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Better ribozymes for trans-splicing and triphosphorylation

  • Author(s): Dolan, Gregory F.
  • et al.
Abstract

Catalytic RNAs (ribozymes) are RNAs that can catalyze a chemical reaction without the use of protein cofactors. This dissertation centered on improving the activities for two different ribozymes: a trans-splicing group I intron ribozyme and a triphosphorylation ribozyme. Trans-splicing group I intron ribozymes can be used in gene therapy and synthetic biology applications, and this work focused on improving the trans-splicing activity of the group I intron ribozyme from Azoarcus, a small and robustly folding ribozyme located in the intervening sequence of pre-tRNAIle. These results showed that although the trans- splicing Azoarcus ribozyme could not mediate a growth phenotype in E. coli cells, the Azoarcus ribozyme is indeed capable of catalyzing the in vitro trans-splicing reaction as efficiently as other trans-splicing ribozymes previously tested. The most efficient trans-splicing Azoarcus ribozymes contained secondary structural contacts that mimicked the contacts made by the natural cis- splicing ribozyme, suggesting that the natural splicing interactions are the preferred trans-splicing interactions for the Azoarcus ribozyme. The second ribozyme improved in this dissertation is the triphosphorylation ribozyme, a ribozyme that triphosphorylates the 5'-hydroxyl of RNAs using trimetaphosphate as a substrate. This ribozyme's activity is important in the context of the RNA world and in the origin of life because RNA polymerization is an energetically unfavorable process in an aqueous environment, and the 5'-triphsophate can supply the energetic driving force for RNA polymerization. A previous graduate student in the Müller laboratory performed an in vitro selection identifying the first triphosphorylation ribozymes. The work herein improved the activity of one of the most active original triphosphorylation ribozymes by performing a 'doped selection.' A ̀doped selection' is a method to identify sequence variants with higher activity by selecting for the best ribozymes from a partially randomized pool consisting of sequence variants of the original ribozyme. This selection yielded a ribozyme with seven mutations and a catalytic rate that was 24-fold higher then the first generation ribozyme. Unlike the original ribozyme, this new ribozyme has measurable activity at prebiotically relevant trimetaphosphate concentrations and has a catalytic rate of 6.8 min⁻¹ at optimal conditions

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