Stem-loop formation drives RNA folding in mechanical unzipping experiments.

Area of Science:

• Biophysics
• Molecular Biology
• Computational Chemistry

Background:

• Accurate RNA hybridization free energies are crucial for predicting RNA structure and function.
• Existing methods struggle to extract thermodynamic data due to irreversible processes in RNA mechanical unfolding.
• Understanding RNA folding pathways is key to deciphering its biological roles.

Purpose of the Study:

• To precisely determine nearest-neighbor base pair (NNBP) RNA free energies using optical tweezers.
• To overcome experimental challenges posed by irreversible force-distance curves (FDCs) in RNA hairpin unzipping.
• To investigate the role of salt ions (sodium and magnesium) on RNA free energy calculations.

Main Methods:

• Mechanical unzipping and rezipping of a 2-kbp RNA hairpin using optical tweezers.
• Development of a tailored pulling protocol and optimized RNA synthesis to achieve fully reversible FDCs.
• Introduction of a barrier energy landscape model to characterize irreversibility and hysteresis.

Main Results:

• Derived 10 NNBP RNA free energies with 0.1 kcal/mol precision.
• Demonstrated the equivalence of sodium and magnesium free-energy salt corrections at the NNBP level.
• Correlated observed hysteresis in FDCs with a barrier energy landscape, linked to competing stem-loop structures.

Conclusions:

• Developed a robust method for obtaining reversible FDCs, enabling precise thermodynamic measurements of RNA.
• Identified stem-loop formation as a general mechanism explaining kinetic trapping and intermediate states in RNA folding.
• Provided fundamental thermodynamic data critical for accurate RNA structure and function prediction.