Determination of base binding strength and base stacking interaction of DNA duplex using atomic force microscope

Abstract

As one of the most crucial properties of DNA, the structural stability and the mechanical strength are attracting a great attention. Here, we take advantage of high force resolution and high special resolution of Atom Force Microscope and investigate the mechanical force of DNA duplexes. To evaluate the base pair hydrogen bond strength and base stacking force in DNA strands, we designed two modes (unzipping and stretching) for the measurement rupture forces. Employing k-means clustering algorithm, the ruptured force are clustered and the mean values are estimated. We assessed the influence of experimental parameters and performed the force evaluation for DNA duplexes of pure dG/dC and dA/dT base pairs. The base binding strength of single dG/dC and single dA/dT were estimated to be 20.0 ± 0.2 pN and 14.0 ± 0.3 pN, respectively, and the base stacking interaction was estimated to be 2.0 ± 0.1 pN. Our results provide valuable information about the quantitative evaluation of the mechanical properties of the DNA duplexes.

Figure 1. Schematic diagram of the unzipping mode and the stretching mode used for measurement of the binding strength of the base pairs.

Figure 2. Force curves showing the rupture force versus relative surface displacement of the cantilever tip.

The blue line and the red line represent the approaching and retracing processes, respectively. (a): sequential rupture of three DNA duplexes; (b): one rupture of single DNA duplex.

Figure 3. Dependence of rupture force on the loading rates for different lengths of dG/dC base pair using the stretching mode.

Figure 4. The clustered rupture force using the algorithm of k-means clustering.

All the rupture forces were obtained under the condition of 100 mM NaCl condition and 10 s holding time. (a) a duplex of 14-dG/dC base pairs using the unzipping mode; (b) a duplex of 14-dG/dC base pairs using the stretching mode.