New Monomethine Cyanine Dye and Its Interaction with Different DNA Forms†

doi:10.7503/cjcu20140562

Abstract

Abstract:

Cyanine dyes have been widely used as fluorescent probes for nucleic acids. A well-known monomethine cyanine dye, thiazole orange(TO), has been reported to bind various forms of nucleic acids, such as RNA, duplex DNA, triplex DNA and especially G-quadruplex DNA. As a nucleic acid light-up probe, TO has been used in a variety of applications. The binding modes between TO and G-quadruplex DNA are complex, may include end-stacking, groove binding and external stacking, the predominant binding mode is reported to be end-stacking. In order to further understand the interaction of monomethine cyanines with diffe-rent nucleic acid forms and develop new probes, we synthesized a new analogue of TO, 1,2-dimethyl-6-{[3-methylbenzo[d]thiazol-2(3H)-ylidene]methyl}pyridine-1-ium(MTP) and investigated its interaction with different DNA forms. The interaction of MTP with c-myc(parallel G-quadruplex), 22AGK⁺(mixed-type G-quadruplex), ss/dsDNA(single/double-stranded DNA) caused notable red-shift and hypochromicity of the absorption spectrum of MTP. MTP exhibited almost no fluorescence in aqueous buffer condition. However, the interaction of MTP with DNA resulted in great enhancement of MTP fluorescence, approximately 130—180-fold for c-myc or 22AGK⁺, 40—60-fold for single/double-stranded(ss/ds) DNA and 15—25-fold for TBA and 22AGNa⁺(antiparallel G-quadruplexes). The apparent dissociation constants(K_d) of MTP and different DNA were in the range of 4.0—17 mmol/L. The fluorescence enhancement was corrected with the binding affinity of MTP and different DNA forms. These results suggest that MTP can be used as a fluorescent probe to distinguish different forms of nucleic acids. The binding stoichiometry showed that two molecules of MTP bound to one molecule of c-myc or 22AGK⁺. The induced CD spectroscopy and G-quadruplex/hemin peroxidase inhibition experiment suggested that the first MTP bound to c-myc through the groove binding model and the second MTP bound to c-myc through the end-stacking model. Compared with TO, MTP has a smaller size; it showed different absorption spectral changes upon binding to DNA and exhibited different fluorescence responses to 22AGK⁺ and 22AGNa⁺, which may suggest the different binding modes of TO and MTP to G-quadruplexes. These results provide important information for the design of the ligands and probes for nucleic acids.

Key words: Cyanine dye, DNA, G-quadruplex, Fluorescent probe, Nucleic acid ligand

CLC Number:

O652.3

TrendMD:

WU Shangrong, JIN Bing, ZHANG Nan, LIU Ying, LIU Xiangjun, LI Songqing, SHANGGUAN Dihua. New Monomethine Cyanine Dye and Its Interaction with Different DNA Forms^†[J]. Chem. J. Chinese Universities, 2014, 35(10): 2085.

Figures/Tables 7

Scheme 1 Synthetic routes of MTP

Table 1 Binding stoichiometry [putative number of binding sites on a given DNA(n)] and apparent dissociation constants(Kd) of the DNA used in this work

DNA	Sequence	n	K_d/(μmol·L^-1)
22AGK⁺	AGGGTTAGGGTTAGGGTTAGGG	2	4.5±0.1
c-myc	TGAGGGTGGGGAGGGTGGGGAA	2	4.3±0.2
22AGNa⁺	AGGGTTAGGGTTAGGGTTAGGG	1	12.3±0.4
TBA	GGTTGGTGTGGTTGG	1	16.7±1.4
ds26	CAATCGGATCGAATTCGATCCGATTG	2	8.3±0.2
ss2	GGGTTACTACGAACTGG	2	6.6±0.7

Fig.1 Absorption spectra of 5 μmol/L MTP in the presence of 5 μmol/L different kinds of DNA(A) and 3 μmol/L MTP in the presence of increasing amounts of c-myc(B) (A) a. Without DNA; b. TBA; c. 22AGNa+; d. 22AGK+; e. c-myc; f. ds26; g. ss2.(B) Concentration of c-myc(μmol·L-1) from a to f: 0, 1.0, 3.0, 6.0, 9.0, 12.

