Design and Production of Asymmetric Hammerhead Ribozymes

Asymmetric hammerhead ribozymes represent a variation of catalytic antisense RNAs that have been introduced in this book as a specialized form of ribozymes in Chapter 14 . Catalytic antisense RNAs are characterized by a relatively long antisense sequence (>30 bases) that allow efficient association with the target RNA. Owing to their long antisense flanks, multiple turnover cleavage of several target RNA molecules cannot be anticipated, but instead, one molecule of catalytic antisense RNA is expected to inactivate one target RNA molecule. In those catalytic antisense RNAs described in Chapter 14 , the catalytic domain of the hammerhead ribozymes was flanked by two relatively long antisense regions. By contrast, asymmetric hammerhead ribozymes contain only one long antisense flank—the one that forms helix III in the hammerhead complex—whereas the other flank that forms helix I is truncated to as little as three nucleotides (1 ). This “design” (see Fig. 1 ) has the advantage that the catalytic antisense RNA is physically dissected into its two functional domains, which control (1) association with the target RNA and (2) cleavage of the target RNA. Such an asymmetric hammerhead ribozyme resembles an antisense RNA with an additional cleavage domain (“bombshell”) at the 5′-terminus. The association of the asymmetric hammerhead ribozyme with its target RNA is entirely controlled by the antisense domain downstream of the catalytic domain. This allows straightforward optimization of the length of helix III for efficient association with the target RNA, a method that is outlined in Chapter 30 . The general strategy for applying asymmetric hammerhead ribozymes is to create first a construct with a helix III-forming region of about 200 nt or more. For further improvement, this antisense domain is truncated, and the resulting catalytic RNAs are experimentally tested for fast hybridization with their target. This will select catalytic RNA with “optimized” binding kinetics.