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In Vivo Evolution of a Trans-Splicing Group I Intron Ribozyme

  • Author(s): Behera, Ishani
  • Advisor(s): Muller, Ulrich F
  • et al.
Abstract

The Tetrahymena group I intron was one of the two first catalytic RNAs (ribozymes) to be discovered. Group I introns are sequences that can exist between exons in pre-mRNAs, that are able to self-splice and remove themselves when forming mature mRNAs in the absence of the spliceosome. The Tetrahymena cis-splicing ribozyme was engineered to accept substrate RNAs in trans. The designed trans-splicing ribozyme was termed the “spliceozyme”. The spliceozyme could in principle be used in therapeutic applications. However, there are two hurdles to overcome; the delivery of the ribozyme into cells and its efficiency once delivered. The focus of this study is to improve the efficiency of the trans-splicing ribozyme in cells by evolving the ribozyme in E. coli. In a previous study, an evolution system in cells was developed for the spliceozyme which was used to generate a clone, W11, that increased product formation. However, this evolution used high ribozyme expression levels and focused on a single splice site. To improve the efficiency and sequence generality, the spliceozyme was evolved at low expression levels at two different splice sites with different flanking sequences. The results of the spliceozyme evolution in bacterial cells showed that specific mutations are able to improve spliceozyme efficiency in bacterial cells. This suggests that further analysis, and perhaps further evolution experiments, could generate spliceozymes with improved efficiency on a broad range of substrate sequences.

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