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

Group I introns are ribozymes (catalytic RNAs) that excise themselves from RNA primary transcripts by catalyzing two successive transesterification reactions. These cis-splicing ribozymes can be converted into trans-splicing ribozymes, which can modify the sequence of a separate substrate RNA, both in vitro and in vivo. Previous work on trans-splicing ribozymes has mostly focused on the 16S rRNA group I intron ribozyme from Tetrahymena thermophila. Here, we test the trans-splicing potential of the tRNA(Ile) group I intron ribozyme from the bacterium Azoarcus. This ribozyme is only half the size of the Tetrahymena ribozyme and folds faster into its active conformation in vitro. Our results showed that in vitro, the Azoarcus and Tetrahymena ribozymes favored the same set of splice sites on a substrate RNA. Both ribozymes showed the same trans-splicing efficiency when containing their individually optimized 5' terminus. In contrast to the previously optimized 5'-terminal design of the Tetrahymena ribozyme, the Azoarcus ribozyme was most efficient with a trans-splicing design that resembled the secondary structure context of the natural cis-splicing Azoarcus ribozyme, which includes base-pairing between the substrate 5' portion and the ribozyme 3' exon. These results suggested preferred trans-splicing interactions for the Azoarcus ribozyme under near-physiological in vitro conditions. Despite the high activity in vitro, however, the splicing efficiency of the Azoarcus ribozyme in Escherichia coli cells was significantly below that of the Tetrahymena ribozyme.

How does a non-coding RNA evolve in cells? To address this question experimentally we evolved a trans-splicing variant of the group I intron ribozyme from Tetrahymena over 21 cycles of evolution in E.coli cells. Sequence variation was introduced during the evolution by mutagenic and recombinative PCR, and increasingly active ribozymes were selected by their repair of an mRNA mediating antibiotic resistance. The most efficient ribozyme contained four clustered mutations that were necessary and sufficient for maximum activity in cells. Surprisingly, these mutations did not increase the trans-splicing activity of the ribozyme. Instead, they appear to have recruited a cellular protein, the transcription termination factor Rho, and facilitated more efficient translation of the ribozyme's trans-splicing product. In addition, these mutations affected the expression of several other, unrelated genes. These results suggest that during RNA evolution in cells, four mutations can be sufficient to evolve new protein interactions, and four mutations in an RNA molecule can generate a large effect on gene regulation in the cell.

In support of the RNA world hypothesis, previous studies identified trimetaphosphate (Tmp) as a plausible energy source for RNA world organisms. In one of these studies, catalytic RNAs (ribozymes) that catalyze the triphosphorylation of RNA 5'-hydroxyl groups using Tmp were obtained by in vitro selection. One ribozyme (TPR1) was analyzed in more detail. TPR1 catalyzes the triphosphorylation reaction to a rate of 0.013 min-1 under selection conditions (50 mM Tmp, 100 mM MgCl2, 22°C). To identify a triphosphorylation ribozyme that catalyzes faster triphosphorylation, and possibly learn about its secondary structure TPR1 was subjected to a doped selection. The resulting ribozyme, TPR1e, contains seven mutations relative to TPR1, displays a previously unidentified duplex that constrains the ribozyme's structure, and reacts at a 24-fold faster rate than the parent ribozyme. Under optimal conditions (150 mM Tmp, 650 mM MgCl2, 40°C), the triphosphorylation rate of TRP1e reaches 6.8 min-1.