Although DNA origami nanostructures have found their way into numerous fields of fundamental and applied research, they often suffer from rather limited stability when subjected to environments that differ from the employed assembly conditions, that is, suspended in Mg2+‐containing buffer at moderate temperatures. Here, means for efficient cryopreservation of 2D and 3D DNA origami nanostructures and, in particular, the effect of repeated freezing and thawing cycles are investigated. It is found that, while the 2D DNA origami nanostructures maintain their structural integrity over at least 32 freeze–thaw cycles, ice crystal formation makes the DNA origami gradually more sensitive toward harsh sample treatment conditions. Whereas no freeze damage could be detected in 3D DNA origami nanostructures subjected to 32 freeze–thaw cycles, 1000 freeze–thaw cycles result in significant fragmentation. The cryoprotectants glycerol and trehalose are found to efficiently protect the DNA origami nanostructures against freeze damage at concentrations between 0.2 × 10−3 and 200 × 10−3 m and without any negative effects on DNA origami shape. This work thus provides a basis for the long‐term storage of DNA origami nanostructures, which is an important prerequisite for various technological and medical applications.
DNA origami nanostructures are widely employed in various areas of fundamental and applied research. Due to the tremendous success of the DNA origami technique in the academic field, considerable efforts currently aim at the translation of this technology from a laboratory setting to real-world applications, such as nanoelectronics, drug delivery, and biosensing. While many of these real-world applications rely on an intact DNA origami shape, they often also subject the DNA origami nanostructures to rather harsh and potentially damaging environmental and processing conditions. Furthermore, in the context of DNA origami mass production, the long-term storage of DNA origami nanostructures or their pre-assembled components also becomes an issue of high relevance, especially regarding the possible negative effects on DNA origami structural integrity. Thus, we investigated the effect of staple age on the self-assembly and stability of DNA origami nanostructures using atomic force microscopy. Different harsh processing conditions were simulated by applying different sample preparation protocols. Our results show that staple solutions may be stored at −20 °C for several years without impeding DNA origami self-assembly. Depending on DNA origami shape and superstructure, however, staple age may have negative effects on DNA origami stability under harsh treatment conditions. Mass spectrometry analysis of the aged staple mixtures revealed no signs of staple fragmentation. We, therefore, attribute the increased DNA origami sensitivity toward environmental conditions to an accumulation of damaged nucleobases, which undergo weaker base-pairing interactions and thus lead to reduced duplex stability.
DNA nanostructures enable the attachment of functional molecules to nearly any unique location on their underlying structure. Due to their single-base-pair structural resolution, several ligands can be spatially arranged and closely controlled according to the geometry of their desired target, resulting in optimized binding and/or signaling interactions. Here, the efficacy of SWL, an ephrin-mimicking peptide that binds specifically to EphrinA2 (EphA2) receptors, increased by presenting up to three of these peptides on small DNA nanostructures in an oligovalent manner. Ephrin signaling pathways play crucial roles in tumor development and progression. Moreover, Eph receptors are potential targets in cancer diagnosis and treatment. Here, the quantitative impact of SWL valency on binding, phosphorylation (key player for activation) and phenotype regulation in EphA2-expressing prostate cancer cells was demonstrated. EphA2 phosphorylation was significantly increased by DNA trimers carrying three SWL peptides compared to monovalent SWL. In comparison to one of EphA2’s natural ligands ephrin-A1, which is known to bind promiscuously to multiple receptors, pinpointed targeting of EphA2 by oligovalent DNA-SWL constructs showed enhanced cell retraction. Overall, we show that DNA scaffolds can increase the potency of weak signaling peptides through oligovalent presentation and serve as potential tools for examination of complex signaling pathways.
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