studies on intramolecular charge transfer fluorescence probe and dna binding characteristics

Studies on Intramolecular Charge Transfer Fluorescence Probeand DNA Binding Characteristics

Xin Yang, Guo-Li Shen, and Ru-Qin Yu1

Institute for Chemometrics and Chemical Sensing Technology, College of Chemistry andChemical Engineering, Hunan University, Changsha 410082, People’s Republic of China

Received February 3, 1999; accepted May 4, 1999

An intramolecular charge transfer fluorescence probe of 49-N,N-dimethylamino-4-amino-chal-cone(DMAC) exhibits characteristics clearly correlated with the polarity of solvents. The interactionof this fluorescence probe with calf thymus DNA has been investigated. Generally, DMAC bound toDNA shows marked changes in fluorescence and absorbance properties compared to the spectralcharacteristics of the free form in solution phase. In the presence of DNA the fluorescence intensityof DMAC is greatly increased with a large bathochromic shift of excitation and emission wave-lengths. A hypochromism in absorption spectrum was also observed. The absorption and fluorescencespectra, salt concentration effect, and KI quenching experiments demonstrate that DMAC moleculeas an intercalator is inserted into the base-stacking domain of DNA double helix, and the interactionof the nucleobases with DMAC molecule causes the increase of fluorescence intensity and hypo-chromism in absorption spectrum. The intrinsic binding constant and the binding site number wereestimated to be 7.043 106 mol L21 in base pairs and 0.065, respectively. TheI /I 0 vs DNAconcentration plot shows a linear range covering 1.983 1026 to 2.083 1024 mol L21 in base pairswhich can be used for determining DNA with a detection limit of 6.03 1027 mol L21 in base pairs(0.6 mg ml21). © 1999 Academic Press

INTRODUCTION

The fluorescence of native DNA is too weak to be measured directly; its analyticalapplication is therefore limited (1). Recent years have seen growing interest in theemployment of small molecules for DNA studies (2–5). Some small molecules, such asacridines, can be used as biological probes; they are also potential parent compounds foranticancer drugs acting on DNA as the target. Ethidium bromide (EB) is one of the mostsensitive fluorescence probes with the binding characteristic reported in detail (6, 7). Inaqueous solution the fluorescence quantum yield of EB is rather small. While binding toDNA it can engender enhanced fluorescence with significant bathochromic shift of theexcitation wavelength. Corinet al. (8) showed that the delayed luminescence spectrum ofproflavin is a very useful tool in exploring dynamic interaction between a small moleculeand DNA. Kumaret al. (9, 10) reported that (9-anthrylmethyl) ammonium chloride(AMAC) can bind to DNA with a relatively high affinity. Once binding to DNA, thefluorescence of AMAC will be quenched seriously by DNA base pairs. Furthermore, thisprocess has the characteristics of strong site selectivity. Zhao (11) and Guo (12) studiedthe phenomenon of fluorescence self-quenching of probe and monomer–dimer equilib-rium using DNA. Braunset al. (13) investigated local dynamics of acridine orange (AO)

1 To whom correspondence should be addressed.

Microchemical Journal62, 394–404 (1999)Article ID mchj.1999.1747, available online at http://www.idealibrary.com on

0026-265X/99 $30.00Copyright © 1999 by Academic PressAll rights of reproduction in any form reserved.

394

in DNA solution. They concluded that the Stokes shift of AO results from the change ofeffective cavity size of the dye caused by the movements of neighboring groups.

Now, some new fluorescence probes with higher sensitivity and better selectivity findapplications in analytical practice. Because of its sensitivity to microenvironment condi-tions, intramolecular charge transfer (ICT) fluorescence attracted more and more attentionin biological and biomedical studies (14, 15). The molecules with ICT fluorescenceusually contain not only electron donor but also electron acceptor groups. Under certainconditions they can generate ICT or twisted intramolecular charge transfer (TICT)fluorescence emission (16). Up to the date, ICT compound has rarely applied as afluorescence probe to determination of DNA. This paper reports the investigation of theinteraction of ICT fluorescence probe 49-N,N-dimethylamino-4-amino-chalcone (DMAC)with DNA in aqueous solutions and the effect of various experimental parameters.

