Site-Directed Spin Labeling of RNA for Distance Measurements by EPR

Electron paramagnetic resonance (EPR) is a structural method and can in addition to X-ray, nuclear magnetic resonance (NMR), and FRET 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 the macromolecular systems. In order to do so free radicals with good stability have to be introduced, most prominently nitroxide radicals. Here, we describe the site-directed spin labeling (SDSL) of DNA and RNA based on the Sonogashira cross-coupling reaction and compare to other methods available. In our method the appropriate building blocks, either 5-iodo-substituted pyrimidine or 2-iodopurine deoxy- or ribo-nucleoside phosphoramidites, were prepared and incorporated by solid-phase synthesis. Following this the protected oligonucleotides were “on column” reacted with the acetylenic nitroxide spin labels and subsequently purified. As a result the so-called “RNA ruler” for distance measurements of double-stranded RNA was developed. We present applications of this technique for DNA duplexes, DNA/RNA hybrids, RNA hairpins, riboswitches, and aptamers in vitro and “in cell.”