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.
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.
Several small-molecule ligands specifically bind and stabilize Gquadruplex (G4) nucleic acid structures, which are considered to be promising therapeutic targets. G4s are polymorphic structures of varying stability, and their formation is dynamic.Here, we investigate the mechanisms of ligand binding to dynamically populated human telomere G4 DNA by using the bisquinolinium based ligand Phen-DC3 and a combination of single-molecule FRET microscopy, ensemble FRET and CD spectroscopies. Different cations are used to tune G4 polymorphism and folding dynamics. We find that ligand binding occurs to pre-folded G4 structures and that Phen-DC3 also induces G4 formation in unfolded single strands. Following ligand binding to dynamically populated G4s, the DNA undergoes pronounced conformational redistributions that do not involve direct ligand-induced G4 conformational interconversion. On the contrary, the redistribution is driven by ligandinduced G4 folding and trapping of dynamically populated short-lived conformation states. Thus, ligand-induced stabilization does not necessarily require the initial presence of stably folded G4s.
Oxidative stress has been connected to aging, cancer and the development of many other diseases. Oxidative damage occurs while cellular oxidizing species concentration increases or antioxidant defenses decrease. One of the most common products of oxidative damage in DNA is 8-oxoguanine (8-oxoG).The frequent presence of 8-oxoguanine is due to the lowest oxidation potential of guanine amongst the four DNA bases. In particular, the guanine rich nature telomeric 3'-overhang sequence makes telomere more prone to the 8-oxoG damage. Therefore it provides an interesting region for studying the consequence of oxidative damage in DNA. We studied the effect of 8-oxoG lesion in human telomeric overhang which consist of repeats TTAGGG that can self-fold into G-quadruplex (GQ). The central guanine of triple-G was replaced
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