Minimized Hammerhead Ribozymes

The hammerhead ribozyme as engineered by Haseloff and Gerlach ( 1 ) consists of the conserved nucleotides C 3 -A 9 and G 12 -C 15.2 connected by nucleotides that form stem-loop II, and nucleotides at the 5′- and 3′-ends, which form helix I and helix III, respectively, in complex with the substrate. Such a ribozyme is shown in Fig. 1 . Most hammerhead ribozymes of this type are constructed with 12 nucleotides in stem-loop II, with four nucleotides in the loop and eight nucleotides forming a 4 bp double helix.   Fig. 1.  An RNA substrate and hammerhead ribozyme in the form described by Haseloff and Gerlach ( 1 ). The substrate is drawn containing the triplet GUC, which is the triplet usually chosen for cleavage. Cleavage occurs on the 3′-side of C 17 as shown by the arrow. The remaining sequence of the substrate determines the identities of each N in the ribozyme. The conserved nucleotides C 3 -A 9 and G 12 -A 15.1 (C 15.2 ) are in the ribozyme. On binding to the substrate, nucleotides in the 5′- and 3′-arms of the ribozyme form helix I and helix III, respectively. The nucleotides joining A 9 and G 12 form helix II and loop II; the sequence of stem-loop II shown here occurs naturally in the satellite RNA (+) strand of tobacco ringspot virus ( 2 ). Generally, most synthetic ribozymes are constructed with 4 bp in helix II and 4 nucleotides in loop II. The nucleotides are numbered according to the scheme of Hertel et. al. ( 3 ).