Trans-acting antigenomic HDV ribozyme for production of in vitro transcripts with homogenous 3′ ends

Abstract

During in vitro run-off transcription with T7 RNA polymerase, transcripts with heterogenous 3′ ends are commonly synthesized. Here, we describe an efficient procedure for correct processing of transcript 3′ ends with the use of antigenomic HDV ribozyme. The procedure involves the extension of nascent transcripts with seven nucleotides complementary to the ribozyme's recognition site and, subsequently, the removal of those nucleotides with the HDV ribozyme acting in trans. Sufficient reaction rates and final cleavage extents of approx. 90% can be obtained with just twofold excess of the ribozyme. The highest concentration of RNA substrate suggested for practical applications turns out to be 3 μM. The procedure is an alternative to the use of ribozymes as cis-cleaving autocatalytic cassettes attached to transcript 3′ ends. © 2012 Springer Science+Business Media, LLC.