Use of Engineered Ribozymes to Catalyze Chimeric Gene Assembly

96-04458 Jarrell第一部分技术本研究旨在开发和推广一种技术，利用自剪接第二组内含子来设计新型重组核糖核酸和脱氧核糖核酸分子，并分析和确定有助于第二组内含子剪接的准确性和效率的序列。它将专注于人类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外显子将以预先选择的顺序一次一个地连接到另一个。与t-PA相关的蛋白质的外显子也将与t-PA外显子一起洗牌。P.I.的初步研究表明，基于第二组内含子的核酶可以被改造成插入到靶RNA的特定位置。当利用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连接到内含子的5‘端，另一种是来自插入位点下游的靶RNA连接到内含子的3’端。这些重组RNA产物可以通过反式剪接无缝连接。基于这些初步发现，设计了实验来剖析剪接反应的序列要求。能够参与一个以上反式剪接反应的重组RNA分子能够被产生并连接到多个其他RNA分子的可能性将被检验。两个不同的Y分支核酶将被设计成在给定的靶RNA分子内的不同位置插入，产生带有内含子序列的重组RNA，内含子序列相应地参与两个不同的反式剪接反应，连接其5‘或3’端。因此，已被提出参与新蛋白质进化的“外显子改组”机制将在体外被微型化。由于核酶可以被设计成在任何所需的序列上精确插入，而且剪接反应是单向的，并将RNA无缝连接在一起，因此可以预期改组外显子的框内融合。第二部分：用于产生重组DNA的非技术传统方法包括DNA分子的定点切割和连接。本研究开发了一种新的方法，通过一个简单的酶促步骤从RNA中产生重组DNA。重组RNA是由“核酶”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以催化RNA分子的切割和连接。P.I.已经证明，其中一种核酶，被称为第二组内含子aI5g，可以被改造成催化特定重组基因的精确组装。本研究旨在为这一新的基于核酶的基因工程系统的优化提供补充信息。

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.