Triarylmethyl Labels: Toward Improving the Accuracy of EPR Nanoscale Distance Measurements in DNAs

摘要

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 nitroxideitroxide 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.