G-quadruplexes (G4s) are non-B form DNA structures containing genomic regions rich in guanine that sometimes fold into and often act as transcriptional regulators. They are frequently located at regulatory sites including promoters, replication origins and telomeres11. Understanding their folding pathways, while important to a fundamental understanding of form and function, has been challenging. G4 quadruplex folding pathways are distinct from not only proteins but also duplex RNA and DNA12. Previous work characterizing folding pathways using FRET, CD or stopped-flow absorbance suggested a complex, multi-step pathway. Researchers from the University of Louisville, some of whom are frequent BioCAT users, performed continuous-flow mixing time-resolved SAXS experiments to directly structurally characterize, for the first time, early steps in the G4 quadruplex folding pathway.
G4 formation is primarily driven by hydrogen bonding, ion coordination and nucleotide pi-stacking and their resulting structures are stabilized (or destabilized) by a combination of factors including pH, temperature, ionic strength and loop length. Unlike proteins or most duplex nucleic acids, common chaotropic agents such as urea or Guanidinium hydrochloride are not effective at unfolding G-quadruplexes. As such, preliminary equilibrium circular dichroism studies of the systems of interest, two human telomere hybrid structures, were used to identify effective denaturation conditions amenable to other characterization experiments including SAXS. Alkaline denaturation was chosen as the method of interest given the relative ease of pH-jump experiments and its agreement with thermal denaturation data. Equilibrium SEC-SAXS experiments, performed at BioCAT, were first used to characterize both the folded and fully unfolded states, with the scattering profile for the folded state agreeing well with an NMR structure. The ensemble optimization method (EOM) was used to generate structural ensembles for structural characterization of the unfolded state, and suggested that although the ensemble was extended and flexible, transient hairpin structures may still comprise a part of the ensemble in solution.
Having successfully characterized the endpoints of the folding pathway, Monsen et al. used the laminar flow time-resolved mixer at BioCAT, which gives access to time ranges from ~1 ms to ~1.5 s, to perform a pH-jump (from 11.5 to 7.2) mixing experiment. These experiments showed a rapid collapse in the sub-ms range, as indicated by a large drop in Dmax values prior to the earliest measured (~1 ms) time point. When taken in context with time-resolved FRET data, this result shows that formation of hairpin structures is very rapid. Analysis of the full time series indicates that a monophasic collapse from the hairpin ensemble to a pre-folded globular ensemble is complete by ~1 second (Figure 1), with relaxation rates consistent with stopped-flow absorbance measurements. However, the longest time points are still larger than the equilibrium folded species, indicating that there are folding events still occurring on much longer timescales.
The unique power of time-resolved SAXS to yield structural parameters enabled these experiments to describe a previously unknown step in G-quadruplex folding, namely a transition from a rapidly formed hairpin ensemble to a long-lived intermediate pre-folded globular state. Monsen et al. use this SAXS data as well as data from their previous work and others to propose a global description of the G-quadruplex folding where initial transient hairpin formation is extremely rapid (much less than 1 ms), followed by hydrophobic interaction-driven collapse into a molten globule which is essentially complete by 1000 ms. Finally, on the long timescales a series of discrete intermediates appear to form and disappear until the system reaches equilibrium.
This experiment demonstrates the ability of time-resolved SAXS to provide structural descriptions of kinetically defined steps in folding pathways and other reactions, in a way that allows conclusions about the nature of the solution state – in this case the hairpin ensembles and partially-folded molten globules – to be discerned. We expect that BioCAT’s specialized and unique capabilities for time-resolved SAXS will continue to be a vital resource and will provide further novel insights into these processes going forward.
See: Monsen, R. C., et al. Early Events in G-quadruplex Folding Captured by Time-Resolved Small-Angle X-Ray Scattering. Nucleic Acids Research 53(3) (2025) DOI: 10.1093/nar/gkaf043. PMCID: PMC11780883