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Nucleic Acids Research Advance Access published online on May 21, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm240
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

A facile method for attaching nitroxide spin labels at the 5' terminus of nucleic acids{dagger}

Gian Paola G. Grant1 and Peter Z. Qin1,2,*

1Department of Chemistry and 2Department of Biological Sciences, University of Southern California, LJS-251, 840 Downey Way, Los Angeles, CA 90089-0744, USA

*To whom correspondence should be addressed. Tel: +1-213-821-2461; Fax: +1-213-740-0930; Email: pzq{at}usc.edu

Received January 8, 2007. Revised March 21, 2007. Accepted April 2, 2007.

In site-directed spin labeling (SDSL), a nitroxide moiety containing a stable, unpaired electron is covalently attached to a specific site within a macromolecule, and structural and dynamic information at the labeling site is obtained via electron paramagnetic resonance (EPR) spectroscopy. Successful SDSL requires efficient site-specific incorporation of nitroxides. Work reported here presents a new method for facile nitroxide labeling at the 5' terminus of nucleic acids of arbitrary sizes. T4-polynucleotide kinase was used to enzymatically substitute a phosphorothioate group at the 5' terminus of a nucleic acid, and the resulting phosphorothioate was then reacted with an iodomethyl derivative of a nitroxide. The method was successfully demonstrated on both chemically synthesized and naturally occurring nucleic acids. The attached nitroxides reported duplex formation as well as tertiary folding of nucleic acids, indicating that they serve as a valid probe in nucleic acid studies.

{dagger}Research reported here was supported by the Petroleum Research Fund (PRF 39623-G4), American Cancer Society (IRG-58-007-45), National Institute of Health (R01 GM069557), and a startup fund from the University of Southern California.


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