Generating in vitro transcripts with homogenous 3' ends using trans-acting antigenomic delta ribozyme.

Abstract

In most in vitro run-off transcription reactions with T7 RNA polymerase, transcripts with heterogeneous ends are commonly obtained. Towards the goal of finding a simple and effective procedure for correct processing of their 3' ends we propose the use of trans-acting antigenomic delta ribozyme. We demonstrate that the extension of nascent transcripts with only seven nucleotides complementary to the ribozyme's recognition site, and subsequently, the removal of those nucleotides with the ribozyme acting in trans, is an efficient procedure for generating transcripts with homogenous 3' ends. This approach was tested on two model RNA molecules: an in vitro transcript of yeast tRNA(Phe) and a delta ribozyme, which processed itself during transcription. The proposed procedure is a simple alternative to the use of ribozymes as cis-cleaving autocatalytic cassettes attached to transcript 3' ends. As there is little possibility that the required additional stretch, only seven nucleotides long, enters into stable interactions with other parts of the transcripts, it can be cleaved off with high efficacy.