RNA LIGASES SELECTED FROM RANDOM RNA SEQUENCES

这项研究计划的广泛、长期目标是一个更好的 了解核糖核酸的结构和催化谱。焦点 位于一组至少65个RNA催化剂(核酶)上，这些催化剂已经 通过新近开发的从随机RNA序列中分离出 称为体外选择和体外培养的分子生物学技术 进化论。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 至少被分成三类。 这项建议的具体目标是： (1)表征这三类化合物的催化和结构特征 连接酶。结构特征将用分子生物学进行检测 方法包括修饰干扰、替代干扰、 以及从简并序列池中进行体外选择。候选分子 对于结晶也将进行设计和筛选。催化剂 这三类连接酶的作用机制将通过研究 硫代取代对活性磷酸盐的影响，pH依赖性 以及用替代物取代镁离子的效果 二价阳离子辅因子。 (2)通过产生连接酶来扩展已知的RNA催化谱系 具有聚合酶样的底物结合特性。选定的池 &gt；65连接酶将作为体外选择和 设计用来分离和进化连接酶的进化程序 用于底物识别的糖-磷酸主干接触。 因为新的核酶类组成了很大一部分 已知的RNA催化基序，这些连接酶的详细特征 将有助于了解RNA分子生物学的基础知识。理解 天然RNA结构和催化剂的性能将在投入到 更广泛的背景下，什么是可能的，当从限制解放 自然进化。除了进一步扩大已知的RNA范围之外 催化，分离具有聚合酶性质的连接酶将有助于 RNA催化自我复制的能力--一个中心假设 目前关于生命早期进化的理论。 三类新连接酶的鉴定及附加连接酶分离 具有聚合酶要求的底物结合特性的连接酶 将提供对潜在的、限制的和可能的 体外筛选技术的改进。这种洞察力，当 再加上对反应范围的更好理解，RNA是 能够催化，对于那些试图利用 选择技术产生新的药物等有益 分子。

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.