Nanomechanics of G-quadruplexes within the promoter of the KIT oncogene

Abstract

G-quadruplexes (G4s) are tetrahelical DNA structures stabilized by four guanines paired via Hoogsteen hydrogen bonds into quartets. While their presence within eukaryotic DNA is known to play a key role in regulatory processes, their functional mechanisms are still under investigation. In the present work, we analysed the nanomechanical properties of three G4s present within the promoter of the KIT proto-oncogene from a single-molecule point of view through the use of magnetic tweezers (MTs). The study of DNA extension fluctuations under negative supercoiling allowed us to identify a characteristic fingerprint of G4 folding. We further analysed the energetic contribution of G4 to the double-strand denaturation process in the presence of negative supercoiling, and we observed a reduction in the energy required for strands separation.

Double-stranded flanking ends affect the folding kinetics and conformational equilibrium of G-quadruplexes forming sequences within the promoter of KIT oncogene

“…On this basis, we associated this component to residuals of random-coiled construct. Indeed, substantial amounts of unfolded are generally detected in G4-folding experiments at the single molecule level ( 26 , 29 , 33 ). The second Gaussian component also has rather construct-independent expectation value, while the third population resolved in the FRET efficiency histograms has somewhat more construct-dependent characteristics.…”

“…Indeed, the latter are expected to be easily disrupted by application of modest forces or torques. An estimation of the free energy gap between the coiled sequence and the folded G4 configuration in the case of KIT promoter, based on magnetic tweezers experiments, has been reported in a recent publication ( 33 ).…”

“…The three G4s forming regions were characterized in the past by a panel of spectroscopic techniques, including UV-visible absorption (UV-Vis) ( 25–27 , 30 , 31 ), circular dichroism (CD) ( 25–27 , 30–32 ), nuclear magnetic resonance (NMR) ( 25 , 27 , 28 , 31 , 32 ). In addition, c-kit1 and c-kit2 were probed by single molecule techniques ( 26 , 29 , 33 ), including fluorescence resonance energy transfer (FRET), a widely applied technique to elucidate the conformational features of biomolecules ( 34–37 ). The c-kit* sequence, being the last one to be identified, has been investigated to a lesser extent, and, to the best of our knowledge, there exists only one study in which its folding has been characterized at the single-molecule level, namely by exploiting magnetic tweezers ( 33 ).…”

“…In addition, c-kit1 and c-kit2 were probed by single molecule techniques ( 26 , 29 , 33 ), including fluorescence resonance energy transfer (FRET), a widely applied technique to elucidate the conformational features of biomolecules ( 34–37 ). The c-kit* sequence, being the last one to be identified, has been investigated to a lesser extent, and, to the best of our knowledge, there exists only one study in which its folding has been characterized at the single-molecule level, namely by exploiting magnetic tweezers ( 33 ).…”

Characterization of G-Quadruplexes Folding/Unfolding Dynamics and Interactions with Proteins from Single-Molecule Force Spectroscopy

“…In vivo, many DNA G4-forming sequences are embedded in dsDNA. To address the formation of G4s in double-stranded DNA, several studies have used G4-forming sequences embedded in double-stranded DNA [ 56 , 88 , 89 ] or long DNA hairpins [ 90 ] ( Figure 2 D,E). Together with the DNA origami technique, the constructed DNA tether in force spectroscopy can contain a G4 structure placed inside a DNA origami nanocage that allows quantification of the folding kinetics of G4s in a confined space [ 91 , 92 , 93 ].…”