Ribozymes twin ribozyme

Conventional and reverse-joined hairpin ribozymes as described above can be combined in one molecule to create a twin ribozyme. Their use is, however, limited by the specific structure. Because folding into the active conformation requires a bend between helix 2 and helix 3 in the conventional hairpin ribozyme, and between helix 1 and helix 4 in the reverse-joined hairpin ribozyme, these helices form a sort of hinge and are, therefore, not susceptible to arbitrary changes. Whereas helix 1 in HP-WT can be extended without distortion of tertiary folding, the situation in the reverse-joined hairpin ribozyme is the opposite, helix 2 can be extended,... 

To design hairpin ribozyme units that can be placed at any position in a twin ribozyme we have developed three-way junction hairpin ribozymes [17] and the branched reverse-joined hairpin ribozymes described below. 

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-lH-tetrazole [18a,b] as activator in the phosphoramidite approach, however, we also succeeded in synthesizing a chemical version of HP-... 

Fig. 5.2.11. Secondary structures of twin ribozymes. The arrows mark the cleavage sites. The substrates are fluorescein-labeled at both ends.

After having shown that combination of two catalytic modules in one molecule results in twin ribozymes with double cleavage activity we wondered whether twin ribozymes can also catalyze RNA ligation and thus be used for catalysis of RNA fragment-exchange reactions. If all fragments (substrates and cleavage products)... 

Still working with arbitrary chosen sequences for proof of principle, in future we aim to apply selection procedures to develop twin ribozymes against RNA targets with genetic relevant mutations. In parallel, our studies on the mechanism of RNA catalysis and RNA structure-function relationships will help us to understand how... 

Worldwide on-going work in the field of nucleic acid-based gene therapy targets the question of the delivery, transport, and in-vivo activity of nucleic acid drugs. The results obtained from these studies will be taken into consideration for the in-vivo design of twin ribozymes after the in-vitro assay has proven successful. 

Wikmark OG, Einvik C, De Jonckheere JF, Johansen SD. Short term sequence evolution and vertical inheritance of the Naegleria twin ribozyme group 1 intron. BMC Evol Biol. 2006 6 39. doi 10.1186/1471-2148-6-39. 

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