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，用于研究调节活细胞和生物体中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）的能力，用于分离以高亲和力结合活性蛋白表面的RNA适体，以及用于实现特异性抑制性适体RNA（iaRNA）的快速、高水平和受控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，从而可以评估其体内的主要功能和作用机制。 该提案将TATA结合蛋白（TBP）作为其联系，TBP是启动子结构和功能的核心参与者。 TBP与一系列合作和竞争转录因子的已知物理相互作用是这些抑制剂的靶点，也是这些机制研究的焦点。 具体目标是： 1)进一步表征所选的针对TBP的高亲和力RNA适体（TBPap适体）的结构和活性，并在体外使用这些适体来解决TBP-TATA相互作用在转录起始和再起始中的机制作用。 2)在体外选择并表征结合TBP不同表面并结合与TBP相互作用的其他转录因子的其他适体。 3)完成表达系统的设计和测试，以研究适体对体内转录机制的影响。4)使用适体剖析TBP及其相互作用因子在体内转录中的作用。 iaRNA的选择和使用应该在基础研究中找到广泛的用途，如在复杂的大分子机器和调节电路的机制的解剖中，以及在治疗应用中，作为特定致病蛋白的抑制剂。 已知许多疾病是由内源基因（如癌症中的致癌基因）的过度表达或外源基因（如病毒感染）的表达引起的。 即使是一般的转录因子TBP，这项研究的目标，在癌症中上调，并有助于致癌转化和肿瘤生长。靶向疾病诱导蛋白的RNA适体的受调节的体内表达将被证明具有实质性的治疗价值。

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.