Probing RNA folding pathways by RNA fingerprinting.

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Studying RNA folding and unfolding dynamics is crucial for understanding RNA function.
• Solution-based methods often struggle to capture transient RNA conformers due to rapid exchange rates.
• Characterizing RNA-protein complexes requires methods that preserve structural integrity.

Purpose of the Study:

• To provide protocols for distinguishing RNA folding and unfolding conformers using native polyacrylamide gel electrophoresis (native PAGE).
• To enable the study of RNA conformers with exchange periods in the minutes or seconds range.
• To facilitate the investigation of catalytic activity of separated RNA conformers and RNA-protein complexes.

Main Methods:

• Utilizing native polyacrylamide gel electrophoresis (native PAGE) for RNA conformational analysis.
• Separating and immobilizing distinct RNA conformers within the gel matrix.
• Assessing catalytic activity of immobilized conformers, with or without elution.

Main Results:

• Native PAGE successfully distinguishes between folding and unfolding RNA conformers.
• The method is applicable to RNA conformers with rapid exchange kinetics.
• Catalytic activities of separated conformers can be reliably assessed.

Conclusions:

• Native PAGE is a powerful technique for analyzing dynamic RNA structures.
• This protocol allows for detailed investigation of RNA folding pathways and functions.
• The method is versatile and applicable to various catalytic RNAs and RNA-protein complexes.