OPTIMIZATION OF INTERMOLECULAR HAIRPIN RNA CATALYSIS

本项目的主要目标是加深对 催化核糖核酸发夹结构域的生化特性研究 为合理设计能切割靶RNA的发夹状核酶 体外和体内。因为小的催化RNA可以结合和切割 以序列特异的方式靶向RNA，它们有可能 为基因反义方法提供了有力的扩展 失活。理解裂解反应的生物化学 将有助于将这一潜力转化为有效的技术 和治疗工具。具体设计和设计的关键问题 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，确保选择 在竞争的、相关的序列中正确的目标，2)定义 防止缓慢速率的核酶对切割产物的亲和力 限制催化周转的产品释放，以及3)选择 在结构复杂的靶标中可获得的切割位点。测量 核酶和底物序列变异对特定步骤的影响 动力学机制中的最佳亲和力 核酶、底物和产物的配体。发夹图书馆 具有不同序列特异性的变体将被用来定位 可访问的目标位置在大的、折叠的目标RNA中。测定动力学 在各种温度下，PH值和离子条件都会开始 定义这类新型生物催化剂的化学机理 也提高了体外优化的预测价值。 体内靶RNA的切割。针对这些的发夹核酶变体 实验将针对酵母GAL4基因So的序列 通过体外实验开发的指南可以进行测试 直接放入酵母中。GAL4基因是一个理想的模型靶点，因为 GAL4转录激活蛋白对血管内皮细胞生长的影响 从GAL调节的启动子转录提供了一种敏感的， 核酶介导的失活的定量检测。瞄准 优化研究将得到实验的补充，以选择和 在体外扩增有功能的发夹变体。此方法论适用于“In 体外进化“将提供有关细胞结构的信息 并可能产生一种发夹变异体，超过 催化效率自然发生的顺序。发夹 催化核糖核酸是这两项研究的首选核酶 理由。首先，初步的生化特征表明， 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的裂解。第二, 发夹结构域与锤头结构域不同，很容易催化RNA 结扎术。通过结扎，有功能的发夹可以获得 可选择的序列以允许选择和扩增最佳 核酶序列变异体。

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.