The Selective Recognition of Thymine in Bulged and G-quadruplex DNA With Modified Zn(II) Macrocyclic Complexes
Siters, Kevin Eugene
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The recognition of DNA secondary structures, which contain non-canonical thymines, using Zn(II) complexes will be presented. In particular, the binding to thymines found in bulge, hairpin and G-quadruplex DNA secondary structures. All the complexes contain a conserved Zn(II)-cyclen (cyclen = 1,4,7,10-tetraazacyclododecane) moiety. Previous studies have shown that the Zn(II)-cyclen moiety forms a coordination bond with deprotonated imine nitrogen (N3) of thymine. The binding of the complexes was improved by appending aromatic pendant groups. This introduces a π-π stacking component between the pendant and the thymine aromatic face. These include pendants with two-fused rings such as quinolinone (Zn(QMC)), quinoline (Zn(CQC)), coumarin (Zn(MCC)) and dansyl (Zn(DSC)). Additional complexes contained three-ring pendants such as: acridine (Zn(ACR)) and anthraquinone (Zn(ATQ)). The bifunctional library has been used to study for binding to primarily G-quadruplexes and bulged DNA. Fluorescence spectroscopy, isothermal titration calorimetry, UV-Vis and surface plasmon resonance were used to characterize the binding events. It was determined that the complexes exhibit moderate binding constants to bulged thymines. Furthermore it was concluded that a planar aromatic pendant is required for tight binding. However, the planar, three-ring containing Zn(ACR) exhibited very poor selectivity towards duplex DNA. Low micromolar dissociation constants were observed for binding to G-quadruplexes, and in particular the human telomeric G-quadruplex. The Zn(II) complexes containing two-fused rings appear to bind selectively to G-quadruplex DNA over duplex and hairpin DNA. The complex Zn(DSC) shows 110-fold selectivity towards the human telomeric G-quadruplex over the Dickerson dodecamer. Additionally, two-ringed complexes exhibit a 2:1 stoichiometry when binding to the human telomeric G-quadruplex. Zn(ATQ), however, exhibits a 3:1 stoichiometry and stabilizes this G-quadruplex by 9 °C. The interaction with these structures is highly pH dependant, suggesting binding to the thymine residues. Pentanucleotides CCTCC, CTTCC and CTCTC were used to further understand the binding mode of Zn(DSC) to the G-quadruplex structures. Fluorescence and ITC studies showed that Zn(DSC) binds to CTTCC with a 1:1 stoichiometry and to the spaced thymines in CTCTC with a 2:1 ratio. This helped us to develop a model in which two Zn(DSC) complexes bind to independent thymines in separate loops in the G-quadruplex. Preliminary studies showed that the Zn(II) complexes can bind to promoter region G-quadruplexes such as c-MYC. Binding studies using surface plasmon resonance suggest that the Zn(II) complexes bind modestly to these structures. Although further testing is being performed to determine the binding in solution. Interestingly, the macrocycles appear to self-assemble when equal concentrations of the macrocycle, ZnCl 2 and human telomeric G-quadruplex are added independent of order. The formation of this adduct is specific to the Zn(II) ion. Co(II), Fe(II), Cu(II) complexes of DSC cannot bind to the human telomeric G-quadruplex. This feature may allow for this system to be used as a Zn(II) sensor.