As critical DNA structures capping the human chromosome ends, the stability and structural polymorphism of human telomeric G-quadruplex (G4) have drawn increasing attention in recent years. This work characterizes the equilibrium transitions of single-molecule telomeric G4 at physiological K⁺ concentration. We report three folded states of telomeric G4 with markedly different lifetime and mechanical stability. Our results show that the kinetically favored folding pathway is through a short-lived intermediate state to a longer-lived state. By examining the force dependence of transition rates, the force-dependent transition free energy landscape for this pathway is determined. In addition, an ultra-long-lived form of telomeric G4 structure with a much stronger mechanical stability is identified.

The differential structural–elastic properties of molecules between their transition and initial (native or denatured) states determine force-dependent transition rates.

The DEAH-box ATP-dependent RHAU helicases specifically unfold RNA and DNA G-quadruplexes (G4s). However, it remains unclear how the RHAU's G4 unfolding activity is coupled to different stages of the ATPase cycle. Here, using a single-molecule manipulation approach, we show that binding of Drosophila RHAU stabilizes an intramolecularly folded parallel DNA G4 against mechanical unfolding in its nucleotide-free and in its AMP-PNP or ADP bound states, while it destabilizes the G4 when coupled to ATP hydrolysis. Importantly, our results show that the ADP·AlF|$_4^-$|-bound RHAU does not stabilize the G4. We also found that both a single-stranded 3΄ DNA tail and the RSM domain of RHAU that binds specifically to the G4 structure, are dispensable for the stabilization of the G4, but both are required for G4 destabilization. Our study provides the first evidence that the unfolding kinetics of a G-quadruplex can be modulated by different nucleotide-bound states of the helicase.

The G-rich Pu39 region of the P1 promoter of the oncogene BCL-2, an apoptosis regulator, can fold into multiple G-quadruplex (G4) structures. Bcl2-2345 and Bcl2-1245 are two major G4 species forming with high thermal stability and distinct topologies in the Pu39 region, but their folding/unfolding kinetics have not yet been investigated. Here, we used magnetic tweezers to measure the mechanical stability and the folding/unfolding kinetics of the Bcl2-2345 and Bcl2-1245 G4 structures. We report that the hybrid-stranded Bcl2-2345 G4 had a lower mechanical stability than the parallel-stranded Bcl2-1245 G4. We observed that the Bcl2-2345 G4 is a kinetically favored structure, whereas the Bcl2-1245 G4, with a slow unfolding rate may function as a kinetic barrier for transcription. We also determined that in addition to the Bcl2-2345 and Bcl2-1245 G4s, other stable DNA secondary structures, such as a hybrid-stranded Bcl2-1234 G4, can also form in the Pu39 sequence. The characterization of the folding/unfolding kinetics of specific G4s reported here sheds light on the participation of G4s during gene transcription and provides information for designing G4-targeting small molecules that could modulate BCL-2 gene expression.

G-quadruplexes (G4s) are stable secondary nucleic acid structures that play crucial roles in many fundamental biological processes. The folding/unfolding dynamics of G4 structures are associated with the replication and transcription regulation functions of G4s. However, many DNA G4 sequences can adopt a variety of topologies and have complex folding/unfolding dynamics. Determining the dynamics of G4s and their regulation by proteins remains challenging due to the coexistence of multiple structures in a heterogeneous sample. Here, in this mini-review, we introduce the application of single-molecule force-spectroscopy methods, such as magnetic tweezers, optical tweezers, and atomic force microscopy, to characterize the polymorphism and folding/unfolding dynamics of G4s. We also briefly introduce recent studies using single-molecule force spectroscopy to study the molecular mechanisms of G4-interacting proteins.