Targeting the inverted CCAAT Box-2 of the topoisomerase IIalpha gene: DNA sequence selective recognition by a polyamide-intercalator as a staggered dimer.

The synthesis and DNA binding characteristics of a polyamide-intercalator conjugate, designed to inhibit NF-Y binding to the ICB-2 site of the topoisomerase IIalpha promoter and up-regulate the expression of the enzyme in confluent cells, are reported. Thermal denaturation and CD titration studies demonstrated binding to the cognate sequence (5'-AAGCTA-3'). Formation of ligand-induced CD bands at approximately 330 nm provided indication that the molecule interacts selectively in the minor groove of DNA. Intercalation was evidenced by a fivefold increase in emission of the intercalator moiety upon binding to the ICB-2 hairpin oligonucleotide. An increase in viscosity of a solution of calf-thymus DNA on addition of the conjugate provided further evidence. The binding affinity of the conjugate was ascertained using SPR (5.6x10(6) M(-1)), which according to a gel shift assay was capable of inhibiting the binding of NF-Y at a concentration of 50 microM. DNaseI footprinting, using the topoIIalpha promoter sequence, highlighted the specificity of the conjugate for the cognate site (5'-AAGCTA-3'). Finally, through Western blot analysis, confluent murine NIH 3T3 cells treated with conjugate were found to have enhanced expression of topoIIalpha. These results suggest that the conjugate can enter the nucleus, bind to its target site, presumably as a stacked dimer, and up-regulate the expression of topoIIalpha by blocking the binding of NF-Y.

Synthesis and evaluation of an intercalator-polyamide hairpin designed to target the inverted CCAAT box 2 in the topoisomerase IIalpha promoter.

The synthesis and DNA-binding properties of a novel naphthalimide-polyamide hairpin (3) designed to target the inverted CCAAT box 2 (ICB2) site on the topoisomerase IIalpha (topoIIalpha) promoter are described. The polyamide component of 3 was derived from the minor-groove binder, 2, and tailored to bind to the 5'-TTGGT sequence found in and flanking ICB2. The propensity of mitonafide 4 to intercalate between G-C base pairs was exploited by the incorporation of a naphthalimide moiety at the N terminus of 2. Hybrid 3 targeted 5'-CGATTGGT and covered eight contiguous base pairs, which included the underlined ICB2 site. DNase I footprinting analysis with the topoIIalpha promoter sequence demonstrated that 3 bound selectively to the ICB2 and ICB3 sites. Thermal-denaturation studies confirmed these results, and the highest degree of stabilization was found for ICB2 and -3 in preference to ICB1 (4.1, 4.6, and 0.6 degrees C, respectively). CD studies confirmed minor-groove binding and suggested a 1:1 binding stoichiometry. Emission-titration experiments established intercalative binding. Surface plasmon resonance results showed strong binding to ICB2 (2.5x10(7) M(-1)) with no observable binding to ICB1. Furthermore, the binding constant of 3 to ICB2 was larger than that of the parent polyamide 2. The increased binding affinity was primarily due to a reduction in the dissociation-rate constant of the polyamide-DNA complex, which can be attributed to the N-terminal naphthalimide moiety. In addition, the binding site of 3 was larger than that of 2, which innately improved sequence selectivity. We conclude that the polyamide-naphthalimide 3 selectively binds to the ICB2 site by simultaneous intercalation and minor-groove binding, and warrants further investigation as a model compound for the regulation of topoIIalpha gene expression.