ISOLATION AND CHARACTERIZATION OF NOVEL RIBOZYMES

新型核酶的分离和表征

• 批准号: 2701689
• 负责人: CHARLES WILSON
• 金额: $ 8.81万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-10 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2701689
• 关键词: alkylation; biochemical evolution; biotin; chemical binding; chemical kinetics; chemical models; chemical structure function; chemical transfer reaction; crosslink; enzyme mechanism; enzyme substrate analog; flow cytometry; fluorescent dye /probe; immunoaffinity chromatography; immunologic assay /test; nucleic acid structure; physical separation; polymerase chain reaction; ribozymes; site directed mutagenesis

What types of chemical reactions can RNA enzymes perform and how do the three dimensional structures of these RNAs specify their function? All naturally-occurring ribozymes catalyze a limited set of reactions involving the RNA phosphate backbone. By developing and applying in vitro selection methods, we will isolate novel classes of RNA enzymes that perform a wide range of new catalytic activities. Characterization of these and previously isolated RNAs will be performed to determine the chemical and structural basis for ribozyme catalysis. Starting from a pool of random sequence RNA molecules, we have identified well-defined RNA motifs that recognize the carboxylation co-factor biotin and the fluorophore sulforhodamine with high affinity. Using the biotin- binding RNA as a starting point for mutagenesis, we have successfully evolved ribozymes that utilize a biotin-based substrate to perform a self- alkylation reaction. Preliminary characterization shows that trivial models to account for the high activity of one of these enzymes are invalid, indicating that more involved catalytic mechanisms are exploited to achieve catalysis. FIRST, we will adapt the selection method used to obtain self-alkylating ribozymes to target other types of ligation chemistry, isolating RNAs that catalyze peptide and glycoside formation. SECOND, we will develop a selection strategy for isolating RNA enzymes by immunoaffinity chromatography. This method should make it possible to obtain ribozymes with many different types of catalytic activities. THIRD, we will use a direct fluorescence detection method to isolate multiple turnover ribozymes that catalyze hydrolysis reactions with fluorigenic substrates. FOURTH, we will characterize the biotin- and sulforhodamine-binding motifs, the self-biotinylating ribozymes, and any additional isolated ribozymes using a combination of genetic, biochemical, and biophysical methods. These studies will aim towards building complete structural models for each RNA that explain how nucleotides interact with one another to direct folding to a unique tertiary structure and how nucleotides and ligands interact to yield high affinity binding. FIFTH, we will conduct a series of enzymological experiments to probe the specific mechanisms used by the self-biotinylating ribozymes to achieve catalysis. IN SUMMARY, these experiments should allow us to isolate many different classes of ribozymes that catalyze a wide range of chemical reactions. These enzymes will then serve as simple model systems for answering basis questions about the nature of ribozyme catalysis.

RNA酶可以执行哪些类型的化学反应以及如何 这些RNA的三维结构指定其功能？ 全部 天然呈现的核酶会催化一组有限的反应 涉及RNA磷酸主链。 通过开发和应用体外 选择方法，我们将隔离新型的RNA酶， 进行广泛的新催化活动。 表征 这些和以前隔离的RNA将进行确定 核酶催化的化学和结构基础。 从随机序列RNA分子池开始，我们已经鉴定了 识别羧化辅助因素生物素的明确定义的RNA图案 以及具有高亲和力的荧光团磺胺素。 使用生物素 - 结合RNA作为诱变的起点，我们已经成功 进化的核酶，利用基于生物素的底物进行自我 烷基化反应。 初步表征表明微不足道 解释其中一种酶的高活性的模型是 无效，表明更多涉及的催化机制被利用 实现催化。 首先，我们将调整用于获得自算法的选择方法 核酶靶向其他类型的连接化学，分离RNA 催化肽和糖苷形成。 第二，我们将开发一个 通过免疫亲和力隔离RNA酶的选择策略 色谱法。 这种方法应该使获得核酶 具有许多不同类型的催化活性。 第三，我们将使用 直接荧光检测方法分离多个周转率 催化与荧光底物的水解反应的核酶。 第四，我们将表征生物素和磺胺素结合 基序，自生物素核酶和任何其他分离的 核酶使用遗传，生化和生物物理的结合 方法。 这些研究将旨在建立完整的结构 每个RNA的模型，解释了核苷酸如何相互作用 直接折叠到独特的三级结构，以及核苷酸和如何 配体相互作用以产生高亲和力结合。 第五，我们将进行 一系列探测特定机制的酶学实验 由自生蛋白基核酶用于催化。 在 总而言之，这些实验应该使我们能够隔离许多不同的 核酶的类别催化了广泛的化学反应。 然后，这些酶将用作简单的模型系统以回答基础 有关核酶催化性质的问题。