Using Fluorescence Resonance Energy Transfer to Investigate Hammerhead Ribozyme Kinetics

Small catalytic RNA motifs, such as the hammerhead or hairpin ribozyme, have been used to design artificial nucleases that can cleave a selected target RNA sequence (
1
–
3
). This was first demonstrated by Uhlenbeck in which the catalytic core of a hammerhead motif was combined with antisense flanking sequences in a
trans
-acting ribozyme (
1
). The mechanism of action of such ribozymes may have as many as 12 steps or more (
see
Fig. 1 ) (
4
). The catalytic turnover of these ribozymes is often limited not by the chemical cleavage of the target RNA, but by either substrate association or product dissociation. Knowledge of the factors that contribute to the rates of association and dissociation of the substrate or product is important in designing more catalytically active hammerhead ribozymes. Although the kinetic parameters that are significant in the cleavage of a target RNA by a
trans
-acting hammerhead ribozyme have been determined using conventional electrophoretic techniques (
5
), obtaining these parameters in real time and in solution may be accomplished using fluorescence resonance energy transfer (FRET).Fig. 1.
Mechanism of RNA substrate cleavage by a
trans
-acting ribozyme (
4
).