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Abstract
In the RNA world, the exchange of sequence patches between two RNAs is an intriguing evolutionary concept, allowing generation of new RNA molecules with novel functionality. Based on the hairpin ribozyme (HPR) with its unique cleavage‐ligation properties, we here demonstrate RNA supported RNA recombination as a possible scenario for the emergence of larger RNA molecules with more complex functionality. A HPR variant designed for the purpose of recombination is capable of cleaving two different RNA molecules, one being a hammerhead ribozyme (HHR) and the other an aptamer (A), and to subsequently recombine and ligate the resulting fragments to a hammerhead ribozyme that is allosterically controlled (HHA) by a cognate ligand. Two such recombination processes involving aptamers for either theophylline or flavine mononucleotide (FMN) are demonstrated with yields of functional recombination product of up to 34 %.
This thesis summarizes the published works by Robert Hieronymus which were done in the group of bioorganic chemistry of Prof. Dr. Sabine Müller. The different works had the goals to design, develop, and test catalytically active RNA systemes that might have been plausible for an early RNA world scenario.
The different RNA systems presented in this thesis were developed via rational design, for which each time the hairpin ribozyme (HPR) was utilized as a design template. The HPR belongs to the group of self-cleaving ribozymes and comes with features that make it a very attractive candidate for the contemplated tasks: It’s small, it’s variable in sequence, and it can cleave or ligate bound RNA substrates depending on the substrate binding strength. Substrates with weak binding to the ribozyme tend to be cleaved while substrates with stronger binding become ligated. This feature was utilized to develop RNA systems with catalytic cascades.
The first of the catalytic RNA systems that is presented in this thesis establishes a HPR mediated recombination system. In a one-pot reaction two RNA strands without function but with pro-functional regions are getting bound and cleaved by the HPR in separate ways. The generated fragments with the pro-functional parts are designed to bind stronger to the HPR than their non-functional counterparts and are ligated in a subsequential reaction by the HPR. The recombination product is a hammerhead ribozyme (HHR), and thus, a self-cleaving ribozyme on its own, whose synthesis can be monitored by the addition and cleavage of a separate RNA substrate.
The second RNA system is also a recombination system mediated by a HPR, but this time it starts with the functional HHR product from the previous system as an educt. Via a similar mechanism as before the recombination is done with another functional RNA: an RNA aptamer (sensoric RNA). The recombination of HHR and aptamer leads to the generation of a hammerhead aptazyme, an HHR whose cleavage functions are now regulated via ligand binding on the aptamer part. This novel system was successfully demonstrated with RNA sequences of theophylline and FMN aptamers as different educts for the recombination reaction.
The HPR in the final work presented here was designed as a self-splicing ribozyme. Here the HPR sequence is located within the intron and is flanked by two exon sequences on both its ends. The developed HPR is able to fold itself in two alternative conformations, both with either one of the intron-exon interfaces located within the formed catalytic site. Subsequently to the first cleavage and dissoziation of one of the exons, the HPR folds into the alternative conformation, which triggers the cleavage reaction of the remaining exon. Once both exons are cleaved off, the fragments are ligated by the HPR, which concludes the catalytic cascade with the healing of the RNA source strand.
The various works presented in this thesis demonstrate nicely the flexibility of the HPR and how well suited it is to be utilized as a template in rational design of RNA systems. Furthermore, it is plausible to assume that the HPR, due to its many features, must have had a place in the early RNA world.