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

Abstract

G-quadruplexes (G4s) are DNA secondary structures that are capable of forming and function in vivo. The propensity of G4s to exhibit extreme polymorphism and complex dynamics is likely to influence their cellular function, yet a clear microscopic picture of their folding process is lacking. Here we employed single-molecule FRET microscopy to obtain a direct view of the folding and underlying conformational dynamics of G4s formed by the human telomeric sequence in potassium containing solutions. Our experiments allowed detecting several folded states that are populated in the course of G4 folding and determining their folding energetics and timescales. Combining the single-molecule data with molecular dynamics simulations enabled obtaining a structural description of the experimentally observed folded states. Our work thus provides a comprehensive thermodynamic and kinetic description of the folding of G4s that proceeds through a complex multi-route pathway, involving several marginally stable conformational states.

Folding of guanine quadruplex molecules–funnel-like mechanism or kinetic partitioning? An overview from MD simulation studies

“…This suggests that the GQ folding is likely different from the folding of small proteins. As we will argue below [24][25][26][27], and as suggested by others [13,14,22,23], the time-scale of GQ folding in those experiments reporting long-time-scale folding [13-16, 22, 23] and long-lived substates [17,22] can only be explained by a kinetic partitioning (KP) mechanism, i.e., a multi-pathway process over a rugged free-energy landscape [1,28].…”

“…The funneled folding landscape is only marginally frustrated by non-native interactions and leads to fast folding events. This is not the case of GQ DNA sequences, where many experiments have revealed folding on a timescale of up to several days [13][14][15][16][17][18][19][20][21][22][23].…”

Environmentally Sensitive Fluorescent Nucleoside Analogues for Surveying Dynamic Interconversions of Nucleic Acid Structures

“…The combination of the two probesa ppears well complementary to FRET approaches for monitoring and characterizing a large number of structural transitions under various environmental stimuli.F RET approaches are invaluable for monitoring large structuralt ransitions,s uch as for instance between different conformations of Holliday junctions and G-quadruplexes. [4,5] Owing to the dependence of FRET on the distance and/ or orientation of the FRET donor-acceptor pair,t he FRET approach can give unique information on the geometry of the initial and final species,a nd sometimes of the intermediate species. On such systems, the approachw ith the environmentally sensitivef luorescent nucleobases describedh ere could be helpful for confirming and completing the description of the mechanistic aspects of these transitions, by selectively monitoring the structural changes at defined locations of the nucleic acids.…”

“…For instance, they were employed to characterize the dynamics and interconversion betweend ifferent conformations of Holliday junctions and G-quadruplexes. [4,5] However,t hey failed to detect DNA/ RNA changes that do not significantly impact the distance between the donora nd acceptor.M oreover,F RET-based experiments require two chromophores and the commonly used ones tend to be bulky (fluorescein, cyanines, rhodamines, Alexas,e tc. ), and can alter the DNA/RNA structure and the binding of proteinsa nd ligands.…”

Structure and function of Pif1 helicase

“…Measurements of G4DNA unfolding are affected by the rate of G4DNA folding. The consensus is that folding of G4DNA structures proceeds slowly to the thermodynamic equilibrium; however, fast folding to various intermediate occurs [84,85]. These reported folding rates differ from those reported in smFRET studies, which indicate very rapid folding [61,86].…”