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Site-directed spin-labeling of nucleotides and the use of in-cell EPR to determine long-range distances in a biologically relevant environment

Nature Protocols volume 8, pages 131–147 (2013)Cite this article

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Abstract

Double electron-electron resonance (DEER) is an electron paramagnetic resonance (EPR) technique used to determine distance distributions in the nanometer range between spin labels by measuring their dipole-dipole interactions. Here we describe how in-cell DEER can be applied to spin-labeled DNA sequences to unravel their conformations in living cells by long-range distance measurements in cellula. As EPR detects unpaired electron spins only, diamagnetic molecules provide no background and do not reduce detection sensitivity of the specific signal. Compared with in-cell NMR spectroscopy, low concentrations of spin-labeled molecules can be used owing to the higher sensitivity of EPR per spin. This protocol describes the synthesis of the spin labels, their introduction in DNA strands, the injection of labeled DNA solutions in cells and the performance of in-cell EPR measurements. Completion of the entire protocol takes ∼20 d.

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Figure 1: Synthesis of TPA-labeled 5′-DMT-protected 2′-deoxyuridine phosphoramidite (10).
Figure 2
Figure 3: DEER experiment.
Figure 4: Schematic representation of data analysis.
Figure 5: CW-EPR spectrum of TPA-labeled phosphoramidite.
Figure 6: HPLC purification of spin-labeled oligonucleotides.
Figure 7: Optical images of stage VI X. laevis oocytes.

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Acknowledgements

We thank M. Spitzbarth and M. Wassmer for preparing figures. Research in M.D.'s laboratory is supported by the Deutsche Forschungsgemeinschaft, the Ministry of Science, Research and the Arts of Baden-Württemberg and the German Excellence Initiative.

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Author notes

  1. Mykhailo Azarkh & Vijay Singh

    Present address: Present addresses: Department of Molecular Physics, Leiden University, Leiden, The Netherlands (M.A.), Department of Chemistry, Stanford University, Stanford, California, USA (V.S.).,

Authors and Affiliations

  1. Department of Chemistry, Konstanz Research School of Chemical Biology and Zukunftskolleg, University of Konstanz, Konstanz, Germany

    Mykhailo Azarkh, Vijay Singh, Oliver Okle, Isabelle T Seemann, Daniel R Dietrich, Jörg S Hartig & Malte Drescher

  2. Department of Biology, Konstanz Research School Chemical Biology and Zukunftskolleg, University of Konstanz, Konstanz, Germany

    Mykhailo Azarkh, Vijay Singh, Oliver Okle, Isabelle T Seemann, Daniel R Dietrich, Jörg S Hartig & Malte Drescher

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Contributions

M.A., J.S.H. and M.D. conceived the experimental strategy. J.S.H., D.R.D. and M.D. supervised the project. V.S. synthesized the labeled DNA sequences, O.O. performed the microinjection and M.A. conducted the EPR experiments and data analysis. All authors contributed to the discussion and writing of the manuscript.

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Correspondence to Malte Drescher.

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Azarkh, M., Singh, V., Okle, O. et al. Site-directed spin-labeling of nucleotides and the use of in-cell EPR to determine long-range distances in a biologically relevant environment. Nat Protoc 8, 131–147 (2013). https://doi.org/10.1038/nprot.2012.136

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