Folding dynamics and conformational heterogeneity of human telomeric G-quadruplex structures in Na⁺solutions by single molecule FRET microscopy

Abstract

G-quadruplex structures can occur throughout the genome, including at telomeres. They are involved in cellular regulation and are potential drug targets. Human telomeric G-quadruplex structures can fold into a number of different conformations and show large conformational diversity. To elucidate the different G-quadruplex conformations and their dynamics, we investigated telomeric G-quadruplex folding using single molecule FRET microscopy in conditions where it was previously believed to yield low structural heterogeneity. We observed four FRET states in Na+ buffers: an unfolded state and three G-quadruplex related states that can interconvert between each other. Several of these states were almost equally populated at low to medium salt concentrations. These observations appear surprising as previous studies reported primarily one G-quadruplex conformation in Na+ buffers. Our results permit, through the analysis of the dynamics of the different observed states, the identification of a more stable G-quadruplex conformation and two transient G-quadruplex states. Importantly these results offer a unique view into G-quadruplex topological heterogeneity and conformational dynamics.

A direct view of the complex multi-pathway folding of telomeric G-quadruplexes

“…As expected, one major population is observed in the FRET histogram (Figure 2A). The peak is centered at E≈0.85, which is characteristic for the chair conformation, as previously found in similar buffer conditions (30). The highest FRET state (E≈0.88) of hTelo has a very similar FRET value, strongly indicating that this E state corresponds to the chair conformation.…”

“…Overall, high potassium concentrations favor the formation of thermodynamically stable hybrid 1 conformation and result in reduction of the observed dynamics (Supplementary Figure S9). Studies in sodium-containing solutions show that G4 folding can be very dynamic and follows a multi-pathway process also in these conditions (30,58). These independent observations obtained for different DNA sequences or solutions conditions may point towards common mechanistic features of G4 folding, as described in this work.…”

“…This multifactorial nature of G4s makes their folding highly complex, possibly involving a number of stable intermediate states and misfolded species and is beyond a simple two-state approximation (21). Several studies have found G4 folding to occur through a simple sequential pathway, involving up to two intermediate states (21–26), however recently more complex folding scenarios have started to emerge (27–30). One important bottleneck in uncovering the G4 folding pathway remains the structural identification of the different folded states appearing during the folding process.…”

A practical guide to studying G-quadruplex structures using single-molecule FRET

“…In addition, similar image integration times (100 ms vs. 150 ms) were used in both studies. Despite these extensive similarities, all molecules were folded into a relatively sharp distribution and no significant dynamics was observed in one study (Long and Stone 2013), while in the other about a third of the molecules did not fold into GQ, and the remaining molecules showed a much broader distribution with rich dynamics (Noer et al 2016). Therefore, there must be a feature in the Noer et al construct that makes it significantly less stable than Long et al construct and also our construct, which also folds into a sharp single peak (Figure 1F).…”

“…The minor variations between the two histograms are within the variation we obtain among different sample chambers that have the same surface. Therefore, a BSA surface, which is significantly easier and less time consuming to prepare compared to a PEG surface, should be adequate in cases where only GQ structure is probed (Long and Stone 2013; Noer et al 2016). If proteins or small molecules are also used in these measurements, then the decision about what type of surface to use should be made on a case by case basis, as proteins and small molecules might show large variations in terms of their propensity to non-specific binding to these surfaces.…”