Time-Dependent DNA Origami Denaturation by Guanidinium Chloride, Guanidinium Sulfate, and Guanidinium Thiocyanate

Abstract

Guanidinium (Gdm) undergoes interactions with both hydrophilic and hydrophobic groups and, thus, is a highly potent denaturant of biomolecular structure. However, our molecular understanding of the interaction of Gdm with proteins and DNA is still rather limited. Here, we investigated the denaturation of DNA origami nanostructures by three Gdm salts, i.e., guanidinium chloride (GdmCl), guanidinium sulfate (Gdm₂SO₄), and guanidinium thiocyanate (GdmSCN), at different temperatures and in dependence of incubation time. Using DNA origami nanostructures as sensors that translate small molecular transitions into nanostructural changes, the denaturing effects of the Gdm salts were directly visualized by atomic force microscopy. GdmSCN was the most potent DNA denaturant, which caused complete DNA origami denaturation at 50 °C already at a concentration of 2 M. Under such harsh conditions, denaturation occurred within the first 15 min of Gdm exposure, whereas much slower kinetics were observed for the more weakly denaturing salt Gdm₂SO₄ at 25 °C. Lastly, we observed a novel non-monotonous temperature dependence of DNA origami denaturation in Gdm₂SO₄ with the fraction of intact nanostructures having an intermediate minimum at about 40 °C. Our results, thus, provide further insights into the highly complex Gdm-DNA interaction and underscore the importance of the counteranion species.

Keywords: DNA nanotechnology; DNA origami; atomic force microscopy; denaturation; guanidinium.

Figure 1

AFM images of DNA origami triangles after incubation in 2 M GdmCl at different times and temperatures. Images have a size and height scale of 3 × 3 µm² and 2 nm, respectively. The white arrows indicate collapsed triangles that disintegrated by rupture at the vertices. For additional AFM images, see Figures S1–S3.

Figure 2

Results of the statistical analysis of the AFM images for 2 M GdmCl. Each data point represents the average of three AFM images with the standard deviations given as error bars.

Figure 3

AFM images of DNA origami triangles after incubation in 2 M Gdm₂SO₄ at different times and temperatures. Images have a size and height scale of 3 × 3 µm² and 2 nm, respectively. The white arrows indicate damaged yet mostly intact triangles with one or two ruptured vertices. For additional AFM images, see Figures S4–S6.

Figure 4

Results of the statistical analysis of the AFM images for 2 M Gdm₂SO₄. Each data point represents the average of three AFM images with the standard deviations given as error bars.

Figure 5

AFM images of DNA origami triangles after incubation in 2 M GdmSCN at different times and temperatures. Images have a size and height scale of 3 × 3 µm² and 2 nm, respectively. The white arrows indicate collapsed triangles with ruptured vertices, while blue arrows indicate triangles with damaged trapezoids or dangling scaffold. For additional AFM images, see Figures S7–S9.

Figure 6

Results of the statistical analysis of the AFM images for 2 M GdmSCN. Each data point represents the average of three AFM images with the standard deviations given as error bars.

Figure 7

Comparison of the fractions of intact DNA origami triangles obtained in the different Gdm salts at different temperatures and as a function of incubation time.