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Genotype-phenotype map of an RNA-ligand complex

Puchta, O.; Sobczyk, G.; Smer-Barreto, V.; Ireland, H.; Vendrell, M.; Oyarzun, D. A.; Bujnicki, J. M. A.; Whyte, G.; Kudla, G.

2020-12-17 synthetic biology
10.1101/2020.12.17.423258 bioRxiv
Show 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.

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