RNA APTAMER PROBES OF TRANSCRIPTIONAL MECHANISMS IN VIVO

体内转录机制的 RNA 适体探针

• 批准号: 6032084
• 负责人: JOHN T LIS
• 金额: $ 19.46万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6032084
• 关键词: DNA footprinting; Drosophilidae; RNA; RNA binding protein; Saccharomyces cerevisiae; crosslink; gene expression; genetic library; genetic promoter element; genetic transcription; immunoprecipitation; intermolecular interaction; nuclear runoff assay; nucleic acid inhibitor; nucleic acid probes; nucleic acid sequence; oligonucleotides; polymerase chain reaction; protein structure function; transcription factor

This proposal describes the development and use of "inhibitory aptamer RNAs" (iaRNA) to study the mechanistic role of transcription factors in vivo. The proposal combines the power of in vitro selection (SELEX) for isolating RNA aptamers that bind to active protein surfaces with high affinity, and new strategies for controlled, rapid, and high level expression of specific inhibitory aptamer RNAs. The aptamers are selected to bind and potentially interfere with specific functions of a target protein. The rapid and controlled expression of particular iaRNAs in cells and whole organisms should allow mechanistic assessment of the primary effects of masking or inhibiting particular transcription factor domains or discrete functional molecular surfaces. The initial target will be the TATA-binding protein (TBP), which has several distinct surfaces that display numerous well-documented in vitro interactions with other general and specific transcription factors as well as DNA. The effects of inhibiting specific surfaces of TBP and promoter function and structure will be followed kinetically using established assays of promoter function and structure including nuclear run-on assays, in vivo footprinting and protein/DNA crosslinking assays. These studies should allow the dissection of the mechanistic roles of TBP in transcription in vivo. The controlled in vivo expression of a set of iaRNAs will be assessed in two well studied model organisms, yeast and Drosophila, that offer particular advantages in evaluating the mechanism and efficacy of inhibition. While the initial focus of this proposal is on TBP, the approach should be applicable to other general and specific transcription factors and more broadly to any target to which a high affinity aptamer can be selected.

该建议描述了“抑制性适体RNA”（IARNA）研究转录因子在体内的机理作用。 该提案结合了体外选择（SELEX）的能力，用于分离与具有高亲和力的活性蛋白质表面结合的RNA适体，以及针对特定抑制性适体RNA的受控，快速和高水平表达的新策略。 选择适体以结合并可能干扰靶蛋白的特定功能。 特定IARNA在细胞和整个生物中的快速和控制表达应允许对掩盖或抑制特定转录因子域或离散功能分子表面的主要作用进行机械评估。 最初的靶标将是TATA结合蛋白（TBP），该蛋白具有几个不同的表面，这些表面与其他一般和特定的转录因子以及DNA显示了许多有据可查的体外相互作用。抑制TBP和启动子功能和结构的特定表面的影响将使用启动子功能和结构的既定测定法进行，包括核跑步测定，体内足迹以及蛋白/DNA交联测定。 这些研究应允许解剖TBP在体内转录中的机械作用。 一组IARNA的体内表达将在两个研究的模型生物分别进行酵母和果蝇中评估，在评估抑制的机制和功效方面具有特殊的优势。 尽管该提案的最初重点是TBP，但该方法应适用于其他一般和特定的转录因子，并且更广泛地适用于可以选择高亲和力适体的任何目标。