Directed Evolution of Nucleic Acid Enzymes

核酸酶的定向进化

• 批准号: 6718376
• 负责人: GERALD F JOYCE
• 金额: $ 31.11万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6718376
• 关键词: DNA; N glycosidase; biotechnology; catalyst; cofactor; directed evolution; endoribonucleases; enzyme activity; ribozymes; synthetic enzyme; synthetic nucleic acid

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.

描述：（由申请人提供）拟议的研究涉及 可用于调节的新型核酸酶的体外进化 生物过程。核添加物具有遗传和催化特性， 使其能够简单地将基因的扩增和突变结合起来， 根据它们相应的催化性质进行选择。 努力将集中在两类催化核酸的开发上 酸：具有N-糖基化酶活性的DNA酶和具有归巢作用的RNA酶 内切核糖核酸酶活性前者将进化为修复特定病变 由突变或氧化损伤引起的DNA损伤。后者将 通过连接两种现有的RNA酶来构建双功能的 一种可以在特定的时间以不可逆的方式插入自身的分子， 在RNA中的位置它将用于进行靶向基因破坏或 在mRNA内的编码区的靶向插入。同时也要注意 旨在理解进化过程本身。比较将 这些分子是由不同程度的 选择压力或其组成亚基的不同复杂性。一个新 采用猝灭流动装置的方法将用于进化ENA酶， 非常快的反应速度。RNA酶也将被开发出来， 极端pH值或温度条件下，揭示了 基于RNA的催化功能最后，一类新的拴系小分子 辅因子将被合成并提供给核酸酶， 催化功能。这些辅因子将由氨基 在寡脱氧核苷酸的末端添加或添加短肽 适配器的.接头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。