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Crystal structures of the Mango-II RNA aptamer reveal heterogeneous fluorophore binding and guide engineering of variants with improved selectivity and brightness. Robert Trachman, Amir Abdolahzadeh, Alessio Andreoni, Razvan Cojocaru, Jay R Knutson, Michaël Ryckelynck, Peter John Unrau, and Adrian Ferré-D’Amaré Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00399 • Publication Date (Web): 16 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018
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Biochemistry
Crystal structures of the Mango-II RNA aptamer reveal heterogeneous fluorophore binding and guide engineering of variants with improved selectivity and brightness. Robert J. Trachman III1, Amir Abdolahzadeh2, Alessio Andreoni1, Razvan Cojocaru2, Jay R. Knutson1, Michael Ryckelynck3, Peter J. Unrau2, Adrian R. Ferré-D’Amaré1* 1
Biochemistry and Biophysics Center, National Heart, Lung and Blood Institute, 50 South Drive MSC 8012, Bethesda, MD 20892-8012, USA
2
Department of Molecular Biology and Biochemistry,
Simon Fraser University, 8888 University Drive,
Burnaby, British Columbia, Canada V5A 1S6 3 Architecture et Réactivité de l'ARN, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, 67084, Strasbourg, France KEYWORDS RNA, aptamer, fluorescence, structure, G-quadruplex, fluorophore, thiazole orange 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, MangoII, an RNA that binds TO1-Biotin with ~1 nM affinity and enhances its fluorescence by >1,500-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 MangoII(A22U), which retains high affinity for TO1-Biotin but now discriminates over 5-fold against TO3-biotin. Moreover, fluorescence enhancement of TO1-Biotin increases 18% while that of TO3-Biotin decreases by 25%. Crystallographic, spectroscopic, and analog 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 improved through structure-guided engineering.
Several aptamers that induce fluorescence of their cognate small molecules by >1000-fold, and that can be used in vivo as RNA analogues of fluorescent proteins have been described.1-3 Among these, RNA Mango is noteworthy because of its small size (
