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Fluorescence Spectroscopic and Calorimetry Based Approaches to Characterize the Mode of Interaction of Small Molecules with DNA

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Abstract

Ethidium bromide displacement assay by fluorescence is frequently used as a diagnostic tool to identify the intercalation ability of DNA binding small molecules. Here we have demonstrated that the method has pitfalls. We have employed fluorescence, absorbance and label free technique such as isothermal titration calorimetry to probe the limitations. Ethidium bromide, a non-specific intercalator, netropsin, a (A-T) specific minor groove binder, and sanguinarine, a (G-C) specific intercalator, have been used in our experiments to study the association of a ligand with DNA in presence of a competing ligand. Here we have shown that netropsin quenches the fluorescence intensity of an equilibrium mixture of ethidium bromide - calf thymus DNA via displacement of ethidium bromide. Isothermal titration calorimetry results question the accepted interpretation of the observed decrease in fluorescence of bound ethidium bromide in terms of competitive binding of two ligands to DNA. Furthermore, isothermal titration calorimetry experiments and absorbance measurements indicate that the fluorescence change might be due to formation of ternary complex and not displacement of one ligand by another.

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Abbreviations

EtBr:

Ethidium bromide

ITC:

Isothermal titration calorimetry

Net:

Netropsin

ct DNA:

Calf thymus DNA

SGR:

Sanguinarine

References

  1. Das BB, Sen N, Roy A, Dasgupta SB, Ganguly A, Mohanta BC, Dinda B, Majumder HK (2006) Differential induction of Leishmania donovani bi-subunit topoisomerase I-DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I. Nucleic Acids Res 34(4):1121–1132

    Article  PubMed  CAS  Google Scholar 

  2. Ting CY, Hsu CT, Hsu HT, Su JS, Chen TY, Tarn WY, Kuo YH, Whang-Peng J, Liu LF, Hwang J (2003) Isodiospyrin as a novel human DNA topoisomerase I inhibitor. Biochem Pharmacol 66(10):1981–1991

    Article  PubMed  CAS  Google Scholar 

  3. Marverti G, Cusumano M, Ligabue A, Di Pietro ML, Vainiglia PA, Ferrari A, Bergomi M, Moruzzi MS, Frassineti C (2008) Studies on the anti-proliferative effects of novel DNA-intercalating bipyridyl-thiourea-Pt(II) complexes against cisplatin-sensitive and -resistant human ovarian cancer cells. J Inorg Biochem 102(4):699–712

    Article  PubMed  CAS  Google Scholar 

  4. Chowdhury AR, Sharma S, Mandal S, Goswami A, Mukhopadhyay S, Majumder HK (2002) Luteolin, an emerging anti-cancer flavonoid, poisons eukaryotic DNA topoisomerase I. Biochem J 366(Pt 2):653–661

    Article  PubMed  CAS  Google Scholar 

  5. Prescott TA, Sadler IH, Kiapranis R, Maciver SK (2007) Lunacridine from Lunasia amara is a DNA intercalating topoisomerase II inhibitor. J Ethnopharmacol 109(2):289–294

    Article  PubMed  CAS  Google Scholar 

  6. Ahmed MS, Ramesh V, Nagaraja V, Parish JH, Hadi SM (1994) Mode of binding of quercetin to DNA. Mutagenesis 9(3):193–197

    Article  PubMed  CAS  Google Scholar 

  7. Solimani R (1996) Quercetin and DNA in solution: analysis of the dynamics of their interaction with a linear dichroism study. Int J Biol Macromol 18(4):287–295

    Article  PubMed  CAS  Google Scholar 

  8. Yan H, Mizutani TC, Nomura N, Takakura T, Kitamura Y, Miura H, Nishizawa M, Tatsumi M, Yamamoto N, Sugiura W (2005) A novel small molecular weight compound with a carbazole structure that demonstrates potent human immunodeficiency virus type-1 integrase inhibitory activity. Antivir Chem Chemother 16(6):363–373

