Flanking sequence effects on hammerhead ribozyme activity in vitro and in vivo

Hammerhead ribozymes (hRz) are small catalytic ribonucleic acids (RNA) with endoribonuclease activity. Cleavage sites for the hRz are NUH triplets, where N represents any nucleotide, and H can be either A, U, or C. However, most NUH triplets are sequestered by the folding structures of a target RNA, making it difficult to be accessible. A library selection protocol utilizing a systematic evolution of ligands by an exponential enrichment (SELEX) approach was developed to identify ribozyme cleavable sites. Four cleavable sites within a model gene (1,163 nt) were identified by the SELEX protocol.; Recognition sequences regulate affinity, specificity, and turnover rates for hRz catalytic cycles. Kinetic studies of symmetric hRz containing varied lengths of recognition sequences (7, 9, 15, or 25 nt) targeted to one of the library-selected sites (GUC₁₄₃) were performed in vitro . The results suggested that the rate-limiting step in vitro is effective binding of the target RNA by hRz not product dissociation, in contradiction to published results. Stably transfected cell populations were generated for analyses of cellular hRz activities. All catalytically active hRz showed equivalent effects on reducing rC9 gene expression in cell culture, regardless of their lengths of flanking sequences. To assess the potential antisense effects of hRz, catalytically inactive hRz (mRz7 and mRz25) were created. In stably transfected cell populations, mRz7 did not reduce rC9 transcript levels, whereas mRz25 consistently showed antisense-mediated reductions of rC9 expression.; A series of hRz7 mutants with single base, two-base, or four-base mismatches in their recognition sequences were generated and tested for their catalytic activity both in vitro and in cultured cells. As a general rule, a nucleotide mismatch closer to the actual cleavage site caused more severe inhibition of catalytic activity of hRz7. In addition, alternative G•U Wobble base pairing can partially maintain the catalytic activity of the hRz7.; The rC9-targeted hRz25 was widely distributed in both nuclei and cytoplasm in transiently transfected fibroblasts, as determined by fluorescence in situ hybridization. Quantitative real time PCR analyses of cellular RNA from nuclei and cytoplasm indicated that about 10% of hRz25 was found in the nucleus, with 90% in the cytoplasm.