Saccharides as Perspective Immobilizers of Nucleic Acids for Room-Temperature Structural EPR Studies

Pulsed Dipolar Electron Paramagnetic Resonance (EPR) spectroscopy is a powerful tool for structural studies of biomolecules and their complexes. This method, whose applicability has been recently extended to room temperatures, requires immobilization of studied biosystem to prevent averaging of dipolar couplings; at the same time, the modification of native conformations by immobilization must be avoided. Disaccharide trehalose perfectly fulfils both purposes for proteins; however its applicability for nucleic acids was questionable and not investigated so far. In this work we provide first demonstration of room-temperature EPR distance measurements on model spin-labeled DNA duplexes immobilized in trehalose, sucrose and glucose. We have analyzed different ways of sample dehydration and have discovered a reliable approach that keeps structural conformation and unity of the complex. The obtained spin-spin distances in all three saccharides agree well with our previous data using ion-exchange sorbent; however among glucose, sucrose and trehalose the latter gives the best accuracy. Finally, room-temperature electron spin dephasing time of triarylmethyl label in trehalose is noticeably longer compared to glucose, sucrose and other immobilizers used previously, thus providing a longer range of available distances. Therefore, saccharides, and especially trehalose, can be efficiently used as easy-to-handle immobilizers of nucleic acids and their complexes, mimicking native conditions and allowing broad range of structural EPR studies at room temperatures.