Ribozymes effects

Lange, W. (1995) Cleavage of BCR/ABL mRNA by synthetic ribozymes-effects on the proliferation rate of K562 cells. Clin. Padiatr., 207, 222-224. 

Kleimnan et al. 2008). In addition, synthetic siRNAs are also subject to degradation in vivo by nuclease activity. Besides side effects and instability, the efficient and specific delivery of the RNAi indncers to the target cell still requires optimization. Here we snmmarize the cnrrent statns of nncleic acid-based antiviral therapentics. The focns will be on antiviral strategies nsing antisense and RNAi technology. Additionally, antiviral ribozymes and aptamers will be discussed briefly, with a focus on recent studies. Gene therapy approaches and delivery systems are the subject of Chapter 11 of this book. 

RNAi and ribozymes represent two additional approaches to gene silencing/down-regulation with therapeutic potential. RNAi is an innate cellular process that achieves silencing of selected genes via an anti-sense mechanism. It shares many characteristics with the antisense-based approach described above, but also some important differences, e.g. in the exact mechanism by which the antisense effect is achieved. 

Fig. 8 Effects of the monovalent Na" cation on the hammerhead ribozyme reaction in the presence of the divalent cation Mn. The reactions were performed under singleturnover conditions ([Ribozyme]>>[Substrate]). They were 15 min-reaction in the presence of 1 mmol/1 Mn ions at various concentrations of Na ions from 0 to 3000 mmol/ 1 or no Mn ions at 3000 mmol/1 Na ions...

In our recent study of reaction kinetics, we observed an unusual phenomenon when we analyzed the activity of a hammerhead ribozyme as a function of the concentration of Na ions on a background of a low concentration of either Mn or Mg ions [82]. At lower concentrations of Na ions, Na ions had an inhibitory effect on ribozyme activity, whereas at higher concentrations, Na ions had a rescue effect. We propose that these observations can be explained if we accept the existence of two kinds of metal-binding site that have different affinities. Our data also support the two-phase folding theory [77-80, Fig. 7], in which divalent metal ions in the ri-bozyme-substrate complex have lower and higher affinities, as proposed by Lilley and coworkers on the basis of their observations of ribozyme complexes in the ground state. 

This inhibitory effect of NaCl can be explained on the basis of the data from Horton et al. [81] that is described above. The Na ions remove Mn ions from the lower affinity site(s), which is somehow involved in the ribozyme activity, from the ribozyme-substrate complex. When Hammann et al. used ions in their NMR analysis, they noticed that the apparent Ka for the lower affinity ions depended on the concentration of Na ions [80]. An increase in the background concentration of NaCl from 10 mmol/1 to 50 mmol/1 weakened the affinity of the Mg ions for the complex. Their observations also reconcile with the observed inhibition by Na ions in our study, with the competitive removal of Mg /Mn ions from the ribozyme-substrate complex. 

It is clear that the analysis of thio effects, rescue experiments and other experiments with derivatives have contributed significantly to our understanding of the mechanism of the action of the large group I intron ribozyme of Tetmhymena. All the available data appear to support the Lewis acid catalysis for activation of the attacking nucleophile and enhancement of the leaving group that is shown in Fig. IIB. 

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