Fig.2 Fluorometric titration of MTP(2 μmol/L) with different concentrations of c-myc(A) and fluorometric titration curves of MTP(2 μmol/L) with different DNA(B) (A) Concentration of c-myc(μmol·L-1) from a to l: 0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 7.0, 10; λex=430 nm. (B) a. TBA; b. 22AGNa+; c. ss2; d. ds26; e. 22AGK+; f. c-myc; λex=430 nm, λem=503 nm.

Fig.3 Job’s plot obtained from fluorometric analysis of mixtures of MTP with c-myc(A) and 22AGK+(B) cMTP+cDNA=4 μmol/L; Xaxis=molar fraction of quadruplex DNA; λex=430 nm, λem=503 nm.

Fig.4 CD spectra of different quadruplex DNA(5 μmol/L) in the absence(a) and presence of MTP(b,c) (A) c-myc; (B) 22AGK+; (C) 22AGNa+; (D) TBA. a. DNA only; b. n(DNA)∶n(MTP)=1∶1; c. n(DNA)∶n(MTP)=1∶2.

Fig.5 Inhibition activity of MTP on the peroxidase activity of G-quadruplex/hemin complex The curves represent the change of the product concentration(absorbance at 415 nm) of G-quadruplex/hemin peroxidase within 30 min. The G-quadruplex/hemin peroxidase reaction in the absence of MTP was used as control. c(ABTS)=0.4 mmol/L; c(H2O2)=0.4 mmol/L. a. 1 μmol/L c-myc+1 μmol/L Hemin; b. 1 μmol/L c-myc+2 μmol/L MTP+1 μmol/L Hemin; c. 1 μmol/L c-myc+10 μmol/L MTP+1 μmol/L Hemin.

