Structural basis for activation of fluorogenic dyes by an RNA aptamer lacking a G-quadruplex motif

Area of Science:

• Biochemistry
• Structural Biology
• RNA Therapeutics

Background:

• RNA aptamers are increasingly used as molecular tools and therapeutics.
• Fluorogenic RNA aptamers offer sensitive detection methods for small molecules.
• DIR2s is a second-generation aptamer selected for binding dimethylindole red (DIR).

Purpose of the Study:

• To elucidate the 3D structure of the DIR2s RNA aptamer.
• To understand the binding mechanism of the oxazole thiazole blue (OTB) fluorophore.
• To characterize the structural basis for DIR2s's fluorogenic properties.

Main Methods:

• In vitro selection of RNA aptamers.
• X-ray crystallography to determine apo and OTB-SO3 bound structures.
• Structural analysis of RNA-ligand interactions.

Main Results:

• Crystal structures of DIR2s (apo and OTB-SO3 bound) were solved at 2.0 Å and 1.8 Å resolution.
• DIR2s adopts a compact, tuning fork-like architecture.
• OTB-SO3 binds in a planar conformation within a claw-like pocket formed by a purine base-triple and an unpaired nucleotide.

Conclusions:

• DIR2s possesses a unique structural motif distinct from other known fluorogenic RNAs.
• The structure provides insights into the design of novel RNA-based sensors and therapeutics.
• The findings highlight the potential of DIR2s for multicolor detection applications.