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Nat Chem Biol. 2019 May;15(5):472-479. doi: 10.1038/s41589-019-0267-9. Epub 2019 Apr 15.

Structure and functional reselection of the Mango-III fluorogenic RNA aptamer.

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Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, Bethesda, MD, USA.
Architecture et Réactivité de l'ARN, Université de Strasbourg, Institut de biologie moléculaire et cellulaire du CNRS, Strasbourg, France.
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, Bethesda, MD, USA.


Several turn-on RNA aptamers that activate small-molecule fluorophores have been selected in vitro. Among these, the ~30 nucleotide Mango-III is notable because it binds the thiazole orange derivative TO1-Biotin with high affinity and fluoresces brightly (quantum yield 0.55). Uniquely among related aptamers, Mango-III exhibits biphasic thermal melting, characteristic of molecules with tertiary structure. We report crystal structures of TO1-Biotin complexes of Mango-III, a structure-guided mutant Mango-III(A10U), and a functionally reselected mutant iMango-III. The structures reveal a globular architecture arising from an unprecedented pseudoknot-like connectivity between a G-quadruplex and an embedded non-canonical duplex. The fluorophore is restrained into a planar conformation by the G-quadruplex, a lone, long-range trans Watson-Crick pair (whose A10U mutation increases quantum yield to 0.66), and a pyrimidine perpendicular to the nucleobase planes of those motifs. The improved iMango-III and Mango-III(A10U) fluoresce ~50% brighter than enhanced green fluorescent protein, making them suitable tags for live cell RNA visualization.

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