Spectroscopic Exploration of Mode of Binding of ctDNA with 3

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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 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, fluorescence quenching experiment, circular dichroism (CD) study, comparative binding study with the known intercalative binder ethidium bromide and thermometric 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.

’ 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 biosensors1 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 comparatively more protected from the external agents compared to those bound through other interactions.6 Electrostatic, hydrogen bonding and hydrophobic interactions generally contribute to the stability of groove binding,7 and intercalative binding 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, suggesting that it acts as a therapeutic agent.9 It is also one of the bestknown prototype molecules undergoing excited state intramolecular proton transfer (ESIPT) and thereby exhibiting dual r 2011 American Chemical Society

fluorescence.10
14 Although ESIPT of 3HF have been extensively 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 fluorometric,16 enzyme hydrolysis,17 HPLC,18 and voltamometric19,20 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 Published: November 30, 2011 639

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Scheme 1. Structures of (A) 3HF, (B) Fisetin, and (C) Quercetin

Figure 1. Absorption spectrum of aqueous buffer solution of 3HF (∼2  10
5 mol dm
3).

if the presence of the additional hydroxyl groups in fisetin and quercetin makes any difference in the probe-DNA binding mode. Steady-state fluorometric measurements have been exploited for the study together with other techniques like comparative ligand binding using the known intercalator ethidium bromide (EtBr),6,21,22 circular dichroism, thermometric experiment for DNA helix melting, etc. While the first part of the study intends to confirm the binding of 3HF with ctDNA, the latter part divulges the mode of binding. All the experimental findings unambiguously establish that 3HF resides in the groove of the DNA helix, contrary to the intercalative mode of binding of fisetin and quercetin (Q) with the same host.

’ EXPERIMENTAL SECTION Materials. 3HF was purchased from Fluka (USA), and quercetin (Q) was received as a kind gift from Professor P. K. Sengupta of Saha Institute of Nuclear Physics, Kolkata. EtBr, ctDNA (molecular wt, 8.4 MDa), urea, KI, and N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] (HEPES) buffer were obtained from SRL (India). Spectroscopic grade 1,4dioxane (Spectrochem, India) and methanol (Merck, India) were used as received. The dyes and ctDNA were also used as received without further purification. Triply distilled water was used for making solutions wherever required. Stock solution of ctDNA was prepared by dissolving solid ctDNA in HEPES buffer (pH = 7) and stored at 4 °C. The purity of ctDNA was verified by monitoring the ratio of absorbance at 260 nm to that at 280 nm (∼1.8). The concentration of the DNA solution (7  10
3 mol dm
3) was determined spectrophotometrically using εDNA = 13 600 mol
1 dm3 cm
1 at 258 nm.5 Concentrated stock solutions of 3HF and quercetin were prepared in methanol. Small aliquots from this stock solution were added to HEPES buffer solutions to give a final fluorophore concentration of ca. 2  10
5 mol dm
3. The concentration of methanol in the final solution was groove binding > electrostatic binding.5,6,23,46,47 Quenching of the fluorescence of 3HF by KI in aqueous buffer solution and in the presence of 1.3  10
3 mol dm
3 ctDNA has been studied 642

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Table 1. Steady-State Fluorescence Anisotropy of 3HF, Quercetin (Q), and EtBr in Different Situations

Figure 8. Stern
Volmer plots for the fluorescence quenching of 3HF by KI in aqueous buffer (red) and ctDNA environments (black). Concentration of 3HF and ctDNA are 2  10
5 mol dm
3 and 1.3  10
3 mol dm
3, respectively. λexc = 345 nm. a

emission

fluorescence

sample

maximum λem (nm)

anisotropy (r)

3HF in aqueous buffer Q in aqueous buffer

500a 520a

0.04 0.04

EtBr in aqueous buffer

620b

0.04

3HF in DNA

500a

0.112

3HF in DNA + EtBr

500a

0.106

3HF in DNA + EtBr

620b

0.23

Q in ctDNA

520a

0.17

Q in ctDNA + EtBr

520a

0.04

Q in ctDNA + EtBr

620b

0.24

λexc = 370 nm. b λexc = 480 nm.

