Abstract
A protocol is described for the incorporation of nitroxide spin-labels into specific 2′-sites within nucleic acids. This labeling strategy facilitates the investigation of nucleic acid structure and dynamics using electron paramagnetic resonance (EPR) spectroscopy and macromolecular complex formation using paramagnetic relaxation enhancement NMR spectroscopy. A spin-labeling reagent, 4-isocyanato TEMPO, which can be prepared in one facile step or obtained commercially, is used for postsynthetic modification of site-specifically 2′-amino-modified nucleic acids. This spin-labeling protocol has been applied primarily to RNA, but is also applicable to DNA. Subsequently, EPR spectroscopic analysis of the spin-labeled nucleic acids allows for the measurements of distances, solvent accessibilities and conformation dynamics. Using the spin-labeling strategy described here, spin-labeled samples can be prepared in 2–4 d.
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Acknowledgements
The authors thank Kristmann Gislason and Pavol Cekan for their help with manuscript preparation. T.E.E. is a Damon Runyon Cancer Research Foundation Postdoctoral Fellow in the laboratory of Adrian R. Ferré-D'Amaré at the Fred Hutchinson Cancer Research Center in Seattle, Washington, USA (DRG-1844-04).
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Edwards, T., Sigurdsson, S. Site-specific incorporation of nitroxide spin-labels into 2′-positions of nucleic acids. Nat Protoc 2, 1954–1962 (2007). https://doi.org/10.1038/nprot.2007.273
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DOI: https://doi.org/10.1038/nprot.2007.273
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