General Design and Construction of RNase P Ribozymes for Gene-Targeting Applications

RNase P ribozyme, such as M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli , cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural ptRNA substrate. When covalently linked with a guide sequence, the M1 ribozyme can function as a sequence-specific endonuclease, M1GS RNA, and cleave any target RNA sequences that basepair with the guide sequence. Using the mRNA coding for the major transcription regulatory protein ICP4 of herpes simplex virus 1 (HSV-1) as the model target, we describe in this chapter the general design and construction of M1GS ribozymes for gene-targeting applications. Specifically, methods are described in detail to determine ideal target regions of an mRNA for M1GS ribozymes and to construct highly active RNase P ribozymes that target these regions. Extensive protocols for in vitro synthesis of the ribozymes and for the cleavage assay of the ribozyme activity are also included. These methods are intended to provide general guidelines for the design and construction of M1GS ribozymes for gene-targeting applications.