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Nucleic Acids Research Advance Access originally published online on September 10, 2006
Nucleic Acids Research 2006 34(17):4722-4730; doi:10.1093/nar/gkl546
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Nucleic Acids Research, 2006, Vol. 34, No. 17 4722-4730
© 2006 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.

Structural Biology

Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe

Qi Cai1, Ana Karin Kusnetzow3, Wayne L. Hubbell3, Ian S. Haworth4, Gian Paola C. Gacho1, Ned Van Eps3, Kálmán Hideg5, Eric J. Chambers4 and Peter Z. Qin1,2,*

1 Department of Chemistry , University of Southern California Los Angeles, CA 90089-0744, USA 2 Department of Biological Sciences, University of Southern California Los Angeles, CA 90089-0744, USA 3 Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California Los Angeles, CA 90095, USA 4 Department of Pharmaceutical Sciences, University of Southern California Los Angeles, CA 90089, USA 5 Institute of Organic and Medical Chemistry, University of Pécs H-7643, Pécs, P.O. Box 99, Hungary

*To whom correspondence should be addressed. LJS-251, 840 Downey Way, Los Angeles, CA 90089 0744, USA. Tel: +1 213 821 2461; Fax: +1 213 740 0930; Email: pzq{at}usc.edu

Received May 5, 2006. Revised June 30, 2006. Accepted July 13, 2006.

In site-directed spin labeling (SDSL), local structural and dynamic information is obtained via electron paramagnetic resonance (EPR) spectroscopy of a stable nitroxide radical attached site-specifically to a macromolecule. Analysis of electron spin dipolar interactions between pairs of nitroxides yields the inter-nitroxide distance, which provides quantitative structural information. The development of pulse EPR methods has enabled such distance measurements up to 70 Å in bio-molecules, thus opening up the possibility of SDSL global structural mapping. This study evaluates SDSL distance measurement using a nitroxide (designated as R5) that can be attached, in an efficient and cost-effective manner, to a phosphorothioate backbone position at arbitrary DNA or RNA sequences. R5 pairs were attached to selected positions of a dodecamer DNA duplex with a known NMR structure, and eight distances, ranging from 20 to 40 Å, were measured using double electron-electron resonance (DEER). The measured distances correlated strongly (R2 = 0.98) with the predicted values calculated based on a search of sterically allowable R5 conformations in the NMR structure, thus demonstrating accurate distance measurements using R5. Furthermore, distance measurement in a 42 kD DNA was demonstrated. The results establish R5 as a sequence-independent probe for global structural mapping of DNA and DNA–protein complexes.


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