Use of Engineered Ribozymes to Catalyze Chimeric Gene Assembly

使用工程核酶催化嵌合基因组装

• 批准号: 9604458
• 负责人: Kevin Jarrell
• 金额: $ 27万
• 依托单位: Trustees of Boston University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-03-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9604458&HistoricalAwards=false
• 关键词: Use; Engineered; Ribozymes; Catalyze; Chimeric

96-04458 Jarrell Part 1. Technical This study is aimed to develop and extend a technology by which self-splicing group II introns can be employed to engineer novel recombinant RNA and DNA molecules, and to dissect and define the sequences that contribute to the accuracy and efficiency of group II intron splicing. It will focus on Y-branched ribozyme insertion between exons of the human t-PA gene. By both random and directed shuffling, t-PA exons will be linked to one another one at a time, in a pre-selected order. Exons from proteins related to t-PA will also be shuffled together with t-PA exons. Preliminary studies by the P. I. show that ribozymes based on the group II intron can be engineered to insert into specific sites in target RNAs. When Y-branched ribozymes are utilized, the insertion reaction creates two products: one in which target RNA from upstream of the insertion site is linked to the 5' end of the intron, and one in which target RNA from downstream of the insertion site is linked to the 3' end of the intron. These recombinant RNA products can be seamlessly rejoined by trans splicing. Based on these initial findings, experiments are designed to dissect the sequence requirements for the splicing reactions. The possibility that recombinant RNA molecules capable of participating in more than one trans splicing reaction can be produced and linked to multiple other RNA molecules will be examined. Two different Y-branched ribozymes will be engineered to insert at different sites within the a given target RNA molecule, yielding a recombinant RNA flanked by intron sequences, which participates accordingly in two different trans splicing reactions, linking its 5' or 3' end. Thus the "exon shuffling" mechanism that has been proposed to have participated in the evolution of new proteins will be micmiced in vitro. Because the ribozymes can be engineered to insert precisely at any desired sequence, and the splicing reactions are unidirectional and link RNAs together seamlessly, in-frame fu sions of shuffled exons can be expected. Part 2. Non-technical Traditional methods used to generate recombinant DNA involve site-specific cutting and joining of DNA molecules. The Present study develops a new method by creating recombinant DNA from RNA via a simple enzymatic step. The recombinant RNA is generated by the activity of "ribozymes". Ribozymes are enzymes that are made of RNA. Certain naturally occurring ribozymes are known to catalyze the cleavage and joining of RNA molecules. The P.I. has demonstrated that one of these ribozymes, referred to as group II intron aI5g, can be engineered to catalyze the precise assembly of particular recombinant genes. This study is designed to provide additional information for optimization of this new ribozyme-based gene engineering system.

96-04458 Jarrell 第 1 部分：技术 这项研究旨在开发和扩展一种技术，通过该技术，可以利用自剪接 II 族内含子来设计新型重组 RNA 和 DNA 分子，并剖析和定义有助于 II 族内含子剪接准确性和效率的序列。它将重点关注人类 t-PA 基因外显子之间的 Y 分支核酶插入。通过随机和定向改组，t-PA 外显子将按照预先选择的顺序一次彼此连接。与 t-PA 相关的蛋白质的外显子也将与 t-PA 外显子一起改组。 P. I. 的初步研究表明，基于 II 组内含子的核酶可以被设计插入靶 RNA 的特定位点。当使用Y分支核酶时，插入反应产生两种产物：一种是来自插入位点上游的靶RNA与内含子的5'端连接，另一种是来自插入位点下游的靶RNA与内含子的3'端连接。这些重组RNA产物可以通过反式剪接无缝地重新连接。 基于这些初步发现，设计实验来剖析剪接反应的序列要求。将检验能够产生能够参与多个转拼反应的重组RNA分子并将其与多个其他RNA分子连接的可能性。两种不同的 Y 分支核酶将被设计为插入给定靶 RNA 分子内的不同位点，产生侧翼为内含子序列的重组 RNA，该重组 RNA 相应地参与两个不同的反式剪接反应，连接其 5' 或 3' 末端。因此，被认为参与新蛋白质进化的“外显子改组”机制将在体外被模拟。因为核酶可以被设计为精确地插入任何所需的序列，并且剪接反应是单向的并将RNA无缝地连接在一起，所以可以预期改组的外显子的框内融合。 第 2 部分. 非技术性 用于生成重组 DNA 的传统方法涉及 DNA 分子的位点特异性切割和连接。本研究开发了一种新方法，通过简单的酶促步骤从 RNA 中产生重组 DNA。 重组RNA是通过“核酶”的活性产生的。核酶是由 RNA 组成的酶。已知某些天然存在的核酶可催化 RNA 分子的裂解和连接。 P.I.已经证明，这些核酶之一（称为 II 组内含子 aI5g）可以被改造为催化特定重组基因的精确组装。 本研究旨在为优化这种新的基于核酶的基因工程系统提供更多信息。