The Reverse-joined Hairpin Ribozyme (HP-RJ)

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 design of reverse-joined hairpin ribozymes is based on the assumption that the bent active conformation of the hairpin ribozyme can be reached from distinct

Fig. 5.2.7. Schematic representation of folding into the hairpin ribozyme active conformation.
Fig. 5.2.8. Secondary structure of the reverse-joined hairpin ribozyme HP-RJ. The arrow denotes the cleavage site. The four helices (H-1 through H-4) are marked by bars.

starting points. Specific contacts between the nucleotides within Loops A and B keep the hairpin ribozyme in the active conformation. It is, therefore, likely that joining the two domains in reverse order also will create an RNA structure that is able to fold into a conformation with loop-loop contact, as long as the capacity of the loop nucleotides for specific interaction is preserved. On the basis of this principle we have designed and synthesized the reverse-joined hairpin ribozyme HP-RJ shown in Figure 5.2.8 [16]. The A6-linker acts as a flexible hinge between helix 1 and helix 4 and is required to bridge the distance of 34 A between the distal ends of helix 1 and helix 4, as observed in the crystal structure [14].

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-WTcan be extended without distortion of tertiary folding, the situation in the reverse-joined hairpin ribozyme is the opposite, helix 2 can be extended, and helix 1 cannot. This enables combination of the two catalytic modules in a single mode as shown in Figure 5.2.11, namely the conventional hairpin ribozyme on the right hand side attached to the reverse-joined unit on the left hand side of the molecule.

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.

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