Long-range distance determination in a DNA model system inside Xenopus laevis oocytes by in-cell spin-label EPR.

Elucidating the structure and dynamics of biomacromolecules, for example, proteins, RNA, or DNA, is crucial for understanding their physiological function. Beside widespread techniques, such as NMR spectroscopy or crystallography, sophisticated electron paramagnetic resonance spectroscopy (EPR) methods in combination with site-directed spin labelingll ) are receiving increasing attention for this purpose. Spin-label EPR (SL-EPR) offers access to structural information on a length scale between approximately 0.5 and 10 nm, and enables the study of dynamics in the picoto microsecond rangeY-B) To date, SLEPR has been carried out in defined buffer solutions. However, recently an in-cell SL-EPR experiment investigating the conformation of ubiquitin was reported;(9) this suggests that the technique can be applied to nucleic acids as well, which is of particular importance since non-canonical DNA conformations are involved in many processes, such as regulatory functions. The development of analytical tools for elucidating native structures inside cells is an ongoing challenge to be addressed by in-cell spectroscopy.(10) Here, we describe long-range distance measurements on a DNA model system in oocytes of Xenopus laevis utilizing in-cell EPR. Compared to in-cell NMR spectroscopy, in-cell EPR has two main advantages. Low concentrations can be used because EPR is much more sensitive per spin than NMR spectroscopy, and since EPR only detects unpaired electron spins no background from diamagnetic molecules is observed. The latter is of particular importance since in contrast to structural studies of isolated macromolecules, in-cell experiments are often hampered by many different cellular components and thereby provide for high background signals. On the other hand, in-cell studies are of greater significance since, in contrast to pure solution experiments, a natural environment is provided, which can be crucial for observing the biologically relevant conformation of biomacromolecules.lll.l2) Double electron-electron resonance (DEER or PELDOR) is a pulsed, two-frequency EPR technique for determining distance distributions (from 1.5 up to 8 nm) between two paramagnetic centers by measuring their dipoledipole interaction. I131S) Diamagnetic biomacromolecules can be site-directly labeled with nitroxides.ll )