title: RNA functionalization strategies and their application to RNA folding dynamics and experimental RNomics
creator: Samanta, Ayan
subject: ddc-570
subject: 570 Life sciences
description: The oversimplified notion of RNA being a mere carrier of sequence information from gene to protein has been repeatedly undermined over the decades by yet another newly discovered function performed by certain RNA species. These new species include in particular RNAs which regulate gene expression in response to a metabolite sensing event. These RNAs — known as riboswitches — elegantly couple metabolite recognition with gene regulation in the apparent absence of protein helpers. Here we first sought to investigate the folding dynamics of the S-adenosyl-L-methionine responsive riboswitch by FRET spectroscopy. This requires the synthesis of full-length riboswitch constructs site-specifically modified with multiple fluorophores. For this challenging task we have established a 5-way splinted-ligation strategy to prepare dual-fluorophore labelled full-length riboswitch constructs in an unprecedented overall yield of 10 %. These constructs have further been subjected to bulk and single molecule FRET spectroscopy for ligand induced folding analysis. We confirmed similar folding dynamics for the aptamer of the complete riboswitch (aptamer + expression platform) as reported earlier for constructs containing the aptamer alone. However, we also observed a few other folding phenomena induced by a chemically slightly different, yet non-cognate metabolite, which cannot be explained by any facts known about this riboswitch to date and require further experiments to reach a final conclusion. During the course of the aforesaid work, we realized the limitations of existing nucleic acid functionalization strategies. Therefore we decided to use bioorthogonal click reactions as part of our labelling strategy. Among various different click reactions, we first had to find the one which best suits our purpose and to optimize its conditions. Having the optimized click reaction conditions at hand, we developed enzymatic strategies to site-specifically functionalize long RNAs with clickable residues. In our nucleic acid labelling strategy a diverse array of different chemical functionalities can be introduced exploiting the modular nature of click chemistry. This does not demand either de novo synthesis or optimizations of enzymatic reaction conditions for each new single compound. Furthermore, we developed a chemical approach using two different mutually orthogonal click reactions for concurrent, site-specific labelling of DNA molecules with multiple fluorophores.  Moreover, we sought to extend this strategy of enzymatic, site-specific transfer of clickable residues to long RNAs towards photochemical transfer of clickable moieties to a target RNA in a mixture of many unrelated sequences. This technique, which we call Affinity-based Chemical RNomics is a chemical approach in experimental RNomics whereby RNA sequences which bind to a given small-molecule metabolite are to be isolated from a total RNA isolate of any organism just by the virtue of its tight binding to its cognate metabolite and without any prior knowledge of its sequence. This method would therefore allow for the discovery of previously unknown riboswitches, currently the only known kind of natural RNA that binds small-molecule metabolites. Since all currently known riboswitches have been discovered by rational approaches, this will considerably extend the chances of discovering new riboswitches.
date: 2012
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/13400/1/Dissertation.pdf
identifier: DOI:10.11588/heidok.00013400
identifier: urn:nbn:de:bsz:16-opus-134003
identifier:   Samanta, Ayan  (2012) RNA functionalization strategies and their application to RNA folding dynamics and experimental RNomics.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/13400/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng