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.

Stability of DNA Origami Nanostructures in Physiological Media: The Role of Molecular Interactions

“…The denaturing effect of Gdm + on DNA origami is furthermore strongly modulated by its counteranion, with Gdm 2 SO 4 and GdmSCN being much weaker and stronger denaturants, respectively. [75,76] This is because these anions themselves are either chaotropic (SCN − ) or kosmotropic (SO 4 2− ) and thus alter the structure of bulk water and especially the hydration shell of DNA, which modulates the heat capacity change of the transition between intact and denatured DNA origami nanostructures. [75] A direct result of this is a nonmonotonous temperature dependence of DNA origami denaturation, that may display local minima and maxima of DNA origami stability at intermediate temperatures.…”

“…[75] A direct result of this is a nonmonotonous temperature dependence of DNA origami denaturation, that may display local minima and maxima of DNA origami stability at intermediate temperatures. [75,76] Many of the observations discussed in this section rely on specific properties of the employed ions, such as the ability of Gdm + to participate both in hydrophobic and electrostatic interactions and to even undergo like-charge ion pairing. [77] Their impact on DNA origami stability in vivo is therefore rather limited.…”

“…[ 75 ] A direct result of this is a nonmonotonous temperature dependence of DNA origami denaturation, that may display local minima and maxima of DNA origami stability at intermediate temperatures. [ 75,76 ]…”

Superstructure-dependent stability of DNA origami nanostructures in the presence of chaotropic denaturants

“…Previous studies investigated the interaction of 2D DNA origami nanostructures with guanidinium (Gdm + ) salts and focused on the effects of different environmental parameters such as Gdm + concentration, 26 temperature, 26 ionic strength, 29 time, 26,28 and counterion species. 27,28 A surprisingly complex picture was revealed that included for instance strong counterion effects, 27,28 non-monotonic temperature dependencies, 27,28 and a counterintuitive destabilizing influence of additional cations. 29 The origin of these observations lies in the structure of the Gdm + cation (see Fig.…”

“…, the so-called Rothemund triangle. 26–29 However, it is well established that DNA origami stability under various conditions depends on the overall shape as well as certain design choices, e.g. , lattice type and crossover arrangement.…”