Early events in G-quadruplex folding captured by time-resolved small-angle X-ray scattering

Area of Science:

• Biophysics
• Structural Biology
• Biochemistry

Background:

• G-quadruplexes (G4) are crucial nucleic acid structures with diverse biological roles.
• Understanding G4 folding pathways is essential for elucidating their function.
• Previous models lacked detailed insights into the initial folding dynamics.

Purpose of the Study:

• To investigate the early folding events of hybrid 1 and hybrid 2 telomeric G-quadruplexes.
• To quantify the kinetics and structural changes during G4 folding.
• To characterize the unfolded state of G-quadruplexes.

Main Methods:

• Time-resolved small-angle X-ray scattering (SAXS) experiments.
• pH-jump initiation of folding.
• Hand-mixing kinetic studies.
• SAXS Ensemble Optimization Method (SAXS-EOM).

Main Results:

• A rapid, monophasic collapse of unfolded G-quadruplexes was observed within 600 ms.
• The collapse significantly reduced the radius of gyration, indicative of compacting.
• The unfolded state exists as a dynamic ensemble of flexible chains with transient hairpins.
• G4 unfolding is complete at alkaline pH but not in LiCl solutions.

Conclusions:

• G-quadruplex folding initiates with a rapid collapse, analogous to molten globule formation in proteins.
• Subsequent slower steps involve conformational searching within the collapsed structure.
• The unfolded state is not a random coil but a dynamic ensemble.
• Established G4 unfolding conditions require re-evaluation.