RNA APTAMER PROBES OF TRANSCRIPTIONAL MECHANISMS IN VIVO

这项建议描述了“抑制性适配子RNAs”(IaRNA)的开发和使用，以研究转录因子在体内的机制作用。该建议结合了体外选择(SELEX)分离与活性蛋白表面高亲和力结合的RNA适体的能力，以及控制、快速和高水平表达特定抑制性适体RNA的新策略。适体被选择来结合并潜在地干扰目标蛋白的特定功能。特定的iaRNAs在细胞和整个生物体中的快速和受控表达，应该可以对掩盖或抑制特定转录因子结构域或离散功能分子表面的主要影响进行机械性评估。最初的目标将是TATA结合蛋白(TBP)，它有几个不同的表面，在体外显示了许多与其他通用和特定转录因子以及DNA的良好相互作用。抑制TBP特定表面和启动子功能和结构的效果将通过已建立的启动子功能和结构分析来动态跟踪，包括核连续分析、体内足迹和蛋白质/DNA交联分析。这些研究应该可以对TBP在体内转录中的机制作用进行剖析。一组iaRNAs的体内受控表达将在两种研究良好的模式生物--酵母和果蝇--中进行评估，这两种模式生物在评估抑制机制和有效性方面具有特殊的优势。虽然这项建议最初的重点是TBP，但该方法应该适用于其他通用和特定的转录因子，更广泛地适用于任何可以选择高亲和力适配子的靶标。

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.