Generation and application of asymmetric hammerhead ribozymes

Most researchers who intend to suppress a particular gene are interested pr imarily in the application of ribozyme technology rather than its mechanist ic details. This article provides some background information and describes a straightforward strategy to generate and test a special design of a ribo zyme: the asymmetric hammerhead ribozyme. This version of a hammerhead ribo zyme carries at its 5' end the catalytic domain and at its 3' end a relativ ely long antisense flank that is complementary to the target RNA. Asymmetri c hammerhead ribozymes can be constructed via polymerase chain reaction amp lification, and rules are provided on how to select the DNA oligonucleotide s required for this reaction. In addition to details on construction, we de scribe how to test asymmetric hammerhead ribozymes for association with the target RNA in vitro, so that RNA constructs can be selected and optimized for fast hybridization with their target RNA. This test can allow one to mi nimize association problems caused by the secondary structure of the target RNA. Additionally, we describe the in vitro cleavage assay and the determi nation of the cleavage rate constant. Testing for efficient cleavage is als o a prerequisite for reliable and successful application of the technology. A carefully selected RNA will be more promising when eventually used for t arget suppression in living cells. (C) 1999 Academic Press.