Biopolym. Cell. 2007; 23(6):529-537.
Molecular Biophysics
Spectrophotometrical study of mechanisms of cytidine analogues and ethidium bromide binding with DNA
1, 2Iermak Ie. L., 2Kruglova O. B., 3Palchykovska L. H., 3Alexeeva I. V.
  1. Kharkiv National University
    4, Svobody Ave., Kharkiv, Ukraine, 61077
  2. A. Usikov Institute of Radio Physics and Electronics, NAS of Ukraine
    12, Proskura Str., Kharkov, Ukraine, 61085
  3. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680


To study the mechanisms of cytidine and its biologically active analogues binding to DNA we analyzed the binding of these ligands to the DNA in the presence of well-known intercalator ethidium bromide (EtBr). Thereto, we carried out the detailed spectrophotometric research of EtBr-DNA mixtures absorption in the presence of cytidine and its analogues in the wide range of wavelengths and DNA concentrations. Cytidine derivatives containing azagroup in the cytosine ring (6AZC, AZAfur, and AZAxyl) compete with EtBr for the DNA binding sites. The binding constants and binding site sizes of the ligand-DNA complexes were calculated via absorption spectra optimization programs COMP and DALSMOD. Unmodified in cytosine ring ligands (cytidine and Ara-C) do not compete with EtBr for the DNA binding sites, however they contribute to the change of concentration dependencies of titration curves in the region of low DNA concentrations.
Keywords: cytidine analogues, DNA, models of binding, spectrophotometry, ethidium bromide


[1] Grant S. Ara-C: cellular and molecular pharmacology. Adv Cancer Res. 1998;72:197-233.
[2] Grem JL, Shoemaker DD, Hoth DF, King SA, Plowman J, Zaharko D, Grieshaber CK, Harrison SD Jr, Cradock JC, Leyland-Jones B. Arabinosyl-5-azacytosine: a novel nucleoside entering clinical trials. Invest New Drugs. 1987;5(4):315-28.
[3] Hoelzer D, Ganser A, Anger B, Seifried E, Heimpel H. Low-dose Ara-C in the treatment of acute leukemia. Cytotoxicity or differentiation induction? Blut. 1984;48(4):233-8.
[4] Capizzi RL, Poole M, Cooper MR, Richards F 2nd, Stuart JJ, Jackson DV Jr, White DR, Spurr CL, Hopkins JO, Muss HB, et al. Treatment of poor risk acute leukemia with sequential high-dose ARA-C and asparaginase. Blood. 1984;63(3):694-700.
[5] Hoshino J, Frahm J, Kr?ger H. Suppression of nuclear ADP-ribosyltransferase activity in Ehrlich ascites tumor cells by 5-azacytidine and its analogs. Biochem Biophys Res Commun. 1987;142(2):468-74.
[6] Oyelere AK, Kardon JR, Strobel SA. pK(a) perturbation in genomic Hepatitis Delta Virus ribozyme catalysis evidenced by nucleotide analogue interference mapping. Biochemistry. 2002;41(11):3667-75.
[7] Zhang X, Kiechle FL. Cytosine arabinoside substitution decreases transcription factor-DNA binding element complex formation. Arch Pathol Lab Med. 2004;128(12):1364-71.
[8] Estey E, Thall P, Beran M, Kantarjian H, Pierce S, Keating M. Effect of diagnosis (refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, or acute myeloid leukemia [AML]) on outcome of AML-type chemotherapy. Blood. 1997;90(8):2969-77.
[9] Nosach LN, Dyachenko NS, Shalamay AS, Alekseeva IV, Kushko LYa, Ozvinchuk II, Zhovnovataya VL, Butenko SL, Petrovskaya IA, Drannik GN. Antiadenovirus and immunostimulating actions of 6-azacylidine. Biopolym Cell. 1996; 12(1):75-85.
[10] Abdullaeva MV, Frolov AF, Alexeeva IV, Palchykovskaja LI, Fedorova N. E. Inhibitory effect of 6-azacytidine on human cytomegalovirus infection in cellular system. Biopolym Cell. 2004; 20(4):337-42.
[11] Galushko SV, Bulkina ZP, Petrusha NA, Shishkina IP Pharmacokinetics of 6-azacytidine. Pharmaceutical Chemistry Journal. 1986; 20(11):753-755.
[12] Palchikovs'ka LG, Garmanchouk LV, Alexeeva IV, Usenko LS, Shestakova TS, Solyanik GI, Shved AD, Chekhun VF. The N1-glycosilic analogues of 6-azacytidine. Cytotoxic effect and influence on transcription in vitro. Biopolym Cell. 2005; 21(5):433-9.
