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.
• Conformational changes in RNA that occur on the timescale of minutes to seconds are challenging to analyze using traditional solution-based methods.
• Characterizing RNA-protein complexes requires methods that can preserve their structure and activity.

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 transient RNA conformational states that are difficult to capture with other techniques.
• To facilitate the investigation of catalytic activities of RNA and RNA-protein complexes in different conformational states.

Main Methods:

• Utilizing native polyacrylamide gel electrophoresis (native PAGE) to separate distinct RNA conformers based on their structural and charge properties.
• Immobilizing separated RNA conformers within the native gel matrix for subsequent analysis.
• Assessing the catalytic activity of immobilized RNA conformers directly in the gel or after elution.

Main Results:

• Demonstrated the ability of native PAGE to resolve and distinguish between folding and unfolding conformers of RNA.
• Showcased the utility of the method for studying RNA conformers with exchange rates in the minute to second range.
• Validated the application of the technique to diverse catalytic RNAs and RNA-protein complexes, preserving their functional integrity.

Conclusions:

• Native polyacrylamide gel electrophoresis is an effective method for analyzing RNA conformational dynamics.
• This technique overcomes limitations of solution-based methods for studying transient RNA states.
• The developed protocols are broadly applicable to various RNA molecules and their associated proteins, aiding in the study of RNA structure-function relationships.