title: A 4:1 stoichiometric binding and stabilization of mitoxantrone-parallel stranded G-quadruplex complex established by spectroscopy techniques.
abstract: Small molecule ligands which specifically bind and stabilize G-quadruplex structures in telomeric ends inhibit the activity of telomerase enzyme, an important marker for cancer. Understanding of the binding mode of ligand-G quadruplex complex is important for evaluating relative efficacy of anti-tumor drugs. The present study is focused on interaction of anti-tumor drug mitoxantrone (MTX) with tetra-molecular parallel stranded G-quadruplex sequence d-TTGGGGT using absorbance, fluorescence and circular dichroism spectroscopy techniques. Absorbance of mitoxantrone shows hypochromism up to MTX (D)/DNA quadruplex (N) ratio ~5, followed by hyperchromism up to D/N=0.21 accompanied by a red shift of 15nm. The fluorescence emission of MTX shows decrease up to D/N ~5 and then increases with red shift of 8nm. The two observed fluorescent lifetimes, 0.17ns (91%) and 0.44ns (9%), indicate dual binding mode. Absence of isobestic and isoemissive point indicates presence of multiple complexes. Circular Dichroism spectra showing positive induced band at 645nm and two exciton bands centered at 619 and 664nm suggest binding of mitoxantrone as a dimer. Proton NMR studies show intermolecular MTX-MTX short contacts confirming existence of stacked dimer of MTX. Thermal melting transitions of DNA saturate at D/N=4 with ΔTm=25°C. The results establish highly specific external groove binding of 4 molecules of mitoxantrone as two dimers at two distinct sites of DNA.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, India.
authors.forename: Tarikere Palakashan
authors.initials: TP
authors.lastname: Pradeep
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, India. Electronic address: ritubfbs@iitr.ac.in.
authors.forename: Ritu
authors.initials: R
authors.lastname: Barthwal
mesh_terms: D002942:Circular Dichroism; D054856:G-Quadruplexes; D008942:Mitoxantrone / Q000737:chemistry*; D066244:Proton Magnetic Resonance Spectroscopy; D013050:Spectrometry, Fluorescence
chemical_list: D008942:Mitoxantrone
keywords: Absorbance; Anti-cancer; Circular dichroism; G-quadruplex DNA; Mitoxantrone; Steady state and fluorescence lifetime