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Z-DNA is the only DNA conformation that has a left-handed helical twist. Although Z-DNA has been implicated in both carcinogenesis and mutagenesis, its specific biological role remains uncertain. We have demonstrated that the formation of C8-arylguanine DNA adducts, derived from arylhydrazines, shifts the B/Z-DNA equilibrium toward the Z-DNA conformation in d(CG)5 sequences. However, our previous work examined the effect of two adducts in the duplex, and it was unclear whether the two base modifications were working together to cause the equilibrium shift toward the Z-DNA conformation. Here we report the synthesis and characterization of a hairpin oligonucleotide sequence (d(CG)5T4(CG)5) containing only one C8-arylguanine modified base. The unmodified hairpin and the previously studied unmodified double-stranded oligonucleotide were conformationally similar, and each required ∼3 M NaCl to yield a B-/Z-DNA ratio of 1:1. The introduction of a single C8-arylguanine modification significantly reduced the NaCl concentration needed to produce a 1:1 B-/Z-DNA ratio in the hairpin. Further, the addition of MgCl2 and spermine to the C8-arylguanine-modified hairpin shifts the B/Z-DNA equilibrium such that the Z form predominated under physiological conditions. NMR and molecular modeling indicated the conformational effects produced by the C8-arylguanine modification occurred locally at the site of modification while CD data demonstrated that the C8-arylguanine-modified base destabilized the B form. Additionally, our data show that adopting the Z-DNA conformation is preferred over denaturation to the single-stranded form. Finally, the conformational effects of the C8-arylguanine modifications were not additive and the introduction of any such modifications drive Z-DNA formation under physiological conditions, which may provide a novel carcinogenesis mechanism where DNA adducts confer their carcinogenicity through a Z-DNA-mediated mechanism.

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The (1)H and (13)C NMR spectra of 10-deoxymethynolide (1), 8.9-dihydro-10-deoxymethynolide (2) and its glycosylated derivatives (3-9) were analyzed using gradient-selected NMR techniques, including 1D TOCSY, gCOSY, 1D NOESY (DPFGSENOE), NOESY, gHMBC, gHSQC and gHSQC-TOCSY. The NMR spectral parameters (chemical shifts and coupling constants) of 1-9 were determined by iterative analysis. For the first time, complete and unambiguous assignment of the (1)H NMR spectrum of 10-deoxymethynolide (1) has been achieved in CDCl(3), CD(3)OD and C(6)D(6) solvents. The (1)H NMR spectrum of 8,9-dihydro-10-deoxymethynolide (2) was recorded in CDCl(3), (CD(3))(2)CO and CD(3)OD solutions to determine the conformation. NMR-based conformational analysis of 1 and 2 in conjugation with molecular modeling concluded that the 12-membered ring of the macrolactones may predominantly exist in a single stable conformation in all solvents examined. In all cases, a change in solvent caused only small changes in chemical shifts and coupling constants, suggesting that all glycosylated methymycin analogs exist with similar conformations of the aglycone ring in solution.

DNA has many different conformations however only Z‐DNA adopts a left‐handed helix. The zigzag phosphate backbone of Z‐DNA brings the negatively charged phosphate residues closer together rendering Z‐DNA a higher energy form of DNA. Z‐DNA has been implicated in carcinogenesis from its role in gene expression and mutagenesis because Z‐DNA formation can stimulate large scale gene deletions, translocations, and rearrangements in vivo. The inability to generate a stable Z‐DNA structure under physiological conditions has made investigation of B‐ to Z‐DNA interconversion difficult since Z‐DNA requires external stimuli to stabilize its formation like chemical modifications or specific proteins known to stabilize Z‐DNA, Z‐DNA binding proteins (ZBP). We have demonstrated that C8‐arylguanine DNA adducts, derived from carcinogenic arylhydrazines, drive Z‐DNA formation and the addition of MgCl2 stabilizes the Z form under physiological conditions. The conformational effects of the C8‐arylguanine mutation were determined by nuclear magnetic resonance (NMR) and circular dichroism (CD). In addition, preliminary results using in silico molecular dynamics and CD have demonstrated that ZBPs prefer to bind to the C8‐arylguanine modified DNA. C8‐Arylguanine can be used as a tool to clarify the mechanism of ZBP mediated B‐ to Z‐DNA transition. The current view fails to explain the ZBP selectivity for the Z form and leads to a contradictory conclusion that ZBPs cannot bind Z‐DNA. C8‐Arylguanine modified oligonucleotides can be used to fully elucidate the binding of ZBPs to DNA, as both the B and Z forms can be generated under physiological conditions, using surface plasmon resonance (SPR). This may reveal the underlying factors that control ZBP‐DNA interactions which could become a potential therapeutic target. (Supported by a fellowship to BCT (HEPC.dsr.09013) and (NSF EPS‐1003907)