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 dŲ . We compare this approach with another established method that employs the rigid spin label Çm for RNA labeling. By using CD spectroscopy, thermal denaturation measurements, CW‐EPR as well as PELDOR we analyzed and compared the influence of dŲ and Çm on a self‐complementary RNA duplex. Our results demonstrate that the conformational diversity of dŲ is significantly reduced near the freezing temperature of a phosphate buffer, resulting in strongly orientation‐selective PELDOR time traces of the dŲ ‐labeled RNA duplex