This is an application for the second renewal of a project which has led to the initial description of hairpin ribozymes and the subsequent development of catalytic RNA specific for HIV-1, which is currently being evaluated as a therapeutic for AIDS. The overall objective of this proposal is to further develop this class of hairpin ribozymes by extending the sequence requirements at the site of cleavage. The initial hairpin ribozyme modeled from the naturally occurring catalatic RNA of tobacco ringspot virus (sTRSV) has a strong preference for cleavage at sites just before a GUC in the target RNA. During the current funding period, the investigator has identified two new ribozymes in the hairpin family which are capable of efficiently cleaving substrate with a GUA after the cleavage site. These originated from the negative strands of the satellite RNAs from chicory yellow mottle virus (sCYMV1) and from arabis mosaic virus (sArMV), and in this proposal, the investigators will develop ribozymes suitable for cleavage of HIV-1 RNA containing these GUA target sites. Specifically, the investigators will determine substrate targeting rules for the sCYMV1 and sARMV ribozymes and pursue the reasons for GUC or GUA specificity in the various hairpin ribozymes. The investigators propose to modify the ribozymes having GUA specificity and, in an attempt to enhance activity and increase stability, they will develop and optimize these ribozymes for cleavage of HIV-1 sequences. By expanding repertoire of ribozymes capable of targeting HIV-1, chances of overcoming viral mutagenic adaptation would be enhanced.
