RNA aptamer probes of transcriptional mechanisms in vivo

描述（由申请人提供）：本研究的长期目标是开发和应用一种新的策略来研究在活细胞和生物体中调节真核基因表达的分子相互作用。这种新策略结合了体外选择（SELEX）的力量，用于分离高亲和力结合活性蛋白表面的RNA适体，以及实现快速，高水平和控制特异性抑制性适体RNA （iaRNAs）表达和活性的新方法。在高等真核生物中使用这种策略提供了一种快速灭活蛋白质靶向结构域的方法，从而可以评估它们在体内的主要功能和作用机制。该提议与TATA结合蛋白（TBP）有关，它是启动子结构和功能的核心参与者。已知的TBP与一系列合作和竞争转录因子的物理相互作用是这些抑制剂的目标，也是这些机制研究的重点。

DESCRIPTION (provided by applicant): The long-term objective of this study is to develop and apply a new strategy for investigating molecular interactions that modulate eukaryotic gene expression in living cells and organisms. This new strategy combines the power of in vitro selection (SELEX) for isolating RNA aptamers that bind to active protein surfaces with high affinity, and new approaches for achieving rapid, high-level and controlled expression and activity of specific inhibitory aptamer RNAs (iaRNAs). The use of this strategy in higher eukaryotes provides a means of rapidly inactivating targeted domains of proteins, thereby allowing an assessment of their primary function and mechanism of action in vivo. The proposal has as its nexus the TATA Binding Protein (TBP), a central player in promoter structure and function. TBP's known physical interactions with a collection of cooperating and competing transcription factors are the targets of these inhibitors and the focus of these mechanistic studies. The specific aims are to: 1) Further characterize the structure and activity of selected high-affinity RNA aptamers against TBP (TBPaptamers) and use of these in vitro to address mechanistic roles of TBP-TATA interaction in transcription initiation and reinitiation. 2) Select and characterize in vitro additional aptamers that bind distinct surfaces of TBP and that bind to other transcription factors that interact with TBP. 3) Complete the design and test of expressions systems to study aptamers against the transcriptional machinery in vivo. 4) Use of aptamers to dissect the roles of TBP and its interacting factors in transcription in vivo. The selection and use of iaRNAs should find broad use in both basic research, as in the dissection of mechanisms of sophisticated macromolecular machines and regulatory circuits, and in therapeutic applications, as inhibitors of specific disease-causing proteins. Many diseases are known to be caused by either over-expression of an endogenous gene (such as an oncogene in cancer), or expression of exogenous genes (as in a virus infection). Even the general transcription factor TBP, a target of this study, is up-regulated in cancers and contributes to oncogenic transformation and tumor growth. The regulated in vivo expression of RNA aptamers that target disease-inducing proteins would prove to be of substantial therapeutic value.