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  • Title: Design and preparation of a multimeric self-cleaving hammerhead ribozyme.
    Author: Ruiz J, Wu CH, Ito Y, Wu GY.
    Journal: Biotechniques; 1997 Feb; 22(2):338-45. PubMed ID: 9043708.
    Abstract:
    The activity of a ribozyme can be impaired by additional sequences at the 5' and 3' termini of the catalytic sequence. To approach this problem, a system was designed that minimizes sequences upstream and downstream from active regions of a hammerhead ribozyme and allows delivery of a large number of active molecules. A self-cleavable multimeric molecule was prepared by placing a ribozyme target sequence (derived from the core region of the hepatitis B virus [HBV]) upstream and downstream from the catalytic sequence. This construct was cloned in tandem into in vitro expression vectors. 32P-UTP-labeled transcripts of the multimeric construct, as well as non-self-cleaved monomeric ribozyme controls, and substrate were synthesized. The multimeric ribozyme molecule efficiently self-cleaved to release monomeric ribozymes lacking any extra upstream and downstream sequences. In addition, monomers were substantially more active against the HBV target RNA than the non-self-cleavable ribozymes. Up to 80% degradation of the target RNA was achieved by a tenfold molar excess of a pentameric construct. We conclude that ribozymes can be produced as a multimeric tandem of self-cleavable molecules, the monomers of which are more active than monomeric ribozymes and highly efficient in cleavage of target transcripts.
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