RNA LIGASES SELECTED FROM RANDOM RNA SEQUENCES

这项研究计划的广泛和长期目标是更好地 理解RNA的结构和催化库。重点 是一组至少65种RNA催化剂（核酶）， 从随机RNA序列中分离，通过最近开发的 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 这些连接酶可以 至少分为三类。 这项建议的具体目标是： (1)表征三类化合物的催化和结构特征 的连接酶。将用分子生物学检查结构特征 包括修饰干扰，取代干扰， 以及从简并序列库中进行体外选择。候选分子 也将进行设计和筛选。催化 三个连接酶类的机制将通过检查 硫代取代在反应性磷酸盐的影响，pH依赖性 的反应，以及替代Mg 2+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催化功能 具有聚合酶样底物结合特性。所选的 >65个连接酶将用作体外选择的起始分子， 设计用于分离和进化连接酶的进化程序， 用于底物识别的糖-磷酸骨架接触。 因为新的核酶类包含了大部分的 已知的RNA催化基序，这些连接酶的详细表征 将有助于RNA分子生物学的基础知识。理解 天然RNA的结构和催化剂将被增强时，投入 更广泛的背景下，什么是可能的，当解放的限制， 自然进化除了进一步扩展已知的RNA范围外， 催化，分离具有聚合酶样性质的连接酶将说明 RNA催化自我复制的能力- 生命早期进化的现代理论 表征三种新类型的连接酶并分离额外的连接酶。 具有聚合酶所要求的底物结合特性的连接酶 将提供洞察的潜力，限制，和可能的 体外筛选技术的改进。这样的见解，当 结合更好地理解RNA是 能够催化，将有助于那些试图利用 选择技术，以产生新的药物和其他有益的 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.