interaction of calf thymus dna with a cationic tetrandrine derivative at the air-water interface

RESEARCHARTICLE

Copyright © 2008 American Scientific PublishersAll rights reservedPrinted in the United States of America

Journal ofBiomedical Nanotechnology

Vol. