Early Events in G-quadruplex Folding Captured by Time-Resolved Small-Angle X-Ray Scattering

Area of Science:

• Biophysics
• Structural Biology
• Molecular Biology

Background:

• G-quadruplex (G4) structures are crucial in telomere maintenance and gene regulation.
• Understanding the folding pathways of G-quadruplexes is key to elucidating their biological functions.

Purpose of the Study:

• To investigate the early folding dynamics of hybrid 1 and hybrid 2 telomeric G-quadruplex structures.
• To characterize the rapid structural changes during G4 formation using time-resolved experiments.

Main Methods:

• Time-resolved small-angle X-ray scattering (TR-SAXS) to monitor structural changes in real-time.
• pH-jump kinetic studies to initiate and track folding.
• SAXS Ensemble Optimization Method (EOM) for modeling the unfolded state.

Main Results:

• A rapid, monophasic collapse of unfolded oligonucleotides was observed, completing in <600 ms.
• The radius of gyration decreased from 20.6 to 12.6 Å during this collapse.
• SAXS EOM analysis revealed the unfolded state as a dynamic ensemble with transient hairpin structures.
• Equilibrium studies confirmed complete G4 unfolding at alkaline pH, but not in LiCl.

Conclusions:

• G-quadruplex folding involves an initial rapid collapse, analogous to molten globule formation in proteins.
• This collapse is followed by a conformational search leading to the formation of native G4 contacts.
• The findings provide new insights into the G4 folding pathway and the nature of the unfolded state.