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Investigation of RNA kissing complexes by native electrospray mass spectrometry : magnesium binding and ion mobility

Abstract : Besides being the molecular intermediate between DNA and proteins, RNA can have many other functions such as gene regulation (riboswitches), gene expression (mRNA and tRNA) or catalysis (ribozymes). RNA function is linked to its structure and its folding dynamics. Cations such as magnesium bind to RNA and are in some instances essential for proper folding and for stability. The need of structural and thermodynamic details about Mg2+ interactions is then of upmost importance in the study of the structurefunction relationships. The first part of our work consists in characterizing the binding equilibria between magnesium and RNA model motifs, called kissing complexes, using native mass spectrometry (MS). MS makes it possible to distinguish individual binding stoichiometries, and the present work consisted in developing a method to quantify each species, taking into account the contribution of nonspecific adducts. We also explored how tandem mass spectrometry (MS/MS) could further help localizing magnesium ions. Further, we explored the structures of RNA complexes in the gas phase using ion mobility mass spectrometry (IMMS), with the aim to detect shape changes upon cation or ligand binding. But in contrast with anticipations, we found that DNA and RNA duplexes as well as RNA kissing complexes undergo a significant compaction at charge states naturally produced by native ESI-MS, which may hide the effect of cations. Our work showcases how mass spectrometry can bring novel information on RNA-cation binding stoichiometries and affinities, but also discusses some limitations of a gas-phase method to probe solution structures.
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https://tel.archives-ouvertes.fr/tel-01717579
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Submitted on : Monday, February 26, 2018 - 3:08:06 PM
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  • HAL Id : tel-01717579, version 1

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Clemence Rabin. Investigation of RNA kissing complexes by native electrospray mass spectrometry : magnesium binding and ion mobility. Agricultural sciences. Université de Bordeaux, 2017. English. ⟨NNT : 2017BORD0892⟩. ⟨tel-01717579⟩

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