A Study of Fragmentation and Spontaneous Covalent Self-assembly of the Azoarcus Ribozyme from Multiple Small Inactive RNA Fragments

摘要

The question about the origins of life often appears as a difficult question to answer. A more reliable candidate molecule for the chemical origins of life would be a molecule that is capable of making copies of itself from simple precursors. With the finding of the catalytic activities of RNA molecules by Thomas Cech and Sid Altman in late 1980s, the term ribozyme was introduced to define an RNA molecule with catalytic activity. The RNA World is a conceptual period in the early stages in the development of life because RNA simultaneously possesses evolvability and catalytic function. An RNA molecule that could evolve in such a fashion is likely to have been one of the Earth's first life forms. The most important problem facing the RNA World is the difficulty of prebiotic synthesis of RNA. Different prebiotic environments could provide the right reaction conditions for synthesis of catalytically active RNA molecules. Most importantly, these environments can support new ways to assemble monomers into polymers.;In order to understand and demonstrate how small inactive RNA oligomers can self-assemble into an autocatalytic ribozyme molecule, here I have used the Azoarcus Group I intron. First, the fragmentation and the self-assembly reactions were done using the natural IGS-tag combination of the Azoarcus group I intron, which is GUG/CAU. The main purpose was to experimentally demonstrate that the ribozyme can be broken down into five or more shorter RNA fragments and these fragments can self-assemble into a catalytically active covalent full-length molecule. Then, with the successful demonstration that five inactive RNA fragments can self-assemble, the next step to test the other possible breaking locations with mutated IGS/tag combinations. A new IGS/tag pair GCG-CGU also successfully demonstrated the five-piece self-assembly reaction. Finally, we tested these reactions in different Mg2+ concentrations to optimize the self-assembly reactions. By focusing not only one single reaction but on a collection of different sequence requirement combinations and with the development of evaporation and subsequent rehydration by spinning down methods this study successfully illustrates that covalent self-assembly from inactive RNA oligomers is possible. Therefore, this thesis work focuses on a more broader aspect of intermolecular interactions in the study of the RNA World, as illustrated in following chapters.