MECHANISM OF SYNTHETIC RIBOZYME ACTION

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The discovery of self-cleaving structures in plant RNA pathogens has opened up a novel approach to the development of new therapeutic agents for the control of diseases caused by the HIV virus and by other retroviral infections. These ribozyme structures, which are classified as possessing either a "hammerhead" or a "hairpin" motif, have the capacity to recognize specific sequences within a target RNA strand and then cleave a particular phosphodiester linkage within each sequence. The rational design of efficient anti-HIV agents that exploit these properties would be greatly facilitated by a detailed understanding of the structures and mechanisms used by these two ribozyme motifs. To this end, a few small ribozyme domains of each motif will be chemically synthesized in large quantities and in states of high purity. The domains will consist of duplexes formed from pairs of oligoribonucleotides in which one member corresponds to the catalytic site and the other to the substrate site. For the latter, chemical modifications will be introduced near the cleavable phosphodiester linkage in each case, in order to prevent self-destructing activity during analysis. The modifications are designed to prevent cleavage while avoiding large conformational changes in the structure that is responsible for cleavage. Modifications will include substitutions for the 2'-hydroxyl group involved in the cleavage reaction, isomerization of the cleavable phosphodiester from the 3'-5' to the 2'-5' configuration, and substitution of a methylene group for the oxygen located between the phosphorus atom of the 3'-5' diester and the 5' carbon of the adjacent nucleotide. Techniques to be used in determining the structures of these synthetic ribozymes will include thermodynamic measurements, X-ray diffraction of duplex crystals, and NMR analysis of duplex solutions.