Conformationally Restricted Isoindoline-Derived Spin Labels in Duplex DNA: Distances and Rotational Flexibility by Pulsed Electron-Electron Double Resonance Spectroscopy

Three structurally related isoindoline-derived spin labels that have different mobilities were incorporated into duplex DNA to systematically study the effect of motion on orientation-dependent pulsed electron-electron double resonance (PELDOR) measurements. To that end, a new nitroxide spin label, U-ExIm, was synthesized and incorporated into DNA oligonucleotides. U-ExIm is the first example of a conformationally unambiguous spin label for nucleic acids, in which the nitroxide N-O bond lies on the same axis as the three single bonds used to attach the otherwise rigid isoindoline- based spin label to a uridine base. Continuous-wave (CW) EPR measurements of U-ExIm confirm a very high rotational mobility of the spin label in duplex DNA relative to the structurally related spin label U-Im, which has restricted mobility due to an intramolecular hydrogen bond. The Xband CW-EPR spectra of U-ExIm can be used to identify mismatches in duplex DNA. PELDOR distance measurements between pairs of the spin labels U-Im, OxU, and U-ExIm in duplex DNA showed a strong angular dependence for U-Im, a medium dependence for OxU, and no orientation effect for U-ExIm. Thus, precise distances can be extracted from U-ExIm without having to take orientational effects into account.