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

Qi Cai, Ana Karin Kusnetzow, Wayne L. Hubbell, Ian S. Haworth, Gian Paola C. Gacho, Ned Van Eps, Kálmán Hideg, Eric J. Chambers, Peter Z. Qin

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100 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)4722-4730
Number of pages9
JournalNucleic acids research
Volume34
Issue number17
DOIs
Publication statusPublished - Oct 2006

ASJC Scopus subject areas

  • Genetics

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    Cai, Q., Kusnetzow, A. K., Hubbell, W. L., Haworth, I. S., Gacho, G. P. C., Van Eps, N., Hideg, K., Chambers, E. J., & Qin, P. Z. (2006). Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe. Nucleic acids research, 34(17), 4722-4730. https://doi.org/10.1093/nar/gkl546