An aqueous environment has been long been postulated as being necessary for the formation of nucleic acid secondary structures. While there has been considerable interest and debate regarding the actual number of water molecules required to maintain a particular nucleic acid structure (1), there are obvious experimental challenges to studying nucleic acid structures in the absence of water (e.g. low nucleic acid solubility). Thus far, complementary DNA strands have been reported to retain duplex structure only in anhydrous modified polyethylene glycol (2). However, DNA duplexes only exhibit marginal stability in other water-free organic solvents (3). An ever expanding set of new polar solvents, such as room temperature ionic liquids (RTIL), provide new opportunities to study nucleic acids in water-free environments. Here, we report that nucleic acids can form duplex, triplex and G-quadruplex secondary structures that undergo reversible thermal denturation in a water-free solvent (e.g. <0.25% water). This so-called deep eutectic solvent (DES) is comprised of one part choline choride and two parts urea. The choline choloride-urea DES has a melting point of only 12˚C, whereas pure choline chloride melts at 302˚C and urea at 133˚C (4). We have found that nucleic acid secondary structures exhibit different relative stabilities in the DES, compared to aqueous media (5). Thermal transition midpoints of the duplex structures in the DES are lower than those measured in water solution, whereas triplex and G-quadruplex structures can be more stable in the DES compared to an aqueous solution with the same ionic strength. Deep eutectic solvents and ionic liquids are currently of tremendous interest as nonvolatile media for a wide range of chemical reactions and processes. Given previous reports that these water-free solvents can support protein enzyme catalysis, it is now feasible that catalytic nucleic acids and protein enzyme-nucleic acid complexes could be used in these solvents.



References:

1. T.V. Maltseva, P. Agback, J. Chattopadhyaya, Nucleic Acids Res. 21, 4246-4252 (1993).
2. A. M. Leone, S. C. Weatherly, M. E. Williams, H. H. Thorp, R. W. Murray, J. Am. Chem. Soc. 123, 218-222 (2003).
3. G. Bonner, A. M. Klibanov, Biotechnol. Bioeng. 68, 339-344 (2003).
4. A. P. Abbott, G. Capper, D. L. Davies, R. K. Rasheed, V. Tambyrajah, Chem. Comm. 70-71 (2003).
5. I. Mamajanov; A. E. Engelhart; H. D. Bean; N. V. Hud, Angew. Chem. Int. Ed. Eng. 49, 6310–6314 (2010).