anisotropy of 3HF (from 0.112 to 0.106). The fluorescence anisotropy of EtBr, however, increases from ∼0.04 in buffered aqueous solution to ∼0.23 in the ctDNA environment, which is obviously due to the intercalation. Had the binding of 3HF with ctDNA been intercalative, the addition of EtBr to the 3HF
DNA system should have been expected to replace 3HF resulting in the release of it from the DNA environment to the bulk aqueous phase. This should lead to a marked reduction in the fluorescence anisotropy of 3HF fluorescence.16 The experimental observation goes strongly against it and indicates that the binding between 3HF and ctDNA is not intercalative in nature. Table 1 indicates the observed steady-state anisotropy (r) values of 3HF in aqueous buffer, 1.3  10
3 mol dm
3 DNA and upon the addition of 57.4  10
6 mol dm
3 EtBr to it. It is pertinent to mention here that fisetin and quercetin, two other well-known flavonoid probes, bind with DNA through intercalation.15
19,25 To make a comparative study of the binding of 3HF and quercetin with the same host (ctDNA), we have extended the comparative binding study with EtBr taking quercetin. Table 1 displays the relevant data. In the presence of 0.77  10
3 mol dm
3 ctDNA, fluorescence anisotropy of quercetin increases to ∼0.17 from its value of 0.04 in the buffer solution. With the addition of ethidium bromide to the ctDNA
quercetin system, the anisotropy value decreases gradually and at a sufficient concentration of EtBr attains the value 0.04 again. The observation clearly indicates that EtBr displaces the intercalated quercetin molecules from the DNA base pairs to the bulk aqueous solution. The same experiment with 3HF and quercetin gives dissimilar observations suggesting strongly that in contrast to the intercalative binding for quercetin, 3HF undergoes groove binding with ctDNA. Circular Dichroism Study. We have also made an endeavor to decipher the binding mode of 3HF with ctDNA exploiting the circular dichroism technique. The secondary structure of DNA is perturbed markedly by the intercalation of small molecules leaving its signature through the changes in the intrinsic CD spectra of DNA.5,48,49 Groove binding, however, does not put so much impact on the CD signal.45 Figure 10 depicts the CD spectrum of ctDNA in HEPES buffer at pH 7, having a positive peak at ∼277 nm and a negative peak at ∼247 nm, characteristic of the right handed B form.5,50 These bands are caused by the stacking interactions between the bases and the helical suprastructure of the polynucleotide that provides an asymmetric environment for the bases.51 Gradual addition of 3HF to the DNA solution does not reveal any significant change in the

Figure 9. Fluorescence spectra of ctDNA bound 3HF with the addition of varying concentrations of EtBr.

following the Stern
Volmer equation F0 ¼ 1 þ KSV ½KI F where F0 and F are the fluorescence intensities of the tautomer in the absence and in the presence of the quencher, respectively, and KSV is the Stern
Volmer quenching constant. Figure 8 presents the Stern
Volmer plot and depicts the relative extent of quenching. The slopes of the plots yield the KSV values in aqueous buffer and ctDNA environments as 50.0 ((0.5) mol
1 dm3 and 17.2 ((0.2) mol
1 dm3, respectively. Appreciable reduction in the quenching efficiency thus rules out the electrostatic binding and indicates that the DNA bound probe is much less accessible to the ionic quencher that resides in the aqueous phase. A lesser degree of reduction in the KSV in the present case (66%) compared to our earlier observation5 (88%) with phenosafranin in ctDNA, where the probe is established to be intercalated, suggests groove binding of 3HF with the DNA. Comparative Binding Study with Ethidium Bromide. To inquire if the binding of 3HF with the ctDNA is of groove or intercalative in nature, we have performed a comparative binding study with ethidium bromide (EtBr), a well-known intercalative DNA binder.6,21,22 Apart from the steady-state fluorescence as depicted in Figure 9, fluorescence anisotropy has also been monitored for the purpose. As already stated, gradual addition of ctDNA increasing the fluorescence anisotropy of the 3HF tautomer implies binding (groove or intercalative) between the host and the guest. Interestingly, with the addition of EtBr to the DNA bound 3HF, there is hardly any meaningful change in the fluorescence 643

