OPTIMIZATION OF INTERMOLECULAR HAIRPIN RNA CATALYSIS

该项目的主要目标是了解 催化RNA的发夹结构域的生物化学性质，以利用 对于发夹核酶的合理设计， 体外和体内。 由于小的催化RNA可以结合和切割一个 RNA以序列特异性方式靶向，它们有可能 为基因反义方法提供了有力的延伸 失活 了解裂解反应的生物化学 将有助于将这种潜力转化为有效的技术 和治疗工具。 设计具体和 有效的核酶包括：1）确定相互作用的强度 在底物和核酶之间， 正确的目标之间的竞争，相关的序列，2）定义 核酶对切割产物的亲和力， 限制催化转换的产物释放，以及3）选择 在结构化的、复杂的靶中的可接近的切割位点。 测量 核酶和底物序列变化对特定步骤的影响 在动力学机制将揭示之间的最佳亲和力 核酶以及底物和产物配体。 发夹文库 具有不同序列特异性的变体将用于定位 在大的折叠的靶RNA中的可接近的靶位点。 测定动力学 在各种温度、PH和离子条件下， 定义这类新型生物催化剂的化学机理 并且还提高了体外优化的预测值， 在体内切割靶RNA。 发夹状核酶变体 实验将针对酵母GAL4基因中的序列， 通过体外实验制定的指导方针可以被测试 直接在酵母中。 GAL4基因是一个理想的模型靶点， GAL4转录激活蛋白对 从GAL调节的启动子的转录提供了一种敏感的， 核酶介导的失活的定量测定。 靶向 优化研究将通过实验来补充， 在体外扩增功能性发夹变体。 这种方法用于“在 “体外进化”将提供有关结构的信息， 催化结构域，并且可以产生超越催化结构域的发夹变体。 天然存在的序列的催化效率。 发夹 催化RNA是这些研究的选择核酶， 原因 首先，最初的生化特征表明， 发夹结构域可能比锤头结构域更适合于 在体内发现的低二价离子浓度下裂解。 第二、 发夹结构域与锤头结构域不同， 结扎 通过连接，功能性发夹可以获得 选择性序列，以允许选择和扩增最佳的 核酶序列变体。

The main goal of this project is to develop an understanding of the biochemical properties of the hairpin domain of catalytic RNA to exploit for the rational design of hairpin ribozymes to cleave target RNAs in vitro and in vivo. Because small catalytic RNAs can bind and cleave an RNA target in a sequence-specific fashion, they have the potential to provide a powerful extension of the antisense method of gene inactivation. Understanding the biochemistry of the cleavage reaction will facilitate translating this potential into an effective technical and therapeutic tool. Critical issues for the design of specific and efficient ribozymes include: 1) defining the strength of the interaction between the substrate and the ribozyme that ensures selection of the correct target among competing, related sequences, 2) defining the affinity of the ribozyme for cleavage products that prevents slow rates of product release from limiting catalytic turnover, and 3) selecting accessible cleavage sites in structured, complex targets. Measuring the effects of ribozyme and substrate sequence variations on specific steps in the kinetic mechanism will reveal the optimum affinity between the ribozyme and substrate and product ligands. Libraries of hairpin variants with different sequence specificities will be used to locate accessible target sites in large, folded target RNAs. Assaying kinetics under a variety of temperature, PH and ionic conditions will begin to define the chemical mechanism for this new class of biological catalysts and also enhance the predictive value of in vitro optimization for cleavage of target RNAs in vivo. Hairpin ribozyme variants for these experiments will be directed against sequences in the yeast GAL4 gene so that guidelines developed through in vitro experiments can be tested directly in yeast. The GAL4 gene is an ideal model target because the dramatic effect of the GAL4 transcription activator protein on transcription from GAL-regulated promoters provides a sensitive, quantitative assay for ribozyme-mediated inactivation. Targeting optimization studies will be complemented by experiments to select and amplify functional hairpin variants in vitro. This methodology for "in vitro evolution" will provide information about the structure of the catalytic domain and may produce a hairpin variant that surpasses the naturally occurring sequence in catalytic efficiency. The hairpin catalytic RNA is the ribozyme of choice for these studies for two reasons. First, initial biochemical characterizations suggest that the hairpin domain may be better suited than the hammerhead domain for cleavage at the low divalent ion concentrations found in vivo. Second, the hairpin domain, unlike the hammerhead domain, readily catalyzes RNA ligation. Through ligation, a functional hairpin can acquire a selectable sequence to permit selection and amplification of optimal ribozyme sequence variants.