期刊
NUCLEIC ACIDS RESEARCH
卷 34, 期 17, 页码 4722-4730出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkl546
关键词
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资金
- NEI NIH HHS [R01 EY005216, R37 EY005216, EY05216] Funding Source: Medline
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 angstrom 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 angstrom, were measured using double electron-electron resonance (DEER). The measured distances correlated strongly (R-2 = 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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