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three molecular systems are very similar, steric factor does not play a role to execute the differential binding interaction of 3HF compared to the other two probes. The differential effect is attributed to the presence of additional hydroxyl groups in fisetin and quercetin but not in 3HF. The hydroxyl group taking part in the ESIPT process can not be involved in the hydrogen bonding with the base pairs of DNA. However, the additional hydroxyl groups present in fisetin and quercetin not involved in the prototropic transformation do participate in hydrogen bonding with the DNA bases to provide extra stability to the stacking of the DNA through intercalative interaction.8 3HF, being the simplest flavone, lacks these hydroxyl groups apart from the one involved in the ESIPT reaction. Lack of hydrogen bonding with the DNA base pairs on one hand and the hydrophobic interaction between the probe and ctDNA on the other prompts 3HF to undergo groove binding with DNA. Whether the probe binds to the minor or the major groove of ctDNA still remains unexplored.

Figure 10. Intrinsic CD spectra of ctDNA with varying concentration of 3HF. Concentrations of 3HF are shown in the legends. [ctDNA] = 8  10
5 mol dm
3.

Figure 11. Thermal melting profile of 3.3  10
5 mol dm
3 native ctDNA (black) and the same treated with 2  10
5 mol dm
3 3HF (red) and 2  10
5 mol dm
3 quercetin (blue).

intrinsic CD spectrum of the DNA indicating that the binding of the probe with ctDNA does not disturb the stacking of the bases. This observation categorically rules out intercalation of 3HF in the DNA helix and thereby implies that the fluorophore binds to the host DNA through groove binding. Helix Melting Study. The binding mode of interaction between 3HF and ctDNA has also been corroborated from the DNA helix melting experiment. Stabilization of the helix because of the intercalation of probes results in an increase in the helix melting temperature of the DNA.17,44 Melting temperature (Tm) is the temperature at which 50% of double stranded DNA becomes single stranded. In the presence of intercalators, the Tm rises from native DNA until all the intercalating sites are saturated.44 On the contrary, groove binding does not lead to an appreciable change in the Tm value.52 We have determined the Tm of the native ctDNA as well as the DNA in the presence of the two probes, 3HF and quercetin, separately (Figure 11). The estimated melting temperatures come out to be 75.9 ((0.3) °C, 75.8 ((0.3) °C, and 80.1 ((0.3) °C for the unbound ctDNA and the DNA bound to 3HF and quercetin, respectively. The experiment reveals that upon binding with quercetin (an intercalator), the Tm of ctDNA is increased by ∼5 °C, but binding with 3HF does not lead to an appreciable change in Tm. An increase in Tm of the DNA as a result of binding with an intercalator is consistent with the literature.17 Inappreciable change in Tm of the ctDNA upon binding with 3HF establishes the groove binding between the two. The series of experiments thus confirms that in spite of being a member of the flavonoid family, 3HF binds with ctDNA through groove binding, contrary to the intercalative binding of fisetin and quercetin with the same host. Since the skeleton of all the

’ CONCLUSIONS The study provides an explicit picture of the binding interaction of 3HF with ctDNA. The extensive work comprising a series of techniques reveals that the intrinsic fluorometric behavior of 3HF is modified remarkably because of its binding with the DNA. Steady-state fluorescence anisotropy study infers that the probe is located in a motionally restricted region. Estimation of the binding constant, fluorescence quenching study, comparative binding study with EtBr, circular dichroism study, and helix melting study unambiguously establish that 3HF binds with ctDNA in a groove binding fashion in contrast to the intercalative binding modes in case of fisetin and quercetin. The differential binding pattern is attributed to the presence of the additional hydroxyl groups in fisetin and quercetin compared to that of the 3HF system that provides extra stabilization to the DNA helix through intercalative binding. The lack of hydrogen bonding with the DNA base pairs, nonplanar geometry of 3HF, and the van der Waal’s and/or hydrophobic interaction between the probe and ctDNA are ascribed responsible for the groove binding of this particular flavonoid with the biomacromolecule. ’ AUTHOR INFORMATION Corresponding Author

*Fax: 91-33-2414-6584. E-mail: [email protected].

