Structure-informed design of an ultrabright RNA-activated fluorophore

Area of Science:

• Molecular Biology
• Biochemistry
• Chemical Biology

Background:

• RNA-based fluorogenic aptamers, like Mango, are vital for RNA imaging.
• A key challenge is creating brighter, more specific, high-affinity aptamer-ligand systems for cellular imaging.

Purpose of the Study:

• To discover an ultrabright fluorophore for the Mango II aptamer system.
• To enhance cellular RNA imaging capabilities through improved aptamer-ligand systems.

Main Methods:

• Utilized a structure-informed, fragment-based small-molecule microarray approach for dye discovery.
• Characterized the new dye, SALAD1 (structure-informed, array-enabled LigAnD 1), for aptamer affinity and fluorescence.
• Determined the binding mode of SALAD1 using high-resolution X-ray crystallography.

Main Results:

• SALAD1 exhibits subnanomolar aptamer affinity and 3.5-fold greater fluorescence than the Mango II-TO1-biotin system.
• Structural analysis revealed improved binding pocket occupancy and a unique interaction with potassium.
• SALAD1 demonstrated enhanced performance in cell-permeable, in-cell confocal RNA imaging.

Conclusions:

• SALAD1 represents a significant advancement in RNA-activated fluorophores for cellular imaging.
• Fragment-based ligand discovery is an effective strategy for developing high-performance RNA ligands.
• The new dye facilitates improved resolution and sensitivity in visualizing RNA within cells.