Ribozyme wild-type

For the 13 hammerhead FIFI16 mutants prepared by the reference 41 researchers, cleavage activity was significantly reduced in all cases. In four cases, at positions C3, A9, Gio.i, and A13, addition of the correct base (U, A, G, or C) rescued the activity of the ribozyme. A9X, for instance, is a ribozyme where the adenine base at position 9 has been removed to generate an abasic nucleotide (X = a nucleotide in which H substitutes for the removed base—see Figure 6.14). This altered ribozyme exhibited a 4 x 10 times lower catalytic rate than that for the wild-type ribozyme (krei = = 0.0004). When... 

Watanabe, T. and Sullenger, B.A. (2000) Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts. I roc. Natl. Acad. Sci. USA, 97, 8490-8494. 

Pyle, A. M., McSwiggen, J. A., and Cech, T. R. (1990). Direct measurement of oligonucleotide substrate binding to wild-type and mutant ribozymes from Tetrahymena. Proc. Natl. Acad. Sci. USA 87, 8187-8191. 

To explore the catalytic potential of three-way junction hairpin ribozymes we have synthesized the ribozyme HP-TJ, with an additional helix at the branch-point of the two domains, and have studied the kinetic properties in comparison to those of the wild-type ribozyme HP-WT. The new type of hairpin ribozyme contains an extra sequence at the 3 side of the ribozyme strand to enable it to hybridize with the 5 side sequence of a 28-mer substrate RNA. Thus, in comparison to the natural hairpin ribozyme, HP-TJ consists of an additional helix 5 between the ribozyme and part of the substrate strand (Figure 5.2.9). 

Figure 5 Upper Active site of the full-length hammerhead RNA using the canonical minimal sequence numbering scheme described in [40] and [42]. Lower Representative hydrogen bonding of the C3 G8 base pair observed from mutant simulations. Experimental relative catalytic rates of mutant versus wild-type minimal sequence ribozymes (kmut/kwt) are shown in parentheses (C3U from [76], G8A from [78], C3U/G8A from [73], and G8I from [34]), and may differ for the full-length sequence.

Figure 2. Effects of ribozyme-expression plasmids specific for cleavage of CBP mRNA on the levels of CBP in a transient-expression assay. Relative levels of CBP were compared by an amplified sandwich ELISA. Normalized levels of CBP in wild-type (WT) F9 cells were taken arbitrarily as 100%. All values are the averages of results from at least three experiments and the standard deviation for each value relative to the value for WT cells is indicated.

Figure 4A. Morphology of F9 cells that synthesized tRNA -ribozymes and rclativelevels of marker proteins that are s[>ecific for the undifferentiated and the differentiated state, respectively. Left panels show phase Contrast photomicrographs of F9 cells that synthesized either the active (upper) or the inactive (middle) CBP-ribozyme after treatmmt with retinoic adds (RA 3 x U) M) and imdifFeientiated wild type (WT) F9 cells (bottom). Right pands show immunostaining of F9 cells with antibodies spedfic for the surtoce antigen, SSEA-1.

Figure 5. Percentages of apoptotic cells in cultures of wild-type (WT) F9 cells and F9 cells that synthesized a CBP-ribozyme in the presence and in the absence of retinoic acids (RA 3 x 10 M). Apoptosis was inhibited in cells that synthesized the active CBP-ribo me. Cell death was assessed by the TUNEL method. ...

Figure 6. Levels of the cdk inhibitor p27 P during the retinoic acid-induced (RA-induced) differentiation of wild-type (WT) and ribozyme-expressing F9 cells. Normalized levels of p27Kipi p9 (-gi]s taken arbitrarily as 1.0. All values are the averages of results...

We tried to create variants of hammerhead ribozymes with deletions in the stem/loop II region and, fortunately, we found that some shortened forms of hammerhead ribozymes had high cleavage activity that was similar to that of the wild-type parental hammerhead ribozyme (R32 Fig.lA). Moreover, the active species appeared to form dimeric structures with a common stem II (Fig. IB). In the active short ribozymes, the linker sequences that replaced the stem/loop II region were palindromic so that two short ribozymes were capable of forming a dimeric structure with a common stem II. In order to distinguish monomeric forms of conventional minizymes that have extremely low activity from our novel dimers with high-level activity, we... 

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