Using a Specific RNA-Protein Interaction To Quench the Fluorescent RNA Spinach

Area of Science:

• Molecular Biology
• Biochemistry
• Biophysics

Background:

• RNA molecules are vital for cellular functions and participate in complex interaction networks.
• Analyzing RNA interactions biochemically is challenging, often requiring fluorescent labeling for techniques like Förster Resonance Energy Transfer (FRET).
• The "Spinach" RNA aptamer is a genetically encodable biological fluorophore tag, but its use in FRET assemblies was previously unexplored.

Purpose of the Study:

• To explore the potential of the Spinach RNA aptamer in Förster Resonance Energy Transfer (FRET) based interaction studies.
• To develop a novel, genetically encodable system for quantitative analysis of RNA-protein interactions.
• To demonstrate the utility of Spinach aptamer quenching for studying RNA-protein interactions under lysate conditions.

Main Methods:

• Investigated the quenching of the Spinach RNA aptamer when in close proximity to acceptor molecules.
• Employed RNA-DNA hybridization to position quenchers or red organic dyes near the Spinach aptamer.
• Quantitatively analyzed RNA-protein interactions using the Pseudomonas aeruginosa phage coat protein 7 (PP7) and its cognate pp7-RNA as a model system.

Main Results:

• Demonstrated that the Spinach aptamer can be quenched by nearby acceptors, a key requirement for FRET-based assays.
• Successfully utilized RNA-DNA hybridization to control the proximity of quenchers to the Spinach aptamer.
• Showcased the quantitative analysis of RNA-protein interactions via Spinach aptamer quenching, functioning effectively even in cell lysates.

Conclusions:

• The Spinach aptamer can be effectively quenched, enabling its use in FRET-based interaction studies.
• This work presents a direct, genetically encodable method for analyzing RNA-protein interactions through Spinach aptamer quenching.
• The developed system offers a valuable tool for studying RNA-protein interactions under various conditions, including complex lysate environments.