T7 Transcript Length Determination Using Enzymatic RNA Sequencing

The T7 transcription method from single-stranded templates with a double-stranded promoter is a common method of preparing RNA for in vitro ribozyme studies (1 ). Although the identification of full-sized ribozyme transcripts IS fairly straightforward after gel separation, the same cannot be said for the shorter substrate transcription products. Substrate gel separation always results in a heterogeneous population of RNA bands that differ in length. Previous research has shown that the shorter than full-size RNAs are the products of aborted transcription that share identical 5′-sequences. The RNAs that are one nucleotide larger than full size are owing to the propensity of T7 RNA polymerase for adding a single uncoded nucleotide to the full-size product (1 ). RNAs that are significantly larger than the coded transcript can also be produced by T7 RNA polymerase via RNA-directed RNA polymerization (2 ). This phenomenon is believed to occur when the initial transcription product can be used as either an intermolecular or intramolecular RNA primer that is subsequently extended by T7 RNA polymerase.