Published: November 30, 2011 r2011 American Chemical Society 639 dx.doi.org/10.1021/jp2094824 | J. Phys. Chem. B 2012, 116, 639–645 ARTICLE pubs.acs.org/JPCB Spectroscopic Exploration of Mode of Binding of ctDNA with 3-Hydroxyflavone: A Contrast to the Mode of Binding with Flavonoids Having Additional Hydroxyl Groups Barnali Jana, Sudipta Senapati, Debanjana Ghosh, Debosreeta Bose, and Nitin Chattopadhyay* Department of Chemistry, Jadavpur University, Kolkata 700 032, India ’ INTRODUCTION Deoxyribonucleic acid (DNA) is an important biological material whose base sequence controls the heredity of life. In recent years, there has been an increasing interest in the use of DNA as the recognition element of affinity biosensors 1 and for the development of effective therapeutic agents in controlling gene expression. 2 Study of the interaction of small molecules with DNA is gaining increasing importance for exploring the structural and functional features of the biomacromolecule to decipher its biophysical processes. 3 Small molecules bind to the DNA double helix by three dominant modes referred to as (i) intercalative binding where the probe intercalates within the nucleic acid base pairs, (ii) groove binding involving van der Waal’s interaction in the deep major groove or the shallow minor groove of the DNA helix, and (iii) electrostatic binding between the negatively charged DNA phosphate backbone and cationic end of the molecules. 4,5 Intercalated probes are com- paratively more protected from the external agents compared to those bound through other interactions. 6 Electrostatic, hydro- gen bonding and hydrophobic interactions generally contri- bute to the stability of groove binding, 7 and intercalative bind- ing is mostly favored by stacking interaction with the adjacent DNA bases. 8 Flavonoids are polyphenolic compounds, which are ubiquitous in plants of higher genera and are abundant in common plant based food items and beverages. 3-Hydroxyflavone (3HF, Scheme 1), a member of this group of natural products, exhibits strong antioxidant activity in the membrane environment, suggest- ing that it acts as a therapeutic agent. 9 It is also one of the best- known prototype molecules undergoing excited state intramole- cular proton transfer (ESIPT) and thereby exhibiting dual fluorescence. 1014 Although ESIPT of 3HF have been exten- sively investigated in a number of model biomembranes, 1214 an understanding of the therapeutic action of 3-hydroxyflavone (3HF) on the molecular basis would require knowledge of its mode of interaction with other biological targets, especially DNA and proteins. Being intramolecular in nature, the ESIPT process is sensitive to solvent polarity and proticity. In more polar/protic solvents, the dissociable proton is intermolecularly hydrogen bonded to the solvent restricting the ESIPT process. When such probes bind to biomacromolecules, the microenvironment around it becomes less polar and less protic. Thus, a change in the environment can be easily probed monitoring the modification of the fluorometric parameters resulting out of the ESIPT process. With this objective, in the present work we demonstrate the use of the intrinsic fluorescence of this probe together with some other techniques to characterize its binding with the double stranded calf thymus DNA (ctDNA). It is pertinent to mention here that flavonoids like fisetin and quercetin (Q) (Scheme 1) have a similar skeleton as that of 3HF differing only in the number of additional hydroxyl functional groups. The available reports on the DNA binding with fisetin and quercetin using electrochemical, 15 fluoro- metric, 16 enzyme hydrolysis, 17 HPLC, 18 and voltamometric 19,20 techniques imply that these probes intercalate into the DNA. Surprisingly, despite being the simplest member of the flavone family, for 3HF, nothing is available in this regard. We have, therefore, been interested in exploring the binding interaction of 3HF with the ctDNA. The perception of the investigation is to see Received: October 1, 2011 Revised: November 24, 2011 ABSTRACT: Binding interaction of 3-hydroxyflavone (3HF), a bioactive flavonoid, with calf-thymus DNA (ctDNA) has been explored exploiting various experimental techniques. The dual fluorescence of 3HF resulting from the excited state intramolecular proton transfer (ESIPT) is modified remarkably upon binding with the biomacromolecule. The determined binding constant, fluores- cence quenching experiment, circular dichroism (CD) study, comparative bind- ing study with the known intercalative binder ethidium bromide and thermo- metric experiment relating to the helix melting of ctDNA confirm the groove binding of 3HF with the DNA. This is in contrast to two other members of the flavonoid group, namely, fisetin and quercetin, where the bindings are established to be intercalative. The structural difference of 3HF from the other two probes with respect to the absence/presence of the additional hydroxyl groups is ascribed to be responsible for the difference in the mode of binding.