There is great interest in DNA nanotechnology, but its use has been limited to aqueous or substantially hydrated media. The first assembly of a DNA nanostructure in a water- free solvent, namely a low-volatility biocompatible deep- eutectic solvent composed of a 4:1 mixture of glycerol and choline chloride (glycholine), is now described. Glycholine allows for the folding of a two-dimensional DNA origami at 20°C in six days, whereas in hydrated glycholine, folding is accelerated (≤3h). Moreover, a three-dimensional DNA origami and a DNA tail system can be folded in hydrated glycholine under isothermal conditions. Glycholine apparently reduces the kinetic traps encountered during folding in aqueous solvent. Furthermore, folded structures can be transferred between aqueous solvent and glycholine. It is anticipated that glycholine and similar solvents will allow for the creation of functional DNA structures of greater complexity by providing a milieu with tunable properties that can be optimized for a range of applications and nanostructures. For more information, see Gállego, Grover, and Hud, Angewante Chemie Int. Ed. Engl. 54, 2015, 6765-6769.