RNA LIGASES SELECTED FROM RANDOM RNA SEQUENCES

从随机 RNA 序列中选择的 RNA 连接酶

• 批准号: 6019076
• 负责人: DAVID P BARTEL
• 金额: $ 13.03万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019076
• 关键词: acidity /alkalinity; biochemical evolution; chemical kinetics; chemical substitution; cofactor; divalent cations; enzyme mechanism; enzyme structure; enzyme substrate; molecular cloning; nucleic acid chemical synthesis; nucleic acid sequence; nucleic acid structure; polymerase chain reaction; polymerization; ribozymes; synthetic enzyme; thioester

The broad, long-term objective of this research plan is a better understanding of the structural and catalytic repertoire of RNA. The focus is on a set of at least 65 RNA catalysts (ribozymes) that have been isolated from random RNA sequences by means of recently developed molecular biology techniques called in vitro selection and in vitro evolution. The new ribozymes catalyze RNA ligation reactions, and based on ligation regioselectivities and substrate-binding modes these ligases can be grouped into at least three classes. The specific aims of this proposal are: (1) To characterize catalytic and structural features of the three classes of ligases. Structural features will be examined with molecular biology methods including modification interference, substitution interference, and in vitro selection from degenerate sequence pools. Candidate molecules for crystallization will also be designed and screened. The catalytic mechanisms of the three ligase classes will be probed by examining the effects of thio substitutions at the reactive phosphate, the pH dependence of the reactions, and the effects of substituting Mg2+ with alternative divalent cation cofactors. (2) To extend the known repertoire of RNA catalysis by generating ligases with polymerase-like substrate-binding properties. The selected pool of >65 ligases will serve as starting molecules for in vitro selection and evolution procedures designed to isolate and evolve ligases that utilize sugar-phosphate backbone contacts for substrate recognition. Because the new ribozyme classes comprise a significant fraction of the known RNA catalytic motifs, the detailed characterization of these Iigases will contribute to basic knowledge of RNA molecular biology. Understanding of natural RNA structures and catalysts will be enhanced when put into the broader context of what is possible when liberated from the constraints of natural evolution. In addition to further extending the known range of RNA catalysis, isolating ligases with polymerase-like properties will speak to the ability of RNA to catalyze self-replication - a central supposition of current theories of the early evolution of life. Characterizing the three new classes of ligases and isolating additional ligases with the demanding substrate-binding properties of a polymerase will provide insights into the potential, the limitations, and possible improvements for the in vitro selection technology. Such insights, when combined with a better understanding of the scope of reactions that RNA is capable of catalyzing, will be useful for those attempting to exploit the selection technology to generate new pharmaceuticals and other beneficial molecules.

该研究计划的广泛长期目标是更好的 理解RNA的结构和催化曲目。重点 在一组至少65个RNA催化剂（核酶）上 通过最近开发的方式从随机RNA序列中分离出来 分子生物学技术称为体外选择和体外 进化。新的核酶催化RNA连接反应，并基于 连接区域选择性和底物结合模式这些连接酶可以 至少分为三类。 该提案的具体目的是： （1）表征三类的催化和结构特征 连接酶。结构特征将通过分子生物学检查 包括修改干扰，替代干扰的方法， 并从退化序列库中选择。候选分子 为了结晶，还将设计和筛选。催化 三种连接酶类的机制将通过检查 硫代取代在反应性磷酸盐，pH依赖性的影响 反应，以及替代替代Mg2+的影响 二价阳离子辅因子。 （2）通过产生连接酶来扩展已知的RNA催化曲目 具有聚合酶样底物结合特性。选定的池 > 65个连接酶将用作体外选择的起始分子， 旨在隔离和进化的连接酶的进化程序 糖磷酸骨链接触以进行底物识别。 因为新的核酶类包括很大一部分 已知的RNA催化基序，这些IIGases的详细表征 将有助于RNA分子生物学的基础知识。理解 当放入天然RNA结构和催化剂的天然RNA结构和催化剂中 从从约束中解放出来的更广泛的背景 自然进化。除了进一步扩展已知的RNA范围 催化，用聚合酶样性质隔离连接酶会说话 RNA催化自我复制的能力 - 生命早期演变的当前理论。 表征三个新的连接酶并隔离其他 与聚合酶苛刻的底物结合特性的连接酶 将提供有关潜力，局限性和可能的​​见解 体外选择技术的改进。这样的见解，什么时候 结合对RNA的反应范围更好地理解 能够催化，对于那些试图利用的人很有用 选择新药和其他有益的技术 分子。