EXPERIMENTAL

Reagents

DMAC was synthesized and purified as previously reported (17). 14.5 g ofp-amino-acetophenone was dissolved in 40 mL of absolute ethyl alcohol. A 50 mL of aqueoussolution containing 5.0 g of NaOH was added to this solution. Thenp-dimethylamino-benzaldehyde solution (16 g ofp-dimethylaminobenzaldehyde in 60 mL of absolute ethylalcohol) was added dropwise. The mixture was conditioned at 25–30°C for 20 h withstirring. The product was precipitated as an orange yellow solid. Water (50 mL) was addedto this mixture, which was put into refrigerator and kept overnight. The filtered precipitatewas washed with water to neutral reaction, dried, and recrystallized from toluene. Thedetected mass spectral peaks were M/e (% relative intensity) 266 (162), 250 (13), 237(19), 222 (17), 174 (28), 146 (26), 121 (100), 92 (67), 77 (23), 65 (85). IR spectra were1600 cm21 (–CAO–); 2906, 2870 cm21 (–CH3–); 3431 cm21 (–NH2–). Stock solution of1023 mol L21 for DMAC was prepared in acetone. Calf thymus DNA was purchased fromHuamei Biotechnological Co. (Beijing) and used as received, the purity checked by theabsorbance ratioA260/A280 of DNA which should be not less than 1.8 in 0.2 mol L21

phosphate buffer solution containing 60 mmol L21 NaCl. DNA concentration, expressedin base pairs, was estimated by spectrophotometry using a molar absorptivity valuee260 of1.313 104 mol L21 cm21 (18). Other chemicals were of analytical reagent grade. Doublydistilled water was used throughout.

Apparatus

All fluorescence measurements were carried out with an M850 fluorescence spectro-photometer (Hitachi, Japan) with polarization attachment assembly. The excitation andemission wavelengths of 474 and 568 nm were used, respectively. The absorption spectrawere recorded with a UV-1100 spectrophotometer (Rayleigh Analytical Instrument Cor-poration, Beijing).

Procedure

All experiments, except where specifically indicated, were carried out at pH 4.29 in aBritton–Robinson buffer containing NaCl (60 mmol L21) and appropriate amount ofDMAC. All test solutions were incubated at 25°C for 10 min. Fluorescence experiments

395INTRAMOLECULAR CHARGE TRANSFER FLUORESCENCE

were performed by titration. The pH values of buffers covering 1.84–11.95 were mea-sured using a Model PHS-3 ion meter (Shanghai). A Model CS501 thermostat(Chongqing) was used for controlling temperature.

RESULTS AND DISCUSSION

Spectral Characteristics of DMAC in Various Solvents

The excitation and emission wavelengths of DMAC in a variety of solvents aresummarized in Table 1. One can see that the maximum excitation and emission wave-lengths exhibit a significant bathochromic shift with increasing polarity of solvent. Sucha phenomenon may be attributed to a large change in the dipole moment of DMAC in theground and excited electronic states. It seems that the spectra are sensitive to local electricfield generated by the environment. The influence of solvent polarity on the Stokes shiftof luminescence molecule can be expressed by the Lippert equation (19)

Dn 5 na 2 n f 52

hcS e 2 1

2e 1 12

n2 2 1

2n2 1 1D ~m* 2 m! 2

a2 1 Constant. (1)

Here,Dn denotes Stokes shift;na andn f are absorption and emission wavelengths (cm21),respectively;h represents Planck’s constant;c designates light speed;e is the solventdielectric constant;n is the index of refraction;a is the radius residence cavity;m* andm are excited and ground state dipole moments, respectively. (e 2 1)/(2e 1 1) representsthe spectral shift produced by two factors, i.e., the solvent dipole reorientation andelectron redistribution in solvent molecules. (n2 2 1)/(2n2 1 1) expresses the spectralshift generated purely by electron redistribution. The difference between these two itemsrepresents the spectral shift caused by solvent molecular reorientation as shown in Fig. 1.

DMAC possesses a remarkable intramolecular charge transfer resulting in the signifi-cant molecular polarization in excited state, since it contains both electron donor groupssuch as dimethylamino group and electron acceptors such as enone group.

TABLE 1Fluorescence Spectral Properties of DMAC in Various Solvents

Solvent lex (nm) lem (nm) Dn (cm21)a eb nc

Water 412 498 4192 80.10 1.3330Water (DNA) 474 568 3491Dimethylformamide 416 510 4431 36.71 1.4305Methanol 428 550 5183 32.70 1.3288Ethanol 426 544 5092 24.55 1.3611Acetone 408 496 4349 20.70 1.3588Ethyl acetate 400 482 4253 6.02 1.3723Toluene 404 460 3013 2.568 1.4961

a Stokes shift.b Dielectric constant.c Index of refraction.

396 YANG, SHEN, AND YU

Continuous bathochromic shift of the excitation spectrum of DMAC was observed withthe addition of DNA. A relatively small hypsochromic shift was exhibited in the emissionspectrum first. A further increase in the DNA concentration could produce a continuousbathochromic shift with the emission spectrum. As a result, the Stokes shift of DMAC inDNA is significantly smaller than that in aqueous solution. In aqueous solution, thefluorescence intensity of DMAC is very weak. Once DNA was introduced, its fluores-cence intensity is enhanced significantly.

Figure 2 shows the fluorescence excitation and emission spectra for the free and boundDMAC in the absence and presence of different amounts of DNA. Upon binding of theDMAC probe to the DNA double helix, the fluorescence of DMAC was found to besignificantly enhanced with first a blue shift and then a red shift of emission peak whilethe concentration of DNA increases. It indicates that DMAC enters DNA-stacking regionwith a lower polarity comparing to that of the bulk solution of DNA.

Absorbance Measurements

The spectral characteristics associated with the binding of DMAC to DNA are revealedin Fig. 3. DNA may cause a progressive shift in absorption spectrum of DMAC, and itsmaximum absorption shifts from 423 to 440 nm. A clear isosbestic point was located at460 nm, indicating the existence of two forms of DMAC, i.e., free and bound ones. Eachform of DMAC has a unique absorption. The pronounced hypochromism was alsoobserved, which indicates a strong intercalation of the DMAC molecule into DNA basepairs (20–23).

FIG. 1. Excitation maxima (F) and stokes shift (E) of DMAC in various solvents vs a polarizability scalerepresented by a function of the index of refractionn.


Influence of Denaturation of DNA

Denatured DNA was produced by heating a native DNA solution in Britton–Robinsonbuffer (pH 4.29) in a water bath at 100°C for 10 min, followed by a rapid cooling in anice bath to 0°C before being brought back to room temperature. DNA split into twostring-like softer polynucleotide chains from original rigid double-helix structure. Thecharacteristic of DMAC bound to native and denatured DNA was presented in Fig. 4. Ascan be seen from this figure, DMAC was bound to denatured DNA in the same way as thatobserved in the native DNA. The difference lies in the degree of increment in thefluorescence. Highly organized double helix structure of native DNA seems not to be thenecessary condition for the interaction with DMAC.

Effect of pH

Figure 5 shows the variation of fluorescence intensity with pH of solution. Surprisingly,fluorescence intensity of DMAC solution changes only slightly when pH value varies.However, once DNA was loaded, a relatively small change in fluorescence intensity wasobserved at pH lower than 3 or higher than 5. A drastic change in fluorescence intensitywas observed at pH 4.29. From the structure of DMAC (see Scheme 1), one might assumethat the DMAC molecule possesses a single positive charge at such a pH due to theprotonation of nitrogen at position 49 forming a positively charged ion, which interactswith the negatively charged DNA phosphate backbone and leads to the increase influorescence intensity.

FIG. 2. The fluorescence excitation (Ex: 1–7) and emission(Em: 19–79) spectra of DMAC. Curve 1, freeDMAC; curve 2–7, DMAC bound with increasing concentration of DNA (mol L21) (2, 1.133 1026; 3, 3.9731026; 4, 8.503 1026; 5, 1.423 1025; 6, 2.273 1025; 7, 3.123 1025).


Effect of Salt Concentration

The interaction between DMAC and DNA is sensitive to various salts and theirconcentration. The effect of various salts is illustrated in Fig. 6. In the presence of cationsthe fluorescence intensity due to the addition of DNA decreases. As mentioned above, atpH 4.29 DMAC is supposed to have a protonated form. One would expect the cationscould compete with DMAC to bind to DNA sites. The ability of divalent cations such asCa21 and Mg21 to decrease the fluorescence increment of DMAC is much stronger thanthat of univalent such as Na1 and K1 (Fig. 6a).

Figure 6b shows the change in the ratio of the fluorescence of DMAC in the absenceof DNA ( I 0) and in the presence of different amounts of DNA (I ) at different concen-trations of NaCl. The effect of NaCl concentration on theI /I 0 value is obvious. With theincrease in the concentration of Na1, on one hand, the electrostatic repelling interactionamong the negative charges of the DNA phosphate backbone decreases. The DNA doublehelix would hold more tightly. Small molecules are not easy to intercalate to the bindingsites in base pairs of DNA. On the other hand, Na1 and DMAC compete to bind to DNAsites. Therefore, more intercalated DMAC molecules dissociate from DNA double helixinto the solution, which can be led to the ratio (I /I 0) of the fluorescence of DMACdecrease.

Effect of Temperature

The fluorescence anisotropy of DMAC interaction with DNA was measured in thetemperature range from 15 to 60°C. The fluorescence anisotropy (A) is often expressedin terms of the ratio of two intensities (24)

FIG. 3. Absorption spectra of DMAC with addition of DNA. Left to the isosbestic point of 460nm: top curvewithout addition of DNA. From second curve to bottom: with increasing amounts of DNA.


FIG. 4. Fluorescence increment (I–I 0) as a function of native and denatured DNA concentration.I 0, Thefluorescence intensity of DMAC in the absence of DNA.I , The fluorescence intensity of DMAC in the presenceof native or denatured DNA.

FIG. 5. Fluorescence intensity as a function of pH.


A 5 ~I vv 2 I vhG!/~I vv 1 2I vhG!, (2)

where I vv and I vh are the fluorescence intensities measured through vertically and hori-zontally oriented polarizers in the excitation beam and emission beam, respectively. Theinstrumental correction factor,G, is equal toI hv/I hh. The result demonstrated in Fig. 7shows that anisotropy increases with an increase in temperature. This phenomenon seemsanomalous as anisotropy should decrease with an increase in temperature, due to fasterrotation, decreased viscosity, and decreased excited state lifetime (25). The mechanism ofthis process needs further investigation.

KI Quenching

To deduce the interaction pattern of the fluorescence probe DMAC with DNA, the KIquenching experiments have been performed. KI with concentration ranging from 0 to0.05 mol L21 was added to DMAC–DNA solutions. Iodide ion is known to be a dynamicor collisional fluorescence quencher. Stern–VolmerK sv estimated can be used to evaluatethe fluorescence quenching efficiency. The interaction pattern of the fluorescence probewith DNA can be deduced from the variation of theK sv with the experimental conditions.When the fluorescence probe intercalates to DNA base pairs, owing to the electrostaticrepelling interaction between DNA phosphate backbone with negative charges and anions,the fluorescence probe is effectively protected, leading to a decrease in quenching. Inaqueous solution, the fluorescence intensity of DMAC is too weak; KI quenching behavioris rather difficult to be measured. With 1.583 1025 mol L21 DNA added to the solution,theK sv was determined to be 200.7 and 222.4 L mol21 at 10 and 25°C, respectively. Whenthe concentration of DNA increases to 8.03 1025 mol L21, the corresponding values ofK sv are 148.7 and 165.7 L mol21 at these two temperatures, respectively. At a fixedtemperature, the increase in the concentration of DNA leads to a decrease in the quenchingconstantK sv. This phenomenon is due to the fact that the collision quenching of theDMAC fluorescence by I2 ions decreases in the presence of high-concentration DNA. Onthe contrary, in the presence of a fixed concentration of DNA, quenching constantincreases with the rise of temperature. Therefore, the interaction pattern of DMAC withDNA should be an intercalation mode (9).

The Intrinsic Binding Constant and Binding Site Number

According to the well-known Scatchard equation [26],

r /c 5 K~n 2 r !, (3)

SCHEME 1


FIG. 6. (a) The fluorescence intensity as a function of salt concentration. (b) The ratio of fluorescenceintensities (I /I 0) at different NaCl concentration as a function of DNA concentration.I 0, The fluorescenceintensity at different salt concentration in the absence of DNA.I , The fluorescence intensity at different saltconcentration with increasing DNA concentration.


wherer is the ratio of bound probe per base pair,n is the binding site number per basepair,K is the intrinsic binding constant, andc is the free probe concentration, the intrinsicbinding constant and the binding site number were calculated by the recorded fluorescencetitration data. The intrinsic binding constant was 7.043 106 mol L21 in base pairs and thebinding site number was about 0.065.

The Analytical Characteristics

The fluorescence system reported here was used to determine traces of DNA rangingfrom 1.983 1026 to 2.083 1024 mol L21 with a detection limit of 6.03 1027 mol L21

in base pairs (0.6mg mL21). This method exhibits good reproducibility, with a relativestandard deviation of 1.21% obtained from eight separate determinations for 4.03 1026

mol L21 DNA.

Conclusion

The fluorescence probe of DMAC, with intramolecular charge transfer characteristics,is sensitive to microenvironment property, and it has been demonstrated to be a valuabletool for exploring interaction of small molecules with DNA, exhibiting good reproduc-ibility. Further work is necessary for a more basic understanding of the mechanism of theircomplex action.

ACKNOWLEDGMENTS

This work was supported by National Natural Science Foundation and the Foundation for TechnologicalDevelopment of Machinery Industry and Science Commission of Hunan Province.

FIG. 7. Fluorescence anisotropy as a function of temperature.


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studies on intramolecular charge transfer fluorescence probe and dna binding characteristics

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