High-level transcription of human mitochondrial tRNATrp based on ribozyme and characterization on its aminoacylation activity

Abstract:

In vitro transcription systems with T7 RNA polymerase (T7 RNAP) were widely used in preparation of RNA because of their simplicity and high efficiency. The transcripts would have additional 5￠ sequence since T7 promoter spans the transcription start site, while deletion of the transcription start site would severely reduce the T7 RNAP transcriptional activity. We successfully developed an in vitro transcription by combining of T7 RNAP high efficient transcription system and highly specific self-splicing technology of ribozymes, in this system, ribozyme self-splices at the designed specific site and releases the aim RNA without affecting transcription efficiency of T7 RNAP, the aminoacylation activity of human mitochondrial tRNATrp (HmtRNATrp (UCA)) is 113.6 pmol/μg. This method with its high efficiency on transcription and good repeatability is very suitable for preparation of accurate RNA in large scale.