APPLICATION OF COMPUTATIONAL TECHNOLOGIES TO RIBOZYME BIOTECHNOLOGY PRODUCTS

Ribozymes are RNA molecules that act enzymatically to cleave other RNA molecules. The cleavage reaction requires the binding of ribozyme to specific sites on the target RNA through (mostly) Watson-Crick base-pa iring interactions. Association of ribozyme with target completes a th ree-dimensional ribozyme/target complex which results in cleavage of t he target RNA. We are employing both computational and experimental ap proaches to identify sites on target RNA molecules that are open to ri bozyme attack and to determine which ribozymes are most active against those sites. Two types of computational technologies are available fo r aiding in the identification of target sites and design of active ri bozymes. First, DNA/RNA sequence analysis software is employed to iden tify sequence motifs necessary for ribozyme cleavage and to look for s equence conservation between different sources of the target organism so that ribozymes with the broadest possible target range can be desig ned. Second, RNA folding algorithms are employed to predict the second ary structure of both ribozyme and target RNA in an attempt to identif y combinations of ribozyme and target site that will successfully asso ciate prior to ribozyme cleavage. The RNA folding algorithms utilize a set of thermodynamic parameters obtained from measurements on short R NA duplexes; while these rules give reasonable predictions of secondar y structure for a small set of highly structured RNAs, they remain lar gely untested for predicting the structure of messenger RNAs. This pap er outlines the current status of designing ribozymes that fold correc tly and of locating target sites that are sufficiently unfolded to all ow ribozyme cleavage.