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Genotype-phenotype map of an RNA-ligand complex

Olga Puchta, Grzegorz Sobczyk, Vanessa Smer-Barreto, Hollie Ireland, Marc Vendrell, View ORCID ProfileDiego A. Oyarzún, Janusz M. Bujnicki, Graeme Whyte, Grzegorz Kudla
doi: https://doi.org/10.1101/2020.12.17.423258
Olga Puchta
1MRC Human Genetics Unit, Institute for Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh, UK
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Grzegorz Sobczyk
1MRC Human Genetics Unit, Institute for Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh, UK
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Vanessa Smer-Barreto
2MRC Institute for Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh, UK
3School of Informatics, The University of Edinburgh, Edinburgh, UK
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Hollie Ireland
1MRC Human Genetics Unit, Institute for Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh, UK
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Marc Vendrell
4Centre for Inflammation Research, The University of Edinburgh, Edinburgh, UK
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Diego A. Oyarzún
3School of Informatics, The University of Edinburgh, Edinburgh, UK
5School of Biological Sciences, The University of Edinburgh, Edinburgh, UK
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  • ORCID record for Diego A. Oyarzún
Janusz M. Bujnicki
6International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland
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Graeme Whyte
7Heriot-Watt University, Edinburgh, Scotland, UK
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Grzegorz Kudla
1MRC Human Genetics Unit, Institute for Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh, UK
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  • For correspondence: gkudla@gmail.com
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Abstract

RNA-ligand interactions play important roles in biology and biotechnology, but they often involve complex three-dimensional folding of RNA and are difficult to predict. To systematically explore the phenotypic landscape of an RNA-ligand complex, we used microarrays to investigate all possible single and double mutants of the 49-nt RNA aptamer Broccoli bound to the fluorophore DFHBI-1T. We collected more than seven million fluorescence measurements in varying conditions, and inferred dissociation rate constants, spectral shifts, and intragenic epistasis. Our results reveal an unexpectedly complex phenotypic landscape, in which mutations near the fluorophore binding pocket modulated magnesium-, potassium- and fluorophore-binding and fluorescence spectra, while distal mutations influenced structural stability and fluorescence intensity. We trained a machine learning model that accurately predicted RNA secondary structure from local epistatic interactions, despite the presence of G-quadruplexes and other noncanonical structures. Our experimental platform will facilitate the discovery and analysis of new RNA-ligand interactions.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted December 17, 2020.
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Genotype-phenotype map of an RNA-ligand complex
Olga Puchta, Grzegorz Sobczyk, Vanessa Smer-Barreto, Hollie Ireland, Marc Vendrell, Diego A. Oyarzún, Janusz M. Bujnicki, Graeme Whyte, Grzegorz Kudla
bioRxiv 2020.12.17.423258; doi: https://doi.org/10.1101/2020.12.17.423258
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Genotype-phenotype map of an RNA-ligand complex
Olga Puchta, Grzegorz Sobczyk, Vanessa Smer-Barreto, Hollie Ireland, Marc Vendrell, Diego A. Oyarzún, Janusz M. Bujnicki, Graeme Whyte, Grzegorz Kudla
bioRxiv 2020.12.17.423258; doi: https://doi.org/10.1101/2020.12.17.423258

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