The Internal Dynamics of Mini c TAR DNA Probed by Electron Paramagnetic Resonance of Nitroxide Spin-Labels at the Lower Stem, the Loop, and the Bulge

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• 作者：Yan Sun ; Ziwei Zhang ; Vladimir M. Grigoryants ; William K. Myers ; Fei Liu ; Keith A. Earle ; Jack H. Freed ; Charles P. Scholes
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：October 30, 2012
• 年：2012
• 卷：51
• 期：43
• 页码：8530-8541
• 全文大小：563K
• 年卷期：v.51,no.43(October 30, 2012)
• ISSN：1520-4995

文摘

Electron paramagnetic resonance (EPR) at 236.6 and 9.5 GHz probed the tumbling of nitroxide spin probes in the lower stem, in the upper loop, and near the bulge of mini c TAR DNA. High-frequency 236.6 GHz EPR, not previously applied to spin-labeled oligonucleotides, was notably sensitive to fast, anisotropic, hindered local rotational motion of the spin probe, occurring approximately about the NO nitroxide axis. Labels attached to the 2鈥?aminocytidine sugar in the mini c TAR DNA showed such anisotropic motion, which was faster in the lower stem, a region previously thought to be partially melted. More flexible labels attached to phosphorothioates at the end of the lower stem tumbled isotropically in mini c TAR DNA, mini TAR RNA, and 蠄₃ RNA, but at 5 掳C, the motion became more anisotropic for the labeled RNAs, implying more order within the RNA lower stems. As observed by 9.5 GHz EPR, the slowing of nanosecond motions of large segments of the oligonucleotide was enhanced by increasing the ratio of the nucleocapsid protein NCp7 to mini c TAR DNA from 0 to 2. The slowing was most significant at labels in the loop and near the bulge. At a 4:1 ratio of NCp7 to mini c TAR DNA, all labels reported tumbling times of >5 ns, indicating a condensation of NCp7 and TAR DNA. At the 4:1 ratio, pulse dipolar EPR spectroscopy of bilabels attached near the 3鈥?and 5鈥?termini showed evidence of an NCp7-induced increase in the 3鈥测€?鈥?end-to-end distance distribution and a partially melted stem.