SiteDirected Spin Labeling of RNA for Distance Me

Authors: Joachim W Engels Christian Grünewald Lena Wicke

Publish Date: 2014

Volume: , Issue: , Pages: 385-407

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Abstract

Electron paramagnetic resonance EPR is a structural method and can in addition to Xray 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 sitedirected spin labeling SDSL of DNA and RNA based on the Sonogashira crosscoupling reaction and compare to other methods available In our method the appropriate building blocks either 5iodosubstituted pyrimidine or 2iodopurine deoxy or ribonucleoside phosphoramidites were prepared and incorporated by solidphase synthesis Following this the protected oligonucleotides were “on column” reacted with the acetylenic nitroxide spin labels and subsequently purified As a result the socalled “RNA ruler” for distance measurements of doublestranded 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”

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