CONSTRUCTION OF A DIRECTED HAMMERHEAD RIBOZYME LIBRARY - TOWARDS THE IDENTIFICATION OF OPTIMAL TARGET SITES FOR ANTISENSE-MEDIATED GENE INHIBITION

Antisense-mediated gene inhibition uses short complementary DNA or RNA oligonucleotides to block expression of any mRNA of interest. A key p arameter in the success or failure of an antisense therapy is the iden tification of a suitable target site on the chosen mRNA, Ultimately, t he accessibility of the target to the antisense agent determines targe t suitability. Since accessibility is a function of many complex facto rs, it is currently beyond our ability to predict, Consequently, ident ification of the most effective target(s) requires examination of ever y site. Towards this goal, we describe a method to construct directed ribozyme libraries against any chosen mRNA, The library contains nearl y equal amounts of ribozymes targeting every site on the chosen transc ript and the library only contains ribozymes capable of binding to tha t transcript. Expression of the ribozyme library in cultured cells sho uld allow identification of optimal target sites under natural conditi ons, subject to the complexities of a fully functional cell. Optimal t arget sites identified in this manner should be the most effective sit es for therapeutic intervention.