Design and Characterization of DX‐Tile DNA Nanostar‐Based Hydrogels

ABSTRACT Pure deoxyribonucleic acid (DNA) hydrogels synthesized via the hybridization of multi‐arm DNA tiles (DNA nanostars) are uniquely programmable and functionalizable biomaterials, suitable for applications ranging from biosensing to cell‐free protein production and soft tissue engineering. However, the full potential offered by DNA molecules in terms of design flexibility and functionalization has not yet been leveraged for pure DNA hydrogels, thus reducing their versatility and broader use. In this study, we introduce multi‐arm double‐crossover (DX)‐tile motifs, often used in wireframe DNA nanoparticles assembly, to enable greater control over the hydrogel's mechanical properties and facilitate functionalization. Specifically, we demonstrate that modifying structural design parameters, such as the arm geometry, length, valency, and linker design, allows for fine control of the elastic modulus and viscoelastic properties of the hydrogels. We also show that their functionalization can be performed without compromising the hydrogels' physical properties and exhibit enhanced mechanical strength and tunable properties, compared to simple duplex‐based DNA hydrogels. Furthermore, these DNA hydrogels demonstrated printability and scalability, which pave the way toward the development of novel formulations and bioinks for the rational design of soft tissue engineering scaffolds and broaden the use of DNA hydrogels for other biomedical applications.