References 32

[1]	Chen X. Y., Peng X. J., Chemistry,2004, 67(9), 1—9
	(陈秀英, 彭孝军. 化学通报, 2004, 67(9), 1—9)
[2]	Ma D. L., He H. Z., Leung K. H., Zhong H. J., Chan D. S. H., Leung C. H., Chem. Soc. Rev., 2013, 42, 3427—3440
[3]	Peng X. J., Wu T., Fan J. L., Wang J. Y., Zhang S., Song F. L., Sun S. G., Angew. Chem. Int. Ed., 2011, 50(18), 4180—4183
[4]	Yang Q.F., Xiang J. F., Yang S., Zhou Q. J., Li Q., Tang Y. L., Xu G. Z.,Chem. Commun., 2009, (9), 1103—1105
[5]	Yang Q. F., Xiang J. F., Yang S., Li Q., Zhou Q. J., Guan A. J., Zhang X. F., Zhang H., Tang Y. L., Xu G. Z., Nucleic Acids Res., 2010, 38(3), 1022—1033
[6]	Lubitz I., Zikich D., Kotlyar A., Biochemistry,2010, 49(17), 3567—3574
[7]	Zheng A. H., Zhu Q., Xiang D. S., He Z. K., Chin. J. Anal. Chem., 2013, 41(3), 325—329
	(郑爱华, 朱庆, 向东山, 何治轲. 分析化学, 2013, 41(3), 325—329)
[8]	Fei X. N., Liu L. J., Zhang B. L., Shi B. J., Yao K. D., Prog. Chem., 2006, 18(6), 801—807
	(费学宁, 刘丽娟, 张宝莲, 石博杰, 姚康德. 化学进展, 2006, 18(6), 801—807)
[9]	Chen X. Y., Guo L., Zheng C. G., Gao H. Y., Wang H. J., Zhang D., Chin. J. Org. Chem., 2012, 32(8), 1445—1449
	(陈秀英, 郭琳, 郑昌戈, 高海燕, 王海军, 张丹. 有机化学, 2012, 32(8), 1445—1449)
[10]	Jin B., Zhang X., Zheng W., Liu X. J., Qi C., Wang F. Y., Shangguan D. H., Anal. Chem., 2014, 86(1), 943—952
[11]	Rye H. S., Quesada M. A., Peck K., Mathies R. A., GIazer A. N., Nucleic Acids Res., 1991, 19(2), 327—333
[12]	Largy E., Hamon F., Teulade-Fichou M. P., Anal. Bioanal. Chem., 2011, 400, 3419
[13]	Monchaud D., Allain C., Teulade-Fichou M. P., Nucleosides Nucleotides Nucleic Acids,2007, 26(10), 1585—1588
[14]	Monchaud D., Allain C., Bertrand H., Smargiasso N., Rosu F., Gabelica V., De C. A., Mergny J. L., Teulade-Fichou M. P., Biochimie,2008. 90(8), 1207—1223
[15]	Largy E., Saettel N., Hamon F., Dubruille S., Teulade-Fichou M. P., Curr. Pharm. Des., 2012. 18(14), 1992—2001
[16]	Allain C., Monchaud D., Teulade-Fichou M. P., J. Am. Chem. Soc., 2006, 128(36), 11890—11893
[17]	Yanga F., Xu X. L., Gong Y. H., Qiu W. W., Sun Z. R., Zhou J. W., Audebert P., Tang J., Tetrahedron., 2007, 63(37), 9188—9194
[18]	Coetzee J., Cronje S., Dobrzanska L., Raubenheimer H. G., Joone G., Nell M. J., Hoppe H. C., Dalton Trans. ,2011, 40(7), 1471—1483
[19]	Karlsson H. J., Lincoln P., Westman G., Bioorganic & Medicinal Chemistry,2003, 11(6), 1035—1040
[20]	Cian A. D., Guittat L., Kaiser M., Sacca B., Amrane S., Bourdoncle A., Alberti P., Teulade F. M. P., Lacroix L., Mergny J. L., Methods,2007, 42(2), 183—195
[21]	Cheng X., Liu X., Bing T., Zhao R., Xiong S., Shangguan D. H., Biopolymers,2009, 91(10), 874—883
[22]	Martino L., Virno A., Randazzo A., Virgilio A., Esposito V., Giancola C., Bucci M., Cirino G., Mayol L., Nucleic Acids Res., 2006, 34(22), 6653—6662
[23]	He Y. J., Neumann R. D., Panyutin I. G., Nucleic Acids Res., 2004, 32(18), 5359—5367
[24]	Ambrus A., Chen D., Dai J. X., Bialis T., Jones R. A., Yang D. Z., Nucleic Acids Res., 2006, 34(9), 2723—2735
[25]	Dai J. X., Carver M., Hurley L. H., Yang D. Z., J. Am. Chem. Soc., 2011, 133(44), 17673—17680
[26]	Su J. Q., Chi B. R., Li X., Liu L., Liu L. M., Qi Y. X., Wang Z. Y., Jin N. Y., Chem. Res. Chinese Universities,2012, 28(3), 465—471
[27]	Ma Y., Huang Z. S., Chem. J. Chinese Universities,2012, 33(10), 2217—2222
	(马彦, 黄志纾. 高等学校化学学报, 2012, 33(10), 2217—2222)
[28]	Burge S., Parkinson G. N., Hazel P., Todd A. K., Neidle S., Nucleic Acids Res., 2006, 34(19), 5402—5415
[29]	Yang X. J., Fang C. L., Mei H. C., Chang T. J., Cao Z. H., Shangguan D. H., Chem. Eur. J., 2011, 51(17), 14475—14484
[30]	Gao N., Wang Y. B., Wei C. Y., Chem. Res. Chinese Universities,2014, 30(3), 495—499
[31]	Boer D. R., Canals A., Coll M., Dalton Trans,2009, 21(3) 399—414
[32]	Cheng X. H., Liu X. J., Bing T., Cao Z. H., Biochem. ,2009, 48(33), 7817—7823