Design and preparation of a multimeric self-cleaving hammerhead ribozyme.
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.
Ruiz
J
J
University of Connecticut Health Center, Farmington, USA.
Wu
C H
CH
Ito
Y
Y
Wu
G Y
GY
D001483:Base Sequence; D015870:Gene Expression; D005822:Genetic Vectors; D006515:Hepatitis B virus / Q000235:genetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D016337:RNA, Catalytic / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012367:RNA, Viral