RNA G-quadruplex folding is a multi-pathway process driven by conformational entropy.

Area of Science:

• Biophysics
• Computational Biology
• RNA Biology

Background:

• The function of regulatory RNAs, such as RNA G-quadruplexes (rG4s), depends critically on their folding kinetics.
• Understanding rG4 folding pathways is essential for deciphering their biological roles.

Purpose of the Study:

• To characterize the folding pathways of a G-quadruplex from telomeric repeat-containing RNA (TERRA25).
• To develop and validate a method for accurately simulating rG4 folding in the presence of ion atmospheres.

Main Methods:

• Combined all-atom molecular dynamics and coarse-grained simulations with circular dichroism experiments.
• Developed a matching procedure for implicit solvent coarse-grained simulations based on explicit water all-atom simulations to capture ionic double layers.
• Utilized three-site-per-nucleotide coarse-grained simulations to resolve folding pathways and intermediate states.

Main Results:

• Achieved quantitative agreement between simulations and experimental data for folded/unfolded state populations across varying salt concentrations and temperatures.
• Identified intermediate states including hairpin, triplex, and double-hairpin structures stabilized by transient Hoogsteen interactions.
• Revealed that folding progresses through two on-pathway intermediates for each pathway.

Conclusions:

• Conformational entropy is a key driver of the branched, multi-pathway folding process observed in rG4s like TERRA25.
• The developed simulation methodology accurately captures rG4 folding kinetics and intermediate states.
• The study provides insights into the folding dynamics of multiple rG4 systems with varying loop lengths.