    PubMed  CAS  Google Scholar 

  9. Brotz-Oesterhelt H, Knezevic I, Bartel S, Lampe T, Warnecke-Eberz U, Ziegelbauer K, Habich D, Labischinski H (2003) Specific and potent inhibition of NAD + -dependent DNA ligase by pyridochromanones. J Biol Chem 278(41):39435–39442

    Article  PubMed  Google Scholar 

  10. Marshall KM, Andjelic CD, Tasdemir D, Concepcion GP, Ireland CM, Barrows LR (2009) Deoxyamphimedine, a pyridoacridine alkaloid, damages DNA via the production of reactive oxygen species. Mar Drugs 7(2):196–209

    Article  PubMed  CAS  Google Scholar 

  11. Jamalian A, Shafiee A, Hemmateenejad B, Khoshneviszadeh M, Madadkar-Sobhani A, Zahra Bathaie S, Akbar Moosavi-Movahedi A (2011) Novel imidazolyl derivatives of 1, 8-acridinedione as potential DNA-intercalating agents. J Iran Chem Soc 8(4):1098–1112

  12. Ahmadi F, Bakhshandeh F (2009) In vitro study of damaging effects of 2,4-dichlorophenoxyacetic acid on DNA structure by spectroscopic and voltammetric techniques. DNA Cell Biol 28(10):527–533

    Article  PubMed  CAS  Google Scholar 

  13. Bera R, Sahoo BK, Ghosh KS, Dasgupta S (2008) Studies on the interaction of isoxazolcurcumin with calf thymus DNA. Int J Biol Macromol 42(1):14–21

    Article  PubMed  CAS  Google Scholar 

  14. Fortune JM, Osheroff N (1998) Merbarone inhibits the catalytic activity of human topoisomerase IIalpha by blocking DNA cleavage. J Biol Chem 273(28):17643–17650

    Article  PubMed  CAS  Google Scholar 

  15. Rao KE, Dasgupta D, Sasisekharan V (1988) Interaction of synthetic analogues of distamycin and netropsin with nucleic acids. Does curvature of ligand play a role in distamycin-DNA interactions? Biochemistry 27(8):3018–3024

    Article  PubMed  CAS  Google Scholar 

  16. Baguley BC (1982) Nonintercalative DNA-binding antitumour compounds. Mol Cell Biochem 43(3):167–181

    Article  PubMed  CAS  Google Scholar 

  17. Lah J, Vesnaver G (2000) Binding of distamycin A and netropsin to the 12mer DNA duplexes containing mixed AT.GC sequences with at most five or three successive AT base pairs. Biochemistry 39(31):9317–9326

    Article  PubMed  CAS  Google Scholar 

  18. Luck G, Triebel H, Waring M, Zimmer C (1974) Conformation dependent binding of netropsin and distamycin to DNA and DNA model polymers. Nucleic Acids Res 1(3):503–530

    Article  PubMed  CAS  Google Scholar 

  19. Wartell RM, Larson JE, Wells RD (1974) Netropsin. A specific probe for A-T regions of duplex deoxyribonucleic acid. J Biol Chem 249(21):6719–6731

    PubMed  CAS  Google Scholar 

  20. Cosa G, Focsaneanu KS, McLean J, McNamee J, Scaiano J (2001) Photophysical properties of fluorescent DNA-dyes bound to single- and double-stranded DNA in aqueous buffered solution. Photochem Photobiol 73(6):585–599

    Article  PubMed  CAS  Google Scholar 

  21. Scaria PV, Shafer RH (1991) Binding of ethidium bromide to a DNA triple helix. Evidence for intercalation. J Biol Chem 266(9):5417–5423

    PubMed  CAS  Google Scholar 

  22. Liang D, Zhang J, Chu B (2003) Study of ethidium bromide effect on dsDNA separation by capillary zone electrophoresis and laser light scattering. Electrophoresis 24(19–20):3348–3355

    Article  PubMed  CAS  Google Scholar 

  23. Motulsky HJ, Neubig RR (2010) Analyzing binding data. Curr Protoc Neurosci Chapter 7:Unit 7 5. doi:10.1002/0471142301

  24. Taquet A, Labarbe R, Houssier C (1998) Calorimetric investigation of ethidium and netropsin binding to chicken erythrocyte chromatin. Biochemistry 37(25):9119–9126

    Article  PubMed  CAS  Google Scholar 

  25. Adhikari A, Hossain M, Maiti M, Suresh Kumar G (2008) Energetics of the binding of phototoxic and cytotoxic plant alkaloid sanguinarine to DNA: isothermal titration calorimetric studies. J Mol Struct 889(1):54–63

    Article  CAS  Google Scholar 

  26. Baguley BC, Falkenhaug EM (1978) The interaction of ethidium with synthetic double-stranded polynucleotides at low ionic strength. Nucleic Acids Res 5(1):161–171

    Article  PubMed  CAS  Google Scholar 

  27. Reichmann M, Rice S, Thomas C, Doty P (1954) A further examination of the molecular weight and size of desoxypentose nucleic acid. J Am Chem Soc 76(11):3047–3053

    Article  CAS  Google Scholar 

  28. Waring MJ (1965) Complex formation between ethidium bromide and nucleic acids. J Mol Biol 13(1):269–282

    Article  PubMed  CAS  Google Scholar 

  29. Selvi BR, Pradhan SK, Shandilya J, Das C, Sailaja BS, Shankar GN, Gadad SS, Reddy A, Dasgupta D, Kundu TK (2009) Sanguinarine interacts with chromatin, modulates epigenetic modifications, and transcription in the context of chromatin. Chem Biol 16(2):203–216

    Article  Google Scholar 

  30. Matthews J, Zacharewski T (2000) Differential binding affinities of PCBs, HO-PCBs, and aroclors with recombinant human, rainbow trout (Onchorhynkiss mykiss), and green anole (Anolis carolinensis) estrogen receptors, using a semi-high throughput competitive binding assay. Toxicol Sci 53(2):326–339

    Article  PubMed  CAS  Google Scholar 

  31. Matthews J, Celius T, Halgren R, Zacharewski T (2000) Differential estrogen receptor binding of estrogenic substances: a species comparison. J Steroid Biochem Mol Biol 74(4):223–234

    Article  PubMed  CAS  Google Scholar 

  32. Weiss JM, Andersson PL, Lamoree MH, Leonards PE, van Leeuwen SP, Hamers T (2009) Competitive binding of poly- and perfluorinated compounds to the thyroid hormone transport protein transthyretin. Toxicol Sci 109(2):206–216

    Article  PubMed  CAS  Google Scholar 

  33. Sigurskjold BW (2000) Exact analysis of competition ligand binding by displacement isothermal titration calorimetry. Anal Biochem 277(2):260–266

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was funded by CBAUNP project of Intramural Funding from Department of Atomic Energy, Govt. of India. Jasdeep Singh did this work as a part of his project work in the M.S.(Pharm.), Pharmacoinformatics program of NIPER-Kolkata, Govt. of India.

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Authors and Affiliations

  1. Biophysics Division, Saha Institute of Nuclear Physics, Block-AF, Sector-I, Bidhan Nagar, Kolkata, 700 064, West Bengal, India

    Amrita Banerjee & Dipak Dasgupta

  2. Kusuma School of Biological Sciences, Indian Institute of Technology-Delhi, Hauz Khas, New Delhi, 110016, India

    Jasdeep Singh

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  1. Amrita Banerjee
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Correspondence to Dipak Dasgupta.

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Banerjee, A., Singh, J. & Dasgupta, D. Fluorescence Spectroscopic and Calorimetry Based Approaches to Characterize the Mode of Interaction of Small Molecules with DNA. J Fluoresc 23, 745–752 (2013). https://doi.org/10.1007/s10895-013-1211-0

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