Cis-Acting Ribozymes for the Production of RNA In Vitro Transcripts with Defined 5 and 3 Ends

The use of in vitro transcribed RNA is often limited by sequence constraints at the 5′-end and the problem of transcript heterogeneity which can occur at both the 5′- and 3′-ends. This chapter describes the use of cis -acting ribozymes, 5′-end hammerhead (HH) and 3′-end hepatitis delta virus (HDV), for direct transcriptional processing to yield target RNAs with precisely defined ends. The method is focused on the use of the pRZ and p2RZ plasmids that are designed to simplify the production of such dual ribozyme templates. These plasmids each bear a 3′-HDV modified with a unique restriction site that allows the ribozyme to remain on the plasmid and, therefore, be omitted from the cloning procedure. The additional steps required to design a unique hammerhead ribozyme tailored to the 5′-end of each target RNA are detailed. In most cases, a transcriptional template bearing a 5′-HH ribozyme and a 3′-HDV ribozyme can be achieved by cloning a single PCR product into either the pRZ or p2RZ vector. Protocols for optimization of transcription yields from these templates and the isolation of the homogeneous target RNA are also described.