Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe.
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 A 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 A, 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.
Cai
Qi
Q
Department of Chemistry, University of Southern California, Los Angeles, CA 90089-0744, USA.
Kusnetzow
Ana Karin
AK
Hubbell
Wayne L
WL
Haworth
Ian S
IS
Gacho
Gian Paola C
GP
Van Eps
Ned
N
Hideg
Kálmán
K
Chambers
Eric J
EJ
Qin
Peter Z
PZ
D004247:DNA / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D015335:Molecular Probes / Q000737:chemistry; D015394:Molecular Structure; D009589:Nitrogen Oxides / Q000737:chemistry*; D012313:RNA / Q000737:chemistry; D013113:Spin Labels
D015335:Molecular Probes; D009589:Nitrogen Oxides; D013113:Spin Labels; D012313:RNA; D004247:DNA; C039900:nitroxyl