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Zn2+ selectively stabilizes FdU-substituted DNA through a unique major groove binding motif.

Ghosh S, Salsbury FR, Horita DA, Gmeiner WH - Nucleic Acids Res. (2011)

Bottom Line: Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect.DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+).A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Program in Molecular Genetics, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.

ABSTRACT
We report, based on semi-empirical calculations, that Zn(2+) binds duplex DNA containing consecutive FdU-dA base pairs in the major groove with distorted trigonal bipyramidal geometry. In this previously uncharacterized binding motif, O4 and F5 on consecutive FdU are axial ligands while three water molecules complete the coordination sphere. NMR spectroscopy confirmed Zn(2+) complexation occurred with maintenance of base pairing while a slight hypsochromic shift in circular dichroism (CD) spectra indicated moderate structural distortion relative to B-form DNA. Zn(2+) complexation inhibited ethidium bromide (EtBr) intercalation and stabilized FdU-substituted duplex DNA (ΔT(m) > 15 °C). Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect. DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+). A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.

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(A) 1H NMR spectra showing no significant intensity change for the imino region for the 3′-FdU hairpin in the presence of Zn2+ or Mg2+; (B) 19F NMR spectra showing that Zn2+, but not Mg2+, results in significant downfield shifts.
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Figure 5: (A) 1H NMR spectra showing no significant intensity change for the imino region for the 3′-FdU hairpin in the presence of Zn2+ or Mg2+; (B) 19F NMR spectra showing that Zn2+, but not Mg2+, results in significant downfield shifts.

Mentions: The effects of Zn2+ complex formation on the structure of the 3′-FdU sequence were analyzed using NMR spectroscopy. 1H NMR spectra were obtained in 90% H2O solution. Imino 1H signals were readily apparent for the 3′-FdU hairpin in the presence of Zn2+, Mg2+ and no divalent metal at both pH 7 (Supplementary Figure S5) and pH 8 (Figure 5). Interestingly, although the pKA for monomeric FdU is ∼7.6, there was little change in relative intensity for the FdU imino 1H between pH 7 and pH 8 either in the no metal sample or in the sample that contained Mg2+. Integration of the FdU imino region was performed relative to a region of carbon bound 1H (5.8–8.5 ppm) that was indistinguishable in all samples and arbitrarily set to 100. The well-resolved G-imino at 12.5 ppm varied from 0.81 to 1.60 in all samples relative to this standard while integral values ranged from 5.7 to 6.0 for the 10 FdU imino 1H in this region. End-fraying causes the almost complete loss of the terminal FdU imino 1H and chemical exchange with water is likely responsible, in part, for the integral values being significantly less than the theoretical value of nine. Thus, for the no metal and Mg2+ samples, the FdU imino are mostly protonated and do not titrate significantly between pH 7 and 8.Figure 5.


Zn2+ selectively stabilizes FdU-substituted DNA through a unique major groove binding motif.

Ghosh S, Salsbury FR, Horita DA, Gmeiner WH - Nucleic Acids Res. (2011)

(A) 1H NMR spectra showing no significant intensity change for the imino region for the 3′-FdU hairpin in the presence of Zn2+ or Mg2+; (B) 19F NMR spectra showing that Zn2+, but not Mg2+, results in significant downfield shifts.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 5: (A) 1H NMR spectra showing no significant intensity change for the imino region for the 3′-FdU hairpin in the presence of Zn2+ or Mg2+; (B) 19F NMR spectra showing that Zn2+, but not Mg2+, results in significant downfield shifts.
Mentions: The effects of Zn2+ complex formation on the structure of the 3′-FdU sequence were analyzed using NMR spectroscopy. 1H NMR spectra were obtained in 90% H2O solution. Imino 1H signals were readily apparent for the 3′-FdU hairpin in the presence of Zn2+, Mg2+ and no divalent metal at both pH 7 (Supplementary Figure S5) and pH 8 (Figure 5). Interestingly, although the pKA for monomeric FdU is ∼7.6, there was little change in relative intensity for the FdU imino 1H between pH 7 and pH 8 either in the no metal sample or in the sample that contained Mg2+. Integration of the FdU imino region was performed relative to a region of carbon bound 1H (5.8–8.5 ppm) that was indistinguishable in all samples and arbitrarily set to 100. The well-resolved G-imino at 12.5 ppm varied from 0.81 to 1.60 in all samples relative to this standard while integral values ranged from 5.7 to 6.0 for the 10 FdU imino 1H in this region. End-fraying causes the almost complete loss of the terminal FdU imino 1H and chemical exchange with water is likely responsible, in part, for the integral values being significantly less than the theoretical value of nine. Thus, for the no metal and Mg2+ samples, the FdU imino are mostly protonated and do not titrate significantly between pH 7 and 8.Figure 5.

Bottom Line: Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect.DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+).A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Program in Molecular Genetics, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.

ABSTRACT
We report, based on semi-empirical calculations, that Zn(2+) binds duplex DNA containing consecutive FdU-dA base pairs in the major groove with distorted trigonal bipyramidal geometry. In this previously uncharacterized binding motif, O4 and F5 on consecutive FdU are axial ligands while three water molecules complete the coordination sphere. NMR spectroscopy confirmed Zn(2+) complexation occurred with maintenance of base pairing while a slight hypsochromic shift in circular dichroism (CD) spectra indicated moderate structural distortion relative to B-form DNA. Zn(2+) complexation inhibited ethidium bromide (EtBr) intercalation and stabilized FdU-substituted duplex DNA (ΔT(m) > 15 °C). Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect. DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+). A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.

Show MeSH
Related in: MedlinePlus