基于核酶技术的人线粒体色氨酸tRNA的高效转录及其活性鉴定

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

摘要:

利用T7 RNA聚合酶(T7 RNA polymerase, T7 RNAP)的体外转录方法因其简便、高效而在RNA制备中得到广泛应用, 但该法由于T7 RNAP的启动子跨越转录起始位点而会导致转录产物多出附加序列; 如果去掉T7启动子的转录起始位点, 则会严重降低T7 RNAP的转录活性。本实验很好地消除了以上弊端, 将T7 RNAP的高效转录系统与核酶高度专一的自剪切技术相结合, 成功构建了一种不影响转录效率的体外转录方法, 而且转录后核酶能够在设计的特定位点进行自剪切并释放出目的RNA, 得到了活性达113.6 pmol/μg的人线粒体色氨酸tRNA(HmtRNATrp (UCA))。该方法转录效率高、重复性好, 适合RNA的大量精确制备。

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.