Directed Evolution of Nucleic Acid Enzymes

描述：(由申请人提供)建议的研究涉及 可用于调节的新型核酸酶的体外进化 生物过程。核加成既具有遗传性质又具有催化性质， 使它们的基因扩增和突变变得简单明了 序列，并根据其相应的催化特性进行选择。 将重点开发两类催化核 酸：具有N-糖基酶活性的DNA酶和具有归巢功能的RNA酶 内切核糖核酸酶活性。前者将进化为修复特定的损伤 由突变或氧化损伤引起的DNA。后者将是 通过将两种现有的RNA酶连接起来形成双功能 能够以不可逆的方式将自身插入到特定位置的分子 RNA中的位置。它将被用来进行有针对性的基因破坏或 在信使核糖核酸中定向插入编码区。也将受到关注 旨在了解进化过程本身。比较将是 在不同程度的分子中形成的 选择压力或其组成亚基的不同复杂性。一种新的 采用急流装置的方法将用于进化ENA酶 反应速度非常快。核糖核酸酶也将在 极端pH或温度的条件，揭示了 基于RNA的催化功能。最后，一类新的链状小分子 辅因子将被合成并提供给核酸添加酶来辅助 在它们的催化功能中。这些辅因子将由一个氨基组成 添加或连接到寡聚脱氧核苷酸末端的短肽 适配器。该适配器将允许辅因子容易地被酶结合， 允许进化利用结合的辅因子用于催化。

DESCRIPTION: (provided by applicant) The proposed research concerns the in vitro evolution of novel nucleic acid enzymes that can be used to modulate biological processes. Nucleic adds have both genetic and catalytic properties, making it straightforward to couple amplification and mutation of their genetic sequence with selection based on their corresponding catalytic properties. Efforts will focus on the development of two classes of catalytic nucleic acids: DNA enzymes with N-glycosylase activity and RNA enzymes with homing endoribonudease activity. The former will be evolved to repair specific lesions of DNA that result from mutation or oxidative damage. The latter will be constructed by joining two existing RNA enzymes to create a bifunctional molecule that can insert itself in an irreversible manner at a specific location within RNA. It will be used to perform targeted gene disruption or targeted insertion of a coding region within mRNA. Attention also will be directed toward understanding the evolution process itself. Comparisons will be made among molecules that arise as a consequence of different degrees of selection pressure or varying complexity of their component subunits. A new approach employing a quench-flow device will be used to evolve ENA enzymes with very fast reaction rates. RNA enzymes also will be developed that operate under conditions of extreme pH or temperature, shedding light on the limits of RNA-based catalytic function. Finally, a new class of tethered small-molecule cofactors will be synthesized and supplied to the nucleic add enzymes to assist in their catalytic function. These cofactors will consist of either an amino add or a short peptide that is attached to the end of an oligodeoxynucleotide adapter. The adapter will allow the cofactor to be bound readily by the enzyme, allowing evolution to exploit the bound cofactor for use in catalysis.