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.
