Design, Synthesis and Characterization of Twin Ribozymes

With the described set of catalytic modules at hand we designed and synthesized several twin ribozymes, three of those are shown as examples in Figure 5.2.11.

For combination in a twin ribozyme both units should specifically interact with a unique substrate sequence. To this end we changed the substrate sequence in the single units and determined the kinetic constants of the cleavage reaction before constructing the twin ribozymes. All single catalytic modules under single turnover conditions had rate constants between 0.2 and 0.5 min-1.

The preparation of twin ribozymes included both chemical and enzymatic procedures. Basically, in our laboratory RNA strands longer than 60 nucleotides are transcribed from synthetic DNA templates using T7 RNA-polymerase. By using 5-benzylmercapto-1H-tetrazole [18a,b] as activator in the phosphoramidite approach, however, we also succeeded in synthesizing a chemical version of HP-

substrate strand ribozyme segment II

ribozyme segment

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