Ribozyme conventional

Specificity of conventional protein enzymes is provided by precise molecular fit. The mutual recognition of an enzyme and is substrate is the result of various intermolecular forces which are almost always strongly dominated by hydrophobic interaction. In contrast, specificity of catalytic RNAs is provided by base pairing (see for example the hammerhead ribozyme in Figure 1) and to a lesser extent by tertiary interactions. Both are the results of hydrogen bond specificity. Metal ions too, in particular Mg2+, are often involved in RNA structure formation and catalysis. Catalytic action of RNA on RNA is exercised in the cofolded complexes of ribozyme and substrate. Since the formation of a ribozyme s catalytic center which operates on another RNA molecule requires sequence complementarity in parts of the substrate, ribozyme specificity is thus predominantly reflected by the sequence and not by the three-dimensional structure of the isolated substrate. 

The conventional hairpin ribozyme is derived from the minus strand of Tobacco ringspot virus satellite RNA [12a,b]. It works in cis (intramolecularly) in nature, but... 

Fig. 5.2.5. Secondary structure of the conventional hairpin ribozyme HP-WT. The arrow denotes the cleavage site. The four helices (H-1 through H-4) are marked by bars.

Reverse-joined hairpin ribozymes have been introduced by Komatsu et al. [15a,b]. This type of ribozyme is derived from the conventional hairpin ribozyme by dissecting the two domains at the hinge between helix 2 and 3 and re-joining helix 4 to helix 1 via a linker comprising six unpaired bases (Figure 5.2.8). 

The reverse-joined hairpin ribozyme catalyzes the same specific reaction as the conventional hairpin ribozyme and has similar kinetic behavior [16]. 

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,... 

Until recently, it was thought that all enzymes are proteins. But this dogma of biochemistry was recently overthrown by the discovery that some types of RNA can function as biocatalysts. They can cut, splice, and assemble themselves without any outside help from conventional enzymes. This discovery of ribozymes, as they are called, had a major impact on theories of the origin of life. The question was Which came first in the primordial soup from which life began, the proteins or the nucleic acids Proteins could be enzymes and catalyze the reactions needed for life, but they could not store genetic information. The reverse was thought to be true for nucleic acids. But, with the discovery of catalytic activity in certain types of RNA, it now seems almost certain that the earth of 4 billion years ago... 

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... 

The novel tRNA -driven heterodimeric maxizymes, which can cleave one substrate at two independent sites simultaneously, can be designed very easily and, thus, they should be very useful tools in vivo. Nevertheless, it remains true that two independent iRNA l-driven parental riboz5mies can also cleave such a substrate at the two different sites, albeit with lower efficiency In the following section, we shall describe tRNA Ldnven heterodimeric maxizymes that specifically cleave a chimeric mRNA with high selectivity while conventional ribozymes failed to do so. 

Figure 5. Types of translocation and the possible sites of cleavage within notmal ABL mRNA and abnormal BCR-ABL fusion mRNAs by conventional ribozymes (A) and the design of the novel maxizyme (B).

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