Computation of nitroxide–nitroxide distances in spin‐labeled DNA duplexes

Abstract

Nanometer distances in nucleic acids can be measured by EPR using two 1‐oxyl‐2,2,5,5‐tetramethylpyrroline radicals, with each label attached via a methylene group to a phosphorothioate‐substituted backbone position as one of two phosphorothioate diastereomers (R_P and S_P). Correlating the internitroxide distance to the geometry of the parent molecule requires computational analysis of the label conformers. Here, we report sixteen 4‐ns MD simulations on a DNA duplex d(CTACTGCTTTAG) .d(CTAAAGCAGTAG) with label pairs at C7/C19, T5/A17, and T2/T14, respectively. For each labeled duplex, four simulations were performed with S_P/S_P, R_P/R_P, S_P/R_P, and R_P/S_P labels, with initial all trans label conformations. Another set of four simulations was performed for the 7/19‐labeled duplex using a different label starting conformation. The average internitroxide distance 〈r_MD〉 was within 0.2 Å for the two sets of simulations for the 7/19‐labeled duplex, indicating sufficient sampling of conformational space. For all three labeled duplexes studied, 〈r_MD〉 agreed with experimental values, as well as with average distances obtained from an efficient conformer search algorithm (NASNOX). The simulations also showed that the labels have conformational preferences determined by the linker chemistry and label–DNA interactions. These results establish computational algorithms that allow use of the 1‐oxyl‐2,2,5,5‐tetramethylpyrroline label for mapping global structures of nucleic acids. © 2007 Wiley Periodicals, Inc. Biopolymers 87: 40–50, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com