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<p>RNA-based fluorogenic aptamers, such as Mango, are uniquely powerful tools for imaging RNA that activate the fluorescence of a weakly or non-fluorescent small molecule when bound. A central challenge has been to develop brighter, more specific and high-affinity aptamer–ligand systems for cellular imaging. Here we report an ultrabright fluorophore for the Mango II system discovered using a structure-informed, fragment-based small-molecule microarray approach. This dye—termed SALAD1 (structure-informed, array-enabled LigAnD 1)—exhibits subnanomolar aptamer affinity and 3.5-fold brighter fluorescence than Mango II-TO1–biotin pair, a widely used fluorogenic system. Performance was improved by modulating RNA-dye molecular recognition without altering the fluorophore’s π-system. High-resolution X-ray structures reveal the binding mode for SALAD1, which exhibits improved pocket occupancy, a more defined binding pose and a unique bonding interaction with potassium. SALAD1 is cell-permeable and facilitates improved in-cell confocal RNA imaging. This work introduces an additional RNA-activated fluorophore demonstrating how fragment-based ligand discovery can be used to create high-performance ligands for RNA targets.</p><p></p>

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Structure-informed design of an ultrabright RNA-activated fluorophore

  • 期刊论文

  • 发表日期：

    2025年5月28日

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  • Mo Yang,
  • Peri R. Prestwood,
  • Luiz F. M. Passalacqua,
  • Sumirtha Balaratnam,
  • Christopher R. Fullenkamp,
  • J. Winston Arney,
  • Kevin M. Weeks,
  • Adrian Ferre-D’Amare,
  • John S. Schneekloth Jr

摘要

RNA-based fluorogenic aptamers, such as Mango, are uniquely powerful tools for imaging RNA that activate the fluorescence of a weakly or non-fluorescent small molecule when bound. A central challenge has been to develop brighter, more specific and high-affinity aptamer–ligand systems for cellular imaging. Here we report an ultrabright fluorophore for the Mango II system discovered using a structure-informed, fragment-based small-molecule microarray approach. This dye—termed SALAD1 (structure-informed, array-enabled LigAnD 1)—exhibits subnanomolar aptamer affinity and 3.5-fold brighter fluorescence than Mango II-TO1–biotin pair, a widely used fluorogenic system. Performance was improved by modulating RNA-dye molecular recognition without altering the fluorophore’s π-system. High-resolution X-ray structures reveal the binding mode for SALAD1, which exhibits improved pocket occupancy, a more defined binding pose and a unique bonding interaction with potassium. SALAD1 is cell-permeable and facilitates improved in-cell confocal RNA imaging. This work introduces an additional RNA-activated fluorophore demonstrating how fragment-based ligand discovery can be used to create high-performance ligands for RNA targets.