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

Post-print (lokagerð höfundar)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.S.Th.S. acknowledges financial support from the Icelandic Research Fund (080041023 and 120001021). T. F. P. acknowledges financial support from the German Research Foundation (CRC902: Molecular Principles of RNA-Based Regulation). We thank H. Gustmann and P. Wenk for help in collecting the PELDOR data, Dr. G. Reginsson for simulations of CW-EPR spectra, and Dr. S. Jonsdottir for assistance in collecting analytical data for structural characterization of the compounds that were prepared.Peer reviewe