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Title: A novel in vitro selection method to aid in the development of ribozyme-based riboswitches
Author: Haines, Matthew Christopher
ISNI:       0000 0004 9350 6149
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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Ribozymes are RNA sequences capable of catalysing chemical reactions. For some ribozymes their catalytic rate is influenced by molecular ligands. Those Ligand-responsive ribozymes whose catalytic activity influences gene expression can be referred to as ribozyme-based riboswitches. These sequences have potential applications as therapeutics and in the fields of enzyme and metabolic engineering. However, their application is restricted by the limited variety of detectable ligands. With the aim of addressing this issue, a novel in vitro selection method for the enrichment of ribozymes was developed. Compared to analogous methods, this method requires approximately half the time to implement and can be scaled up to 96-well format. Furthermore, the avoidance of size selection steps facilitates larger insertion and deletion mutations, ensuring more diverse regions of the sequence space are explored during selection. In testing and developing this method, new and existing theophylline-activated ribozymes were enriched. This process was assisted by a model, developed to aid the optimisation of selection. As confirmed with NGS data, the model accurately described the dynamics of key sequences for the majority of the selection experiment. Following the enrichment and identification of theophylline-activated ribozymes, several of these sequences were tested for their ability to activate gene expression in E. coli. In contrast to the suggestions of previous reports, all tested theophylline-activated ribozymes increased GFP expression in response to ligand concentrations. Although disputed, this result suggests the existence of a relationship between the in vitro responses of ribozyme sequences and their ability to function in vivo. Though ribozyme-based riboswitches with new ligand-specificities were not characterised during this work, the methods and framework outlined, coupled with the demonstration that selected sequences can regulate gene expression suggests novel ribozyme-based riboswitches should be identified with future work.
Supervisor: Baldwin, Geoffrey ; Stan, Guy-Bart ; Oyarzun, Diego Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral