Mapping the Thermal Behavior of DNA Origami Nanostructures

Abstract

Understanding the thermodynamic properties of complex DNA nanostructures, including rationally designed two- and three-dimensional (2D and 3D, respectively) DNA origami, facilitates more accurate spatiotemporal control and effective functionalization of the structures by other elements. In this work fluorescein and tetramethylrhodamine (TAMRA), a Förster resonance energy transfer (FRET) dye pair, were incorporated into selected staples within various 2D and 3D DNA origami structures. We monitored the temperature-dependent changes in FRET efficiency that occurred as the dye-labeled structures were annealed and melted and subsequently extracted information about the associative and dissociative behavior of the origami. In particular, we examined the effects of local and long-range structural defects (omitted staple strands) on the thermal stability of common DNA origami structures. The results revealed a significant decrease in thermal stability of the structures in the vicinity of the defects, in contrast to the negligible long-range effects that were observed. Furthermore, we probed the global assembly and disassembly processes by comparing the thermal behavior of the FRET pair at several different positions. We demonstrated that the staple strands located in different areas of the structure all exhibit highly cooperative hybridization but have distinguishable melting temperatures depending on their positions. This work underscores the importance of understanding fundamental aspects of the self-assembly of DNA nanostructures and can be used to guide the design of more complicated DNA nanostructures, to optimize annealing protocol and manipulate functionalized DNA nanostructures.

Progress toward Understanding the Interactions between DNA Nanostructures and the Cell

“…Besides macrophages, note that most cell types discussed here may be considered phagocytic, including cancer cells and human umbilical vein endothelial cells (HUVECs) . Note that, as opposed to serum stability, we do not comprehensively address the notion of thermal stability here as the melting point of tile‐based and origami‐based DNA nanostructures can be readily designed to be higher than 37 °C, the temperature at which most cellular uptake studies take place, by adjusting the number and position of sticky ends in the structure.…”

Modelling DNA origami self-assembly at the domain level

“…One of the simplest possible perturbations is to reduce the concentration of staple strands [27]. In Fig.…”

“…In particular, such an approach cannot describe cooperative interactions whereby the presence of a bound staple affects the binding of other staples to the scaffold. Recent evidence suggests that cooperative interactions affect origami folding significantly [23,26,27], leading to sharp formation transitions as the temperature is lowered and contributing to hysteresis in heating and cooling experiments.…”

Enhancing DNA Crystal Durability through Chemical Crosslinking

“…First, most DNA constructs are composed of relatively short DNA duplexes that are prone to thermal denaturation. Although no studies have systematically examined thermal stability of DNA crystals, a number of studies of DNA tile and origami architectures have found that thermal denaturation in solution depends on a number of factors, including average duplex length, geometry, and crossover density . Further, because DNA is polyanionic, most DNA crystals require relatively high monovalent and/or divalent cations for crystallization and post‐crystallization stability .…”