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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.

Author information

1
Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, Bethesda, MD, USA.
2
Architecture et Réactivité de l'ARN, Université de Strasbourg, Institut de biologie moléculaire et cellulaire du CNRS, Strasbourg, France.
3
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
4
Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, Bethesda, MD, USA. adrian.ferre@nih.gov.

Abstract

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.

PMID:
30992561
DOI:
10.1038/s41589-019-0267-9

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