German Edition: DOI: 10.1002/ange.201412354 DNA Nanotechnology International Edition: DOI: 10.1002/anie.201412354 Folding and Imaging of DNA Nanostructures in Anhydrous and Hydrated Deep-Eutectic Solvents** Isaac Gµllego, Martha A. Grover, and Nicholas V. Hud* Abstract: 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 8C in six days, whereas in hydrated glycholine, folding is accelerated ( 3 h). 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 complex- ity by providing a milieu with tunable properties that can be optimized for a range of applications and nanostructures. Assemblies based on DNA programming [1] provide a method to create custom-designed shapes [2] at the nano- meter scale. Common implementations are DNA origami, [2b,c] in which a long DNA strand (scaffold) is folded by hundreds of complementary base-paired oligonucleotides (staples), and systems based on single-stranded tails (SSTs), [2d,e] in which short DNA strands that contain four domains are able to fold into a target shape. DNA nanostructures have been success- fully utilized to create two-dimensional [2b] and three-dimen- sional [2c] devices with applications in lithography, [3] photon- ics, [4] electronics, [5] and the fabrication of inorganic materi- als. [6] Despite the ever-expanding applications of DNA nano- technology, [7] to date, DNA nanostructure designs and applications have been limited to aqueous [1b] or substantially hydrated milieus. [8] Reduced water activity or transfer to organic solvents typically results in alterations of the DNA helical structure or even the loss of base pairing. In aqueous solution, DNA duplexes adopt the B-form helix. Therefore, all DNA devices have thus far been designed according to B-form helical parameters. [2b, 9] Previously, our laboratory demonstrated that DNA can form a stable duplex in reline, [10] an anhydrous deep-eutectic solvent (DES) composed of choline chloride and urea in a 1:2 ratio. [11] This DES is from a family of non-aqueous solvents with improved properties for nanotechnology applications [12] in comparison with water-based media, including enhanced electrodeposi- tion [13] of nanometallic and semiconducting materials, improved inorganic nanoparticle shape control and stabil- ity, [14] and low volatility. However, DNA duplexes adopt a more A-form helical structure in reline (Figure 1 a), and the degree of structural change is sequence-dependent. [10] Thus, reline would not be compatible with existing DNA nano- Figure 1. CD analysis of a 32-mer DNA duplex in aqueous and non- aqueous solvents. a) CD spectra of the 32-mer DNA duplex at 20 8C in aqueous solution and in the non-aqueous solvents glycholine and reline. b) CD spectra of the 32-mer in glycholine after thermal denaturation by heating to 80 8C (red) and cooling to 20 8C (blue) in 2 8C steps (gray) over a total time of 165 min. The complete recovery of the CD spectrum upon cooling to 20 8C (compared with the spectrum before heating; green) demonstrates the reversibility of thermal duplex denaturation in glycholine. [*] Dr. I. Gµllego, Prof. N. V. Hud School of Chemistry and Biochemistry Parker H. Petit Institute for Bioengineering and Bioscience Georgia Institute of Technology E-mail: hud@chemistry.gatech.edu Prof. M. A. Grover School of Chemical & Biomolecular Engineering Parker H. Petit Institute for Bioengineering and Bioscience Georgia Institute of Technology (USA) [**] We thank B. Cafferty and C. He for discussions, Prof. Y. Ke for providing six-helix bundle and nine-helix triangle DNA and advice on TEM experiments, L. Bottomley for use of their atomic-force microscope, and B. V. Breedveld for viscosity measurements. This work was supported by the NSF and the NASA Astrobiology Program under the NSF Center for Chemical Evolution (CHE- 1004570). Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201412354. A ngewandte Chemi e 1 Angew. Chem. Int. Ed. 2015, 54,1–6  2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers! Ü Ü