Red and green denote free and d(T₂AG₄)₄ bound fisetin respectively in 10 mM Tris buffer, pH 7.4 with 25 mM NaCl.

<p>A: Fluorescence emission spectra (λ_ex = 370 nm); B: Fluorescence excitation spectra (λ_em = 530 nm); and C: Absorption spectra of fisetin (∼ 7.5 µM); in the presence of increasing concentrations of DNA (0― red, rest green; 1–, 5–·–, 10–··–, 20 µM–···–). A_inset: Variation of the ratio of the intensity of tautomer vs normal isomers of fisetin (I_{T at 530}/I_{N at 470}) with increasing [DNA]. C_inset highlights the absorption spectra of fisetin bound 5 µM DNA (green) between 200–300 nm. Pink (…….) spectra and marker in Figures A, B, C and A-inset provide the fluorescence emission, excitation, absorption spectra and I_{T at 530}/I_{N at 470} of 7.5 µM fisetin in 20 µM (T₂AG₄)₄ in the presence of 7.5 µM of ethidium bromide (EtBr) respectively. The blue and black dotted lines in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065383#pone-0065383-g003" target="_blank">Figure 3B</a> represent the excitation spectra of EtBr ( = 600 nm) with and without fisetin in 5 µM DNA.</p

Structures of (a) G-quartet, four guanines can hydrogen bond in a square arrangement to form a G-quartet.

<p>There are two hydrogen bonds on each side of the square; (b) i. Tetramolecular parallel, ii. Bimolecular antiparallel structure with parallel adjacent strands, iii. Unimolecular antiparallel with alternating parallel strands, iv. Unimolecular parallel structure with three double chain reversal loops, v. Unimolecular antiparallel structure with parallel adjacent strands and a diagonal loop, vi. Unimolecular mixed structure with three parallel and one antiparallel strands. Structures b_iv-vi have been observed for the human telomeric repeat <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065383#pone.0065383-Huppert1" target="_blank">[4]</a>; (c) Fisetin; (d) Ground and excited (denoted by *) states of normal (N) and tautomer (T) forms of a flavonol.</p

Prospect of Bioflavonoid Fisetin as a Quadruplex DNA Ligand: A Biophysical Approach

<div><p>Quadruplex (G₄) forming sequences in telomeric DNA and c-<i>myc</i> promoter regions of human DNA are associated with tumorogenesis. Ligands that can facilitate or stabilize the formation and increase the stabilization of G₄ can prevent tumor cell proliferation and have been regarded as potential anti-cancer drugs. In the present study, steady state and time-resolved fluorescence measurements provide important structural and dynamical insights into the free and bound states of the therapeutically potent plant flavonoid fisetin (3,3′,4′,7-tetrahydroxyflavone) in a G₄ DNA matrix. The excited state intra-molecular proton transfer (ESPT) of fisetin plays an important role in observing and understanding the binding of fisetin with the G₄ DNA. Differential absorption spectra, thermal melting, and circular dichroism spectroscopic studies provide evidences for the formation of G₄ DNA and size exclusion chromatography (SEC) proves the binding and 1∶1 stoichiometry of fisetin in the DNA matrix. Comparative analysis of binding in the presence of EtBr proves that fisetin favors binding at the face of the G-quartet, mostly along the diagonal loop. Time resolved fluorescence anisotropy decay analysis indicates the increase in the restrictions in motion from the free to bound fisetin. We have also investigated the fingerprints of the binding of fisetin in the antiparallel quadruplex using Raman spectroscopy. Preliminary results indicate fisetin to be a prospective candidate as a G₄ ligand.</p></div

Fluorescence decay parameters of fisetin tautomer (Fis (T))^a and fisetin normal (Fis (N))^b species in water and in the presence of QD DNA.

<p>Fluorescence decay parameters of fisetin tautomer (Fis (T))^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065383#nt101" target="_blank">a</a>} and fisetin normal (Fis (N))^b species in water and in the presence of QD DNA.</p

A: Typical Raman spectra of 1) blue-1 mM Fisetin, 2) red-1 mM (T₂AG₄)₄ DNA, 3) green-1 mM (T₂AG₄)₄ DNA conjugated with 1 mM fisetin, 4) pink-10 mM Tris buffer, pH 7 with 25 mM NaCl in which all the above solutions are prepared, B: 1 mM T₁₅ DNA oligonucleotide dissolved in water.

<p>Here ab, tb, g, ag, f denote adenine base, thymine base, guanine tetrad, antiparallel guanine tetrad and fisetin respectively.</p

3HF and 7HF rescues renal proximal tubule cells from nicotine exposure-associated cytotoxicity by inhibiting ROS production.

<p>(A) Chemical structure of 3-hydroxyflavone (3HF) and 7-hydroxyflavone (7HF). (B) NRK52E cells were pretreated with 20 μM 3HF or 7HF overnight prior to treatment with 200 μM NIC and cell viability was determined 24 hours later. Control cells were left untreated, or treated with 200 μM NIC, 20 μM 3HF or 7HF. Values were expressed as % of untreated control. n = 3; *p<0.05 compared to untreated control. Dotted line represents untreated control value. (C) NRK52E cells were pretreated with 20 μM 3HF or 7HF overnight and 200 μM NIC-mediated ROS production was determined. Control cells were left untreated, or treated with 200 μM NIC, 20 μM 3HF or 7HF. Values are expressed as % of untreated control. n = 3; *p<0.05 compared to untreated control or as indicated. Dotted line represents untreated control value.</p

Average values of RMSD, SASA and no. of H-bonds values of 1DH3 and 2DYH proteins in free and 3HF / 7HF bound systems.

<p>Average values of RMSD, SASA and no. of H-bonds values of 1DH3 and 2DYH proteins in free and 3HF / 7HF bound systems.</p

Docking summary of 7HF and 3HF with 2DYH and 1DH3 proteins.

<p>Docking summary of 7HF and 3HF with 2DYH and 1DH3 proteins.</p

Molecular dynamics simulations.

<p>(A) Time dependence of RMS deviation of distance (RMSD) between alpha-carbon atoms from the crystal structure as a function of simulation time for free 1DH3 and conjugated 1DH3. (B) Time evolution of the solvent accessible surface area (SASA) during 18 ns of MD simulation of 1DH3, 1DH3 with 3HF and 1DH3 with 7HF. (C) Time dependence of RMS deviation of distance (RMSD) between alpha-carbon atoms from the crystal structure as a function of simulation time for free 2DYH and conjugated 2DYH. (D) Time evolution of the solvent accessible surface area (SASA) during 18 ns of MD simulation of 2DYH, 2DYH with 3HF and 2DYH with 7HF. Color codes are given on the figures.</p

3HF and 7HF activates distinct antioxidant genes via distinct signaling pathways.

<p>(A) NRK52E cells were transfected with a MnSOD promoter luciferase plasmid as described in Materials and Methods. A set of cells were infected with the M1CREB adenovirus overnight or treated with 10 μM H89 1 hr prior to treatment with 20 μM 3HF or 7HF. Luciferase activities were determined 24 hours later. n = 3; *p<0.05 compared to control or as indicated. Dotted line represents control value. (B) NRK52E cells were transfected with an HO-1 promoter luciferase plasmid as described in Materials and Methods. A set of cells were co-transfected with 20 nM Nrf2 siRNA or infected with the dnMEK adenovirus 24 hours prior to treatment with 20 μM 3HF or 7HF. Luciferase activities were determined 24 hours later. n = 3; *p<0.05 compared to control or as indicated. Dotted line represents control value. (C) NRK52E cells were transfected with a CRE luciferase plasmid as described in Materials and Methods. A set of cells were infected with a M1CREB adenovirus 24 hours or treated with 10 μM H89 1 hr prior to treatment with 20 μM 3HF or 7HF. Luciferase activities were determined 24 hours later. n = 3; *p<0.05 compared to control or as indicated. Dotted line represents control value. (D) NRK52E cells were transfected with an ARE luciferase plasmid as described in Materials and Methods. A set of cells were co-transfected with 20 nM Nrf2 siRNA or infected with the dnMEK adenovirus 24 hours prior to treatment with 20 μM 3HF or 7HF. Luciferase activities were determined 24 hours later. n = 3; *p<0.05 compared to control or as indicated. Dotted line represents control value.</p