Synthesis and evaluation of an intercalator-polyamide hairpin designed to target the inverted CCAAT box 2 in the topoisomerase IIalpha promoter
| Autor: | Jerome Kluza, John A. Hartley, Caroline O'Hare, Karen L. Buchmueller, Jim S. Sexton, Peter B. Uthe, Daniel Hochhauser, Lloyd V. Flores, Andrew M. Staples, W. David Wilson, Cameron M. Howard, Hilary Mackay, Moses Lee |
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| Rok vydání: | 2006 |
| Předmět: |
Models
Molecular Base pair Stereochemistry Allosteric regulation CAAT box DNA Footprinting DNA footprinting Nucleic Acid Denaturation Biochemistry Moiety Binding site Promoter Regions Genetic Molecular Biology Base Sequence Chemistry Circular Dichroism Organic Chemistry DNase-I Footprinting Temperature Titrimetry DNA Surface Plasmon Resonance Binding constant Protein Structure Tertiary Nylons DNA Topoisomerases Type II Spectrometry Fluorescence Molecular Medicine |
| Zdroj: | Chembiochem : a European journal of chemical biology. 7(11) |
| ISSN: | 1439-4227 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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