid complementary DNA double stranded RNA genetic library genetic regulation genetic screening genetic transcription high throughput technology intermolecular interaction method development molecular cloning molecular genetics neoplastic cell culture for noncancer research nucleic acid quantitation /detection tissue /cell culture transfection

项目摘要

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop methods for characterizing stable base-pairing interactions between complementary RNA molecules in vivo. These methods will be used to characterize the presence of double-stranded RNA within in mammalian cells in order to analyze mechanisms for antisense regulation. Although antisense regulation has been well-documented and mechanistically explored in several prokaryotic systems relatively little is known regarding the mechanism by which antisense RNAs alter the expression of complementary transcripts in eukaryotes. However, there is accumulating evidence that many regions of the mammalian genome expressed overlapping that are transcribed from both strands of the chromosome. In some instances this transcription may give rise to antisense regulation in which expression of a target transcript is repressed by base pairing with complementary antisense RNA present. The proposed study has two specific goals: (1) to develop methods for the isolation, identification and characterization of doublestranded RNA formed in vivo; and (2) to prepare libraries of amplified cDNAs from isolated dsRNA that will be screened using either random or targeted strategies. A key feature of this strategy is that single-stranded RNAs will be modified prior to isolation to prevent adventitious annealing. To facilitate identification of condition that are optimal for isolation of native double-stranded RNA, a synthetic RNA hairpin will express in transfected embryonic carcinoma cells to serve as a target sequence in Specific Aim 1. Finally, libraries will be screened for complementary overlapping sequences using both gene specific probes and high throughput methods to characterize the population of stable double-stranded RNAs. The results of these studies will provide insight into the formation and stability of RNA-RNA base-paired interactions in vivo, and the methods and information needed for further studies of the functional roles of such interactions.

描述（由申请人提供）：本提案的总体目标是开发用于表征体内互补RNA分子之间稳定碱基配对相互作用的方法。这些方法将用于表征哺乳动物细胞内双链RNA的存在，以分析反义调控的机制。虽然反义调控已被充分记录和机制探讨在几个原核系统相对知之甚少的机制，反义RNA改变互补转录本在真核生物中的表达。然而，越来越多的证据表明，哺乳动物基因组的许多区域表达重叠，从染色体的两条链转录。在某些情况下，这种转录可以引起反义调节，其中靶转录物的表达通过与存在的互补反义RNA的碱基配对而被抑制。该研究有两个具体目标：（1）开发用于分离、鉴定和表征体内形成的双链RNA的方法;（2）从分离的dsRNA制备扩增cDNA文库，这些cDNA将使用随机或靶向策略进行筛选。这种策略的一个关键特征是单链RNA在分离之前将被修饰以防止偶然退火。为了便于鉴定分离天然双链RNA的最佳条件，合成RNA发夹将在转染的胚胎癌细胞中表达以充当特异性Aim 1中的靶序列。最后，将使用基因特异性探针和高通量方法筛选文库中的互补重叠序列，以表征稳定双链RNA的群体。这些研究的结果将提供深入了解RNA-RNA碱基配对相互作用在体内的形成和稳定性，以及进一步研究这种相互作用的功能作用所需的方法和信息。