’ ACKNOWLEDGMENT Financial support from the DST and UGC (through CAS program), Government of India, is gratefully acknowledged. Research fellowships from C.SIR (B.J. and D.G.) and DST (D.B.) are gratefully acknowledged. Thanks are due to Professor P. K. Sengupta of SINP, Kolkata, for his kind gift of quercetin and Dr. Suman Das of our department for extending help during the CD measurements. ’ REFERENCES (1) McGown, L. B.; Joseph, M. J.; Pitner, J. B.; Vonk, G. P.; Linn, C. P. Anal. Chem. 1995, 67, 663A–668A. (2) Dervan, P. B. Bioorg. Med. Chem. 2001, 9, 2215–2235. (3) Osborne, S. E.; Ellington, A. D. Chem. Rev. 1997, 97, 349–370. (4) Saenger, W. Principles of Nucleic Acid Structure; Springer-Verlag: New York, 1983. 644

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(43) Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71, 2703–2707. (44) Das, S.; Kumar, G. S. J. Mol. Struct. 2008, 872, 56–63. (45) Sahoo, D.; Bhattacharya, P.; Chakravorti, S. J. Phys. Chem. B 2010, 114, 2044–2050. (46) Berman, H. M.; Young, P. R. Annu. Rev. Biophys. Bioeng. 1981, 10, 87–114. (47) Kumar, C. V.; Asuncion, E. H. J. Chem. Soc., Chem. Commun. 1992, 6, 470–472. (48) Jain, S. S.; Polak, M.; Hud, N. V. Nucleic Acids Res. 2003, 31, 4608–4615. (49) Mergny, J. L.; Duval-Valentin, G.; Nguyen, C. H.; Perrouault, L.; Faucon, B.; Rougee, M.; Montenay-Garestier, T.; Bisagni, E.; Helene, C. Science 1992, 256, 1681–1684. (50) van Holde, K.; Johnson, W. C.; Ho, P. S. Principles of Physical Biochemistry; Prentice Hall: New York, 1998. (51) Long, Y. F.; Liao, Q. G.; Huang, C. Z.; Ling, J.; Li, Y. F. J. Phys. Chem. B 2008, 112, 1783–1788. (52) Kumar, C. V.; Turner, R. S.; Asuncion., E. H. J. Photochem. Photobiol., A 1993, 74, 231–238.

(5) Sarkar, D.; Das, P.; Basak, S.; Chattopadhyay, N. J. Phys. Chem. B 2008, 112, 9243–9249. (6) Lerman, L. S. J. Mol. Biol. 1961, 3, 18–30. (7) Teng, M. K.; Usman, N.; Frederick, C. A.; Wang, A. H. J. Nucleic Acids Res. 1988, 16, 2671–2690. (8) Pyle, A. M.; Rehmann, J. P.; Meshoyrer, R.; Kumar, C. V.; Turro, N. J.; Barton, J. K. J. Am. Chem. Soc. 1989, 111, 3051–3058. (9) Sengupta, B.; Banerjee, A.; Sengupta, P. K. FEBS Lett. 2004, 570, 77–81. (10) McMorrow, D.; Kasha, M. J. Phys. Chem. 1984, 88, 2235–2243. (11) Sengupta, P. K.; Kasha, M. Chem. Phys. Lett. 1979, 68, 382–385. (12) Shyamala, T.; Mishra, A. K. Photochem. Photobiol. 2004, 80, 309–315. (13) Guharay, J.; Chaudhuri, R.; Chakrabarti, A.; Sengupta, P. K. Spectrochim. Acta, Part A 1997, 53, 457–462. (14) Chaudhuri, S.; Banerjee, A.; Basu, K.; Sengupta, B.; Sengupta, P. K. Int. J. Biol. Macromol. 2007, 41, 42–48. (15) Zhu, Z.; Li, C.; Li, N. Q. Microchem. J. 2002, 71, 57–63. (16) Sengupta, B.; Banerjee, A.; Sengupta, P. K. J. Photochem. Photobiol., B 2005, 80, 79–86. (17) Alvi, N. K.; Rizvi, R. Y.; Hadi, S. M. Biosci. Rep. 1986, 6, 861–868. (18) Binsack, R.; Boersma, B. J.; Patel, R. P.; Kirk, M.; White, C. R.; Darley-Usmar, V.; Barnes, S.; Zhou, F.; Parks, D. A. Alcohol.: Clin. Exp. Res. 2001, 25, 434–443. (19) Korbut, O.; Buckova, M.; Labuda, J.; Grundler, P. Sensors 2003, 3, 1–10. (20) Oliveira-Brett, A. M.; Diculescu, V. C. Bioelectrochemistry 2004, 64, 133–141. (21) Lerman, L. S. Proc. Natl. Acad. Sci. U.S.A. 1963, 49, 94–102. (22) Lerman, L. S. J. Cell. Comp. Physiol. 1964, 64, 1–18. (23) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Plenum: New York, 2006. (24) Chaudhuri, S.; Basu, K.; Sengupta, B.; Banerjee, A.; Sengupta, P. K. Luminescence 2008, 23, 397–403. (25) Ahmed, M. S.; Ramesh, V.; Nagaraja, V.; Parish, J. H.; Hadi, S. M. Mutagenesis 1994, 9, 193–197. (26) Strandjord, A. J. G.; Barbara, P. F. J. Phys. Chem. 1985, 89, 2355–2361. (27) Sengupta, B.; Sengupta, P. K. Biochem. Biophys. Res. Commun. 2002, 299, 400–403. (28) Sengupta, B.; Sengupta, P. K. Biopolymers 2003, 72, 427–434. (29) Das, P.; Chakrabarty, A.; Haldar, B.; Mallick, A.; Chattopadhyay, N. J. Phys. Chem. B 2007, 111, 7401–7408. (30) Chakrabarty, A.; Mallick, A.; Haldar, B.; Das, P.; Chattopadhyay, N. Biomacromolecules 2007, 8, 920–927. (31) Das, P.; Chakrabarty, A.; Mallick, A.; Chattopadhyay, N. J. Phys. Chem. B 2007, 111, 11169–11176. (32) Macgregor, R. B.; Weber, G. Nature 1986, 319, 70–73. (33) Bose, D.; Ghosh, D.; Das, P.; Girigoswami, A.; Sarkar, D.; Chattopadhyay, N. Chem. Phys. Lipids 2010, 163, 94–101. (34) Mahata, A.; Sarkar, D.; Bose, D.; Ghosh, D.; Girigoswami, A.; Das, P.; Chattopadhyay, N. J. Phys. Chem. B 2009, 113, 7517–7526. (35) Kosower, E. M.; Dodiuk, H.; Tanizawa, K.; Ottolenghi, M.; Orbach, N. J. Am. Chem. Soc. 1975, 97, 2167–2178. (36) Reichardt, C. Molecular Interactions; Ratajazak, H., Orville Thomas, W. J., Eds.; Wiley: New York, 1982; Vol. 3. (37) Chattopadhyay, N.; Serpa, C.; Pereira, M. M.; Seixas de Melo, J.; Arnaut, L. G.; Formosinho, S. J. J. Phys. Chem. A 2001, 105, 10025–10030. (38) Bose, D.; Sarkar, D.; Chattopadhyay, N. Photochem. Photobiol. 2010, 86, 538–544. (39) Mallick, A.; Chattopadhyay, N. Photochem. Photobiol. 2005, 81, 419–424. (40) Mallick, A.; Haldar, B.; Chattopadhyay, N. J. Phys. Chem. B 2005, 109, 14683–14690. (41) Lambert, B.; Lepecq, J. B. In DNA-Ligand Interactions, from Drugs to Proteins; Guschlbauer, W., Saenger, W., Eds.; Plenum: New York, 1986. (42) Hackl, E. V.; Blagoi, Y. P. J. Inorg. Biochem. 2004, 98, 1911–1920. 645

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