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

Abstract

In this article, we summarize the knowledge and best practices learned from bulk and single molecule measurements to address some of the frequently experienced difficulties in single molecule Förster Resonance Energy Transfer (smFRET) measurements on G-quadruplex (GQ) structures. The number of studies that use smFRET to investigate the structure, function, dynamics, and interactions of GQ structures has grown significantly in the last few years, with new applications already in sight. However, a number of challenges need to be overcome before reliable and reproducible smFRET data can be obtained in measurements that include GQ. The annealing and storage conditions, the location of fluorophores on the DNA construct, and the ionic conditions of the experiment are some of the factors that are of critical importance for the outcome of measurements, and many of these manifest themselves in unique ways in smFRET assays. By reviewing these aspects and providing a summary of best practices, we aim to provide a practical guide that will help in successfully designing and performing smFRET studies on GQ structures.

Dynamic DNA binding, junction recognition and G4 melting activity underlie the telomeric and genome-wide roles of human CST

“…The setup was as described above, except the Cy3-labeled oligonucleotide harbored 4 telomeric repeats adjacent to the region annealed to the Cy5-labeled anchor oligonucleotide (Figure 5A ). The Cy3 and Cy5 labels were positioned on either side of the telomeric repeats so that they would be in close proximity and give a high FRET signal when the DNA is folded into a G4 structure ( 28 , 43 – 45 ). Unfolding of the G4 structure would increase the distance between the dyes resulting in a decrease in FRET efficiency.…”

“…To assess DNA folding, we analyzed FRET signals from the 3 G4-Cy3 substrate in the absence of CST using a range of ionic conditions known to either stabilize (150 mM NaCl) or destabilize G4 structure (0 salt or 50 mM LiCl) ( 19 , 28 , 39 , 43 ). In initial experiments, we included 150 mM NaCl in the imaging buffer and FRET signals were quantified from >4000 individual molecules and plotted as a FRET histogram.…”

“…In 0 mM salt the high FRET peak disappeared and was replaced by a lower FRET peak ( E ∼0.3) characteristic of 24 nt flexible ssDNA. The destabilization of DNA folding in 50 mM LiCl and complete unfolding in 0 mM salt provide strong support for G4 formation ( 43 ).…”

Extreme mechanical diversity of human telomeric DNA revealed by fluorescence-force spectroscopy

“…Single-molecule studies are advantageous because of the ability to characterize individual subpopulations in a heterogeneous sample (18)(19)(20)(21). For example, an early smFRET (single-molecule FRET) study of human telomeric GQ molecules revealed four folded states that differ in FRET efficiencies or in lifetimes and two unfolded states of different lifetimes (22), and GQ conformational diversity in the absence of mechanical stress has been well documented in other smFRET studies (23)(24)(25)(26)(27)(28)(29)(30). Force-based single-molecule measurements showed that human telomeric GQ molecules in 100 mM K + unfold at ∼20 pN (31) with a hint of three folded states that differ in lifetimes (32).…”

Site-Specific Fluorophore Labeling of Guanosines in RNA G-Quadruplexes

“…42 The estimated distance between the mean fluorophore positions in our internally labeled RNA structures (∼17 Å) is less than that typically used for single-molecule FRET ( R ≈ 0.58 R 0 ) to distinguish highly polymorphic G-quadruplexes (e.g., telomeric DNA G-quadruplexes). 28 We then selected two well-characterized G-quadruplexes HpG4-2 43,44 and HpQd 45 (hereafter designated Q A and Q B , respectively). Both have been found to equilibrate with alternative hairpin structures.…”