RNA APTAMER PROBES OF TRANSCRIPTIONAL MECHANISMS IN VIVO

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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.