Detection of Ribozyme Cleavage Products Using Reverse Ligation-Mediated PCR (RL-PCR)

Among the multiple publications that describe the use of ribozymes to knock out gene expression, only a few have directly proven that the ribozymes were catalytically active in vivo (1 –3 ). Most studies rely on indirect evidence, such as a decrease in the level of the target RNA, and the comparison of the ribozyme with a mutant version that has lost cleavage activity. However, demonstration of the in vivo activity of a ribozyme requires detection of its cleavage products. For example, a ribozyme hybridized to its target RNA could promote its degradation indirectly, through cellular, double-strand specific RNases (4 ). Furthermore, a mutant ribozyme could be inactive for reasons independent of the loss of catalytic activity (e.g., the mutation could induce a change in the 3-D structure of the ribozyme, resulting in less efficient hybridization to the target RNA).