Wuebben, Christine, et al. Nucleic acids research, 2020, 48(18), 10518-10526.
Here, site-directed spin labeling (SDSL) combined with pulsed electron-electron double resonance (PELDOR or DEER) is used to study the structure of the P1 and P2 hairpins of the Guanidine-II riboswitch in solution and how they change upon binding of guanidine groups (Gdm+).
PELDOR Measurements
PELDOR measurements were performed on 25 μM RNA samples, using a buffer of 10 mM HEPES, 10 mM MgCl2, and 10 mM KCl at pH 7.5, in D2O plus 20% ethylene glycol-d6 (v/v). The samples underwent annealing by heating at 95°C for 5 minutes, followed by cooling on ice, either without Gdm+ (-Gdm+) or with 40 mM Gdm+ (++Gdm+).
The pumping pulse frequency was adjusted to the peak intensity of the nitroxide signal, with π/2- and π-pulse durations of 12 ns and 24 ns, respectively, including a two-step phase cycle for the π/2-pulse. The pump pulse duration was optimized, and the initial τ was set to 260 ns. Deuterium modulation was minimized by taking eight time traces with a 16 ns increment in τ. The detection window, with a width of 40 ns, was aligned with the echo maximum. To ensure a satisfactory signal-to-noise ratio, the signal was averaged over 3 to 24 hours, with a shot repetition time of 3 ms.
Shah, Binal N., et al. Crystal growth & design, 2011, 11(5), 1493-1501.