Post-synthetic Spin-Labeling of RNA through Click Chemistry for PELDOR Measurements

Site-directed spin labeling of RNA based on click chemistry is used in combination with pulsed electron-electron double resonance (PELDOR) to benchmark a nitroxide spin label, called here dU. We compare this approach with another established method that employs the rigid spin label Cm for RNA labeling. By using CD spectroscopy, thermal denaturation measurements, CW-EPR as well as PELDOR we analyzed and compared the influence of dU and Cm on a self-complementary RNA duplex. Our results demonstrate that the conformational diversity of dU is significantly reduced near the freezing temperature of a phosphate buffer, resulting in strongly orientation-selective PELDOR time traces of the dU-labeled RNA duplex.