title: Synthesis and evaluation of an intercalator-polyamide hairpin designed to target the inverted CCAAT box 2 in the topoisomerase IIalpha promoter.
abstract: 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.
authors.affiliation: Department of Chemistry, Furman University, Greenville, SC 29613, USA.
authors.forename: Lloyd V
authors.initials: LV
authors.lastname: Flores
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Staples
authors.forename: Hilary
authors.initials: H
authors.lastname: Mackay
authors.forename: Cameron M
authors.initials: CM
authors.lastname: Howard
authors.forename: Peter B
authors.initials: PB
authors.lastname: Uthe
authors.forename: Jim S
authors.initials: JS
authors.lastname: Sexton
authors.forename: Karen L
authors.initials: KL
authors.lastname: Buchmueller
authors.forename: W David
authors.initials: WD
authors.lastname: Wilson
authors.forename: Caroline
authors.initials: C
authors.lastname: O'Hare
authors.forename: Jerome
authors.initials: J
authors.lastname: Kluza
authors.forename: Daniel
authors.initials: D
authors.lastname: Hochhauser
authors.forename: John A
authors.initials: JA
authors.lastname: Hartley
authors.forename: Moses
authors.initials: M
authors.lastname: Lee
mesh_terms: D001483:Base Sequence; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009691:Nucleic Acid Denaturation; D009757:Nylons / Q000138:chemical synthesis / Q000737:chemistry*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D013696:Temperature; D017186:Titrimetry
chemical_list: D009757:Nylons; D004247:DNA; D004250:DNA Topoisomerases, Type II