Attachment of nitroxide spin labels to nucleic acids for EPR.

In addition to X-ray, NMR, and FRET, electron paramagnetic resonance (EPR) can be applied to elucidate the structure of different macromolecular systems and determine local surroundings of paramagnetic centers in DNA and RNA. This technique permits structural characterization as well as dynamic structural changes of macromolecular systems. To do so, free radicals with good stability must be introduced. Here, the site-directed spin labeling of DNA and RNA based on the Sonogashira cross-coupling reaction is described. First, the appropriate building blocks, either 5-iodo-substituted pyrimidine deoxy- or ribo-nucleoside phosphoramidites are prepared and incorporated by solid-phase synthesis. Following this, the protected oligonucleotides are "on column" reacted with the acetylenic nitroxide spin labels and subsequently purified. Applications of this technique for duplexes, hairpins, and riboswitches in vitro and in cell are described.