Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness

Biochemistry
Robert J TrachmanAdrian R Ferré-D'Amaré

Abstract

Several RNA aptamers that bind small molecules and enhance their fluorescence have been successfully used to tag and track RNAs in vivo, but these genetically encodable tags have not yet achieved single-fluorophore resolution. Recently, Mango-II, an RNA that binds TO1-Biotin with ∼1 nM affinity and enhances its fluorescence by >1500-fold, was isolated by fluorescence selection from the pool that yielded the original RNA Mango. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin and TO3-Biotin, and found that despite their high affinity, the ligands adopt multiple distinct conformations, indicative of a binding pocket with modest stereoselectivity. Mutational analysis of the binding site led to Mango-II(A22U), which retains high affinity for TO1-Biotin but now discriminates >5-fold against TO3-biotin. Moreover, fluorescence enhancement of TO1-Biotin increases by 18%, while that of TO3-Biotin decreases by 25%. Crystallographic, spectroscopic, and analogue studies show that the A22U mutation improves conformational homogeneity and shape complementarity of the fluorophore-RNA interface. Our work demonstrates that even after extensive functional selection, aptamer RNAs can be further improve...Continue Reading

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Citations

Feb 14, 2019·Nucleic Acids Research·Qianqian ZhaiDengguo Wei
Nov 20, 2019·Nucleic Acids Research·Christian SteinmetzgerClaudia Höbartner
Jul 3, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Mubarak I UmarChun Kit Kwok
Apr 25, 2019·Protein Science : a Publication of the Protein Society·Lynda Truong, Adrian R Ferré-D'Amaré
Nov 30, 2018·Chemistry : a European Journal·Christian SteinmetzgerClaudia Höbartner
Mar 11, 2020·Nature Communications·Adam D CawteDavid S Rueda
Aug 10, 2019·Acta Crystallographica. Section F, Structural Biology Communications·Robert J TrachmanAdrian R Ferré-D'Amaré
Apr 18, 2019·Nature Chemical Biology·Robert J TrachmanAdrian R Ferré-D'Amaré
Sep 19, 2020·Chemical Society Reviews·Ronald Micura, Claudia Höbartner
Aug 20, 2019·Quarterly Reviews of Biophysics·Robert J Trachman, Adrian R Ferré-D'Amaré
Jan 28, 2020·Journal of Materials Chemistry. B, Materials for Biology and Medicine·Puchakayala SwethaJian-Hui Jiang
Jan 24, 2021·RNA·Michael T Banco, Adrian R Ferré-D'Amaré
Mar 22, 2021·The Journal of Biological Chemistry·Eric Westhof, Neocles B Leontis
Apr 4, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Ousama Al ShanaaMarina Padkina
Apr 20, 2021·Critical Reviews in Analytical Chemistry·Huangmei Zhou, Sanjun Zhang
Mar 25, 2019·Methods : a Companion to Methods in Enzymology·Alexis AutourMichael Ryckelynck
Mar 31, 2019·Methods : a Companion to Methods in Enzymology·Irina Randrianjatovo-Gbalou, Marc Delarue
Jun 12, 2021·Nature Communications·Mateusz MieczkowskiClaudia Höbartner
Jun 18, 2021·Molecular Omics·Mohammad Faysal Al MazidJun-Seok Lee
Jan 1, 2022·Biophysical Journal·Balasubramanian HarishCatherine A Royer

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