Structure-fluorescence activation relationships of a large Stokes shift fluorogenic RNA aptamer

Area of Science:

• Molecular Biology
• Biochemistry
• Spectroscopy

Background:

• The Chili RNA aptamer (FLAP) is a 52-nucleotide RNA molecule that induces fluorescence in chromophore derivatives.
• It forms stable complexes with ligands, exhibiting bright, highly Stokes-shifted fluorescence emission.

Purpose of the Study:

• To analyze the interactions between the Chili RNA aptamer and conditionally fluorescent ligands.
• To reveal key structure-fluorescence activation relationships (SFARs).

Main Methods:

• Spectroscopic techniques
• Calorimetric methods (Isothermal Titration Calorimetry)
• Biochemical assays
• NMR spectroscopy

Main Results:

• Ligands fall into two categories with emission maxima around 540 nm or 590 nm, binding with nanomolar to low-micromolar affinities.
• Isothermal titration calorimetry detailed enthalpic and entropic contributions to binding for a unique cationic ligand.
• NMR revealed G-quadruplex formation exclusively upon ligand binding, indicating its role in fluorescence activation.

Conclusions:

• The study elucidates molecular features responsible for the large Stokes shift and strong fluorescence enhancement in RNA-chromophore complexes.
• These findings provide insights into the design of novel fluorescent RNA-based sensors and probes.