
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.09.05.611539
preprint
1
Article
Early Events in G-quadruplex Folding Captured by Time-Resolved Small-Angle X-Ray Scattering
Monsen Robert C. http://orcid.org/0000-0002-6283-3249

Sabo T. Michael
Gray Robert
Hopkins Jesse B. http://orcid.org/0000-0001-8554-8072

Chaires Jonathan B. http://orcid.org/0000-0001-5477-945X

08 9 2024
2024.09.05.611539https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.09.05.611539
nihpp-2024.09.05.611539.pdf
Abstract

Time-resolved small-angle X-ray experiments (TR-SAXS) are reported here that capture and quantify a previously unknown rapid collapse of the unfolded oligonucleotide as an early step in G4 folding of hybrid 1 and hybrid 2 telomeric G-quadruplex structures. The rapid collapse, initiated by a pH jump, is characterized by an exponential decrease in the radius of gyration from 20.6 to 12.6 Å. The collapse is monophasic and is complete in less than 600 ms. Additional hand-mixing pH-jump kinetic studies show that slower kinetic steps follow the collapse. The folded and unfolded states at equilibrium were further characterized by SAXS studies and other biophysical tools, to show that G4 unfolding was complete at alkaline pH, but not in LiCl solution as is often claimed. The SAXS Ensemble Optimization Method (EOM) analysis reveals models of the unfolded state as a dynamic ensemble of flexible oligonucleotide chains with a variety of transient hairpin structures. These results suggest a G4 folding pathway in which a rapid collapse, analogous to molten globule formation seen in proteins, is followed by a confined conformational search within the collapsed particle to form the native contacts ultimately found in the stable folded form.
==== Body
pmc
