A thermodynamic overview of naturally occurring intramolecular DNA quadruplexes

Abstract

Loop length and its composition are important for the structural and functional versatility of quadruplexes. To date studies on the loops have mainly concerned model sequences compared with naturally occurring quadruplex sequences which have diverse loop lengths and compositions. Herein, we have characterized 36 quadruplex-forming sequences from the promoter regions of various proto-oncogenes using CD, UV and native gel electrophoresis. We examined folding topologies and determined the thermodynamic profile for quadruplexes varying in total loop length (5-18 bases) and composition. We found that naturally occurring quadruplexes have variable thermodynamic stabilities (DeltaG(37)) ranging from -1.7 to -15.6 kcal/mol. Overall, our results suggest that both loop length and its composition affect quadruplex structure and thermodynamics, thus making it difficult to draw generalized correlations between loop length and thermodynamic stability. Additionally, we compared the thermodynamic stability of quadruplexes and their respective duplexes to understand quadruplex-duplex competition. Our findings invoke a discussion on whether biological function is associated with quadruplexes with lower thermodynamic stability which undergo facile formation and disruption, or by quadruplexes with high thermodynamic stability.

Figure 1.

CD spectra of quadruplexes (10 μM) obtained in 10 mM sodium cacodylate buffer, 100 mM KCl, pH 7.4.

Figure 2.

UV cooling profile of quadruplexes monitored at 295 nm along with nonlinear least square curve fitting (in red). The experiment was performed in 10 mM sodium cacodylate buffer, pH 7.4, 100 mM KCl. The cooling rate was 0.15°C/min. The thermodynamic parameters obtained by van't Hoff analysis are provided in the inset.

Figure 3.

Representative DSC curves for different quadruplexes in 10 mM sodium cacodylate buffer, pH 7.4, 100 mM KCl.

Figure 4.

Representative graphs showing effect of loop length on thermodynamic stability (ΔG₃₇) of quadruplex.

Figure 5.

Plots showing dependence of relative thermodynamic stability of duplex and quadruplex on total loop length. The encircled points display lower ΔΔG₃₇ values suggesting a significant contribution from competing quadruplex structures.