title: 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.
abstract: 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'-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 ψ(3) RNA, but at 5 °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' and 5' termini showed evidence of an NCp7-induced increase in the 3'-5' end-to-end distance distribution and a partially melted stem.
authors.affiliation: Department of Chemistry, University at Albany, 1400 Washington Avenue, Albany, NY 12222, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Sun
authors.forename: Ziwei
authors.initials: Z
authors.lastname: Zhang
authors.forename: Vladimir M
authors.initials: VM
authors.lastname: Grigoryants
authors.forename: William K
authors.initials: WK
authors.lastname: Myers
authors.forename: Fei
authors.initials: F
authors.lastname: Liu
authors.forename: Keith A
authors.initials: KA
authors.lastname: Earle
authors.forename: Jack H
authors.initials: JH
authors.lastname: Freed
authors.forename: Charles P
authors.initials: CP
authors.lastname: Scholes
mesh_terms: D001483:Base Sequence; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009589:Nitrogen Oxides / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D013113:Spin Labels; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; C071884:NCP7 protein, Human immunodeficiency virus 1; D009589:Nitrogen Oxides; D009841:Oligonucleotides; D013113:Spin Labels; D054301:gag Gene Products, Human Immunodeficiency Virus; C039900:nitroxyl