[13] Samijlenko SP, Alexeeva IV, Palchykivs'ka LH, Kondratyuk IV, Stepanyugin AV, Shalamay AS, Hovorun DM. 1H NMR investigation on 6-azacytidine and its derivatives. Spectrochim Acta - Part A: Mol Biomol Spectrosc. 1999; 55(5):1133-41.
[14] Palchikovska LH, Platonov MO, Alexeeva IV, Shved AD. Mechanism of 6-azacytosine N1-glycosides interaction with catalytic site of phage T7 DNA-dependent RNA-polymerase: model quantum-chemical study. Biopolym Cell. 2005; 21(6):559-61.
[15] M?ller W, Crothers DM. Interactions of heteroaromatic compounds with nucleic acids. 1. The influence of heteroatoms and polarizability on the base specificity of intercalating ligands. Eur J Biochem. 1975;54(1):267-77.
[16] Bresloff JL, Crothers DM. DNA-ethidium reaction kinetics: demonstration of direct ligand transfer between DNA binding sites. J Mol Biol. 1975;95(1):103-23.
[17] Nechipurenko IuD. [Cooperation effects in binding of large ligands to DNA. II. Contact interactions between adsorbed ligands]. Mol Biol (Mosk). 1984;18(4):1066-80.
[18] McGhee JD, von Hippel PH. Theoretical aspects of DNA-protein interactions: co-operative and non-co-operative binding of large ligands to a one-dimensional homogeneous lattice. J Mol Biol. 1974;86(2):469-89.
[19] Kruglova EB, Yermar EL. Competitive binding of two ligands: aktinotsinovogo derivative and caffeine polyribocytidylic acid in different conformational states. Vestnik Kharkovs Univ. Biofiz Vestnik. 2004; 1-2 (14):32-7.
[20] Kruglova EB. [Unusual shapes of absorption isotherms in three-component systems]. Biofizika. 2007;52(5):822-4.
[21] Kruglova EB, Gladkovskaia NA, Maleev VIa. [The use of the spectrophotometric analysis for the calculation of the thermodynamic parameters in actinocin derivative-DNA systems]. Biofizika. 2005;50(2):253-64.
[22] Maleev V, Semenov M, Kruglova E, Bolbukh T, Gasan A, Bereznyak E, et al. Spectroscopic and calorimetric study of DNA interaction with a new series of actinocin derivatives. J Mol Struct. 2003;645(2-3):145–58.
[23] Harley FR, Burgess C, Alcock RM. Solution equilibria. London: Ellis Horwood, 1980. 360 p.
[24] Vardevanyan PO, Antonyan AP, Parsadanyan MA, Davtyan HG, Karapetyan AT. The binding of ethidium bromide with DNA: interaction with single- and double-stranded structures. Exp Mol Med. 2003;35(6):527-33.
[25] Minasyan SH, Tavadyan LA, Antonyan AP, Davtyan HG, Parsadanyan MA, Vardevanyan PO. Differential pulse voltammetric studies of ethidium bromide binding to DNA. Bioelectrochemistry. 2006;68(1):48-55.
[26] Vardevanyan PO, Antonyan AP, Manukyan GA, Karapetyan AT. Study of ethidium bromide interaction peculiarities with DNA. Exp Mol Med. 2001;33(4):205-8.
[27] Benevides JM, Thomas GJ Jr. Local conformational changes induced in B-DNA by ethidium intercalation. Biochemistry. 2005;44(8):2993-9.
[28] Yuzaki K, Hamaguchi H. Intercalation-induced structural change of DNA as studied by 1064 nm near-infrared multichannel Raman spectroscopy. J Raman Spectrosc. 2004;35(12):1013–5
[29] Utsuno K, Tsuboi M, Katsumata S, Iwamoto T. Viewing of complex molecules of ethidium bromide and plasmid DNA in solution by atomic force microscopy. Chem Pharm Bull (Tokyo). 2001;49(4):413-7.
[30] Vladescu ID, McCauley MJ, Rouzina I, Williams MC. Mapping the phase diagram of single DNA molecule force-induced melting in the presence of ethidium. Phys Rev Lett. 2005;95(15):158102.
[31] Eckel R, Ros R, Ros A, Wilking SD, Sewald N, Anselmetti D. Identification of binding mechanisms in single molecule-DNA complexes. Biophys J. 2003;85(3):1968-73.
[32] Garbett NC, Hammond NB, Graves DE. Influence of the amino substituents in the interaction of ethidium bromide with DNA. Biophys J. 2004;87(6):3974-81.