Cooperative stabilization of Zn²⁺:DNA complexes through netropsin binding in the minor groove of FdU-substituted DNA

Abstract

The simultaneous binding of netropsin in the minor groove and Zn²⁺ in the major groove of a DNA hairpin that includes 10 consecutive FdU nucleotides at the 3′-terminus (3′FdU) was demonstrated based upon NMR spectroscopy, circular dichroism (CD), and computational modeling studies. The resulting Zn²⁺/netropsin: 3′FdU complex had very high thermal stability with aspects of the complex intact at 85 °C, conditions that result in complete dissociation of Mg²⁺ complexes. CD and ¹⁹F NMR spectroscopy were consistent with Zn²⁺ binding in the major groove of the DNA duplex and utilizing F5 and O4 of consecutive FdU nucleotides as ligands with FdU nucleotides hemi-deprotonated in the complex. Netropsin is bound in the minor groove of the DNA duplex based upon 2D NOESY data demonstrating contacts between AH2 ¹H and netropsin ¹H resonances. The Zn²⁺/netropsin: 3′FdU complex displayed increased cytotoxicity towards PC3 prostate cancer (PCa) cells relative to the constituent components or separate complexes (e.g. Zn²⁺:3′FdU) indicating that this new structural motif may be therapeutically useful for PCa treatment.

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Keywords:

• DNA
• fluoropyrimidine
• metal:DNA interactions
• netropsin
• prostate cancer

Acknowledgments

This work was supported by DOD PCRP 093606 (WHG), National Institutes of Health CA102532 (WHG), 2P30 CA12197-25, and the North Carolina Biotechnology Center. FRS acknowledges partial support from NIH CA12937. All calculations were performed on the DEAC cluster which is supported by WFUS Information systems and the WFU Provost’s office. The authors acknowledge Dr. Marcus Wright for help with acquisition of ³¹P and ¹⁹F NMR.