Kaempferol and curcumin as fluorescent probes for DNA topologies: Integrated spectroscopic and computational study

Kaempferol (3,4',5,7-tetrahydroxyflavone, KMP) and curcumin (diferuloylmethane, CUR) are naturally occurring polyphenolic compounds with broad therapeutic potential, including anticancer, antioxidant, and anti-inflammatory properties. Among their molecular targets, DNA plays a central role, particularly through interactions with non-canonical DNA structures such as G-quadruplexes (G₄) and i-motifs (C₄), which form in guanine- and cytosine-rich genomic regions, respectively. These structures regulate telomere maintenance, gene expression, and genomic stability, making them attractive drug targets. In this study, we investigate the binding behavior of KMP and CUR with G₄, C₄, and duplex calf thymus DNA (calf thymus (CT)-DNA) using an integrated spectroscopic and computational approach. Circular dichroism, UV-visible absorption, and fluorescence spectroscopy were used to monitor ligand-induced structural and photophysical changes. CUR exhibited pronounced solvatochromism, with emission maxima shifting according to solvent polarity and DNA topology and showed the strongest fluorescence enhancement in G₄ DNA. KMP displayed excited state intramolecular proton transfer (ESIPT), with the highest tautomeric emission observed in G₄ structures. However, G₄ also facilitated ground-state anion formation at the 3-OH group of KMP, which suppressed ESIPT by interfering with intramolecular hydrogen bonding between C(4) = O and 3-OH. ESIPT was least prominent in C₄, and moderate in duplex DNA, where anion formation was less favored. Displacement assays using ethidium bromide (EtBr) provided functional insight into the competitive binding dynamics, confirming groove and loop binding for both ligands in G₄ and C₄ DNA, while KMP also exhibited intercalative binding in duplex DNA. Molecular docking and molecular dynamics simulations corroborated these findings, revealing stable ligand-DNA complexes and specific interaction modes. This comprehensive approach highlights CUR as a polarity-sensitive reporter and KMP as a thermally and structurally responsive ESIPT fluorophore. Together, they represent promising tools for probing DNA topology and developing targeted molecular diagnostics or therapeutic strategies centered on nucleic acid structure recognition.