Resolving the nanometer-scale structure of biomolecules in natural conditions still remains a challenging task. We report the first distance measurement in nucleic acid at physiological temperature using electron paramagnetic resonance (EPR). The model 10-mer DNA duplex has been labeled with reactive forms of triarylmethyl radicals and then immobilized on a sorbent in water solution and investigated by double quantum coherence EPR. We succeeded in development of optimal triarylmethyl-based labels, approach for site-directed spin labeling and efficient immobilization procedure that, working together, allowed us to measure as long distances as ~4.6 nm with high accuracy at 310 K (37 °C).

Triarylmethyl (trityl, TAM) based spin labels represent promising alternative to nitroxides for EPR distance measurements in biomolecules. Herewith, we report synthesis and comparative study of series of model DNA duplexes, 5′-spin-labeled with TAMs and nitroxides. We have found that the accuracy (width) of distance distributions obtained by Double Electron-Electron Resonance (DEER/PELDOR) strongly depends on the type of radical. Replacement of both nitroxides by TAMs in the same spin-labeled duplex allows narrowing of the distance distributions by a factor of three. Replacement of one nitroxide by TAM (orthogonal labeling) leads to a less pronounced narrowing, but at the same time gains sensitivity in DEER experiment due to efficient pumping on narrow EPR line of TAM. Distance distributions in nitroxide/nitroxide pairs are influenced by the structure of linker: the use of a short amine-based linker improves the accuracy by a factor of two. At the same time, negligible dependence on the linker length is found for distribution width in TAM/TAM pairs. Molecular dynamics calculations indicate greater conformational disorder of nitroxide labels compared to TAM ones, thus rationalizing the experimentally observed trends. Thereby, we conclude that double spin-labeling using TAMs allows obtaining narrower spin-spin distance distributions and potentially more precise distances between labeling sites compared to traditional nitroxides.

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