一种不对称菁染料及其与不同结构DNA的相互作用

doi:10.7503/cjcu20140562

高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (10): 2085.doi: 10.7503/cjcu20140562

一种不对称菁染料及其与不同结构DNA的相互作用

吴尚荣^1,², 金冰², 张楠², 柳影², 刘祥军², 李松青¹(), 上官棣华²()

1. 湘潭大学化学学院, 湘潭 411100
2. 北京分子科学国家实验室, 中国科学院化学研究所活体分析化学院重点实验室, 北京 100190

收稿日期:2014-06-20 出版日期:2014-10-10 发布日期:2014-09-19
作者简介:联系人简介: 李松青, 男, 副教授, 主要从事光谱分析研究. E-mail: lisq65@126.com.上官棣华, 男, 博士, 研究员, 博士生导师, 主要从事生化分析研究. E-mail: sgdh@iccas.ac.cn
基金资助:
国家“九七三”计划项目(批准号: 2011CB935800, 2013CB933700)和国家自然科学基金(批准号: 21275149, 21375135, 21321003)资助

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

WU Shangrong^1,², JIN Bing², ZHANG Nan², LIU Ying², LIU Xiangjun², LI Songqing^1,^*(), SHANGGUAN Dihua^2,^*()

1. Colloge of Chemistry, Xiangtan University, Xiangtan 411100, China
2. Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems,Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China

Received:2014-06-20 Online:2014-10-10 Published:2014-09-19
Contact: LI Songqing,SHANGGUAN Dihua E-mail:lisq65@126.com;sgdh@iccas.ac.cn
Supported by:
Supported by the National Basic Research Program of China(Nos.2011CB935800, 2013CB933700) and the National Natural Science Foundation of China(Nos.21275149, 21375135, 21321003)

摘要/Abstract

摘要：

为考察小分子配基与不同核酸结构的结合机理, 发展新的核酸探针分子, 合成了一种新型一次甲基不对称菁染料(MTP). 吸收光谱、荧光光谱及圆二色光谱研究结果表明, MTP可与平行和混合平行G-四链体DNA(如c-myc和22AGK⁺)较强地结合, 并引起130~180倍的荧光增强; 其与单/双链DNA作用较弱, 导致40~60倍荧光增强; 而与反平行G-四链体DNA(如TBA和22AGNa⁺)的作用最弱, 只引起15~25倍荧光增强; 以上结果表明MTP可作为荧光探针分子用于区别不同结构的核酸分子. 结合机理研究表明, 平行和混合平行G-四链体DNA优先通过沟槽结合模式结合1分子MTP, 再通过末端堆积模式结合另1分子MTP.

关键词: 菁染料, 核酸, G-四链体, 荧光分子探针, 核酸配基

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

中图分类号:

O652.3

TrendMD:

吴尚荣, 金冰, 张楠, 柳影, 刘祥军, 李松青, 上官棣华. 一种不对称菁染料及其与不同结构DNA的相互作用. 高等学校化学学报, 2014, 35(10): 2085.

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^†. Chem. J. Chinese Universities, 2014, 35(10): 2085.

图/表 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.

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一种不对称菁染料及其与不同结构DNA的相互作用

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

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一种不对称菁染料及其与不同结构DNA的相互作用

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

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