RT Journal Article
SR Electronic
T1 Genotype-phenotype map of an RNA-ligand complex
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.12.17.423258
DO 10.1101/2020.12.17.423258
A1 Puchta, Olga
A1 Sobczyk, Grzegorz
A1 Smer-Barreto, Vanessa
A1 Ireland, Hollie
A1 Vendrell, Marc
A1 Oyarzún, Diego A.
A1 Bujnicki, Janusz M.
A1 Whyte, Graeme
A1 Kudla, Grzegorz
YR 2020
UL http://biorxiv.org/content/early/2020/12/17/2020.12.17.423258.abstract
AB 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 StatementThe authors have declared no competing interest.