Synthesis of a Cytidine Phosphoramidite with Protected Nitroxide Spin Label for EPR Experiments with RNA

Abstract

Spin labeling of oligonucleotides with nitroxides is hampered by their intrinsic instability under conditions of solid‐phase synthesis and enzymatic ligation. Although nitroxide decomposition can be avoided in some cases by postsynthetic introduction or by special reaction conditions, a more general solution would be reversible protection of the radical. We have recently developed such a method based on photolabile protection groups for DNA oligonucleotides and demonstrated their application in EPR spectroscopy. Here, we extend this method to RNA oligonucleotides. By improving the synthetic procedures, the yield of the coumarin‐protected phosphoramidite could be increased by a factor of 12. Effective recovery of the nitroxides on a duplex RNA enables pulsed EPR experiments to be performed directly after irradiation and air oxidation. Data at Q‐band frequency is shown and distances measured with PELDOR (pulsed electron‐electron double resonance) spectroscopy agree well with the calculated values.