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.