FLEth RNA intercalating probe is a convenient reporter for small interfering RNAs

Area of Science:

• Biochemistry
• Molecular Biology
• RNA Therapeutics

Background:

• Short interfering RNA (siRNA) is crucial for RNA interference (RNAi) and gene silencing.
• Developing tools to monitor siRNA behavior and interactions is essential for understanding RNAi mechanisms and optimizing therapeutic applications.
• Existing methods for tracking siRNA-protein interactions can be complex and lack real-time monitoring capabilities.

Purpose of the Study:

• To develop and characterize a novel fluorescence-based probe (FLEth) for detecting duplex siRNA.
• To investigate the utility of FLEth in reporting on protein-RNA interactions, specifically with viral RNA silencing suppressors.
• To explore FLEth's behavior in cellular environments and its potential for tracking siRNA delivery.

Main Methods:

• Synthesis of a phenanthridine derivative covalently linked to a fluorescein moiety (FLEth).
• Fluorescence spectroscopy, including fluorescence resonance energy transfer (FRET), to study FLEth-siRNA complex formation and dissociation.
• Binding assays to determine dissociation constants (Kd) of FLEth with siRNA.
• Co-incubation experiments with siRNA, FLEth, and the p19 protein to assess protein-RNA interactions.
• Cellular uptake studies using liposomes and fluorescence microscopy in Huh 7.5 cells.

Main Results:

• FLEth acts as a sensitive fluorescence-based probe for duplex siRNA, exhibiting a FRET signal upon complexation.
• At least two FLEth molecules bind to a 21-nucleotide duplex siRNA, with reported dissociation constants.
• FLEth can report on the fraction of unbound siRNA when interacting with the Carnation Italian ringspot virus protein p19, without directly binding the siRNA-p19 complex.
• FLEth binds siRNA within liposome delivery systems but dissociates upon cellular entry, localizing to nucleoli.

Conclusions:

• FLEth is a valuable tool for detecting duplex siRNA and monitoring protein-RNA interactions in vitro.
• The probe's behavior provides insights into siRNA binding dynamics and its dissociation suggests it does not interfere with the core RNA silencing machinery.
• FLEth's ability to report on unbound siRNA and its cellular localization offer potential for studying siRNA delivery and function within cells.