Inactivation of gene expression using ribonuclease P and external guide sequences.

Publisher Summary Experiments performed in the late 1970s indicated that antisense oligonucleotides could be used to affect gene expression. Their activity is often associated with their ability to induce RNase H-mediated cleavage of the target RNA via formation of a DNA/RNA hybrid. The discovery that RNAs could act as enzymes (a function previously ascribed only to proteins) by catalyzing self-cleavage or cleavage of another RNA molecule has extended antisense-targeted technology. Most methods that employ self-cleaving ribozymes, such as those that utilize the hammerhead or hairpin ribozymes, are based on the fact that the structural domain of these enzymes can be divided into two separate entities (or oligonucleotides): one is the catalytic moiety and the other, containing the cleavage site, is referred to as the substrate. The sequence of these two oligonucleotides allows them to form hydrogen (and/or non-hydrogen)-bonded interactions, resulting in the formation of the structural domain capable of carrying out the cleavage reaction. In general, a particular sequence in the target RNA is designated as the substrate and the ribozyme (antisense RNA) is custom designed to form an active complex with the substrate through conventional Watson-Crick hydrogen bonding. The sequence encoding the enzyme is cloned between the appropriate promoter and transcription termination signal, and is delivered to the cells. Another variation of the antisense concept, which is discussed in this chapter, is based on the substrate-recognition properties of the ubiquitous enzyme ribonuclease P (RNase P). To understand the development of such a technology, it is important to be familiar with some of the properties of RNase P.