Optimization and application of the group I ribozyme trans-splicing reaction.
Group I ribozymes are naturally occurring catalytic RNAs that are able to excise themselves as introns (group I introns) from a precursor RNA, and to ligate the flanking exons. Group I ribozymes can be engineered to act in trans by recognizing a separate RNA molecule in a sequence specific manner, and to covalently link an RNA sequence to this separate RNA molecule. This ribozyme transesterification reaction has potential in molecular biology and in medicine as a new approach to gene therapy. Here we describe detailed optimized protocols where trans-splicing group I ribozymes are applied in mapping accessible sites in target messenger RNA, and in messenger RNA-repair by correcting mutations.
Einvik
Christer
C
Department of Molecular Biotechnology--RNA Research Group, Institute of Medical Biology, University of Tromsø, Norway.
Fiskaa
Tonje
T
Lundblad
Eirik W
EW
Johansen
Steinar
S
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004260:DNA Repair / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D015698:Genomic Library; D007438:Introns / Q000235:genetics; D008322:Mammals; D009154:Mutation; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000145:classification / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014162:Transfection; D014764:Viral Proteins
D017931:DNA Primers; D012322:RNA Precursors; D016337:RNA, Catalytic; D012333:RNA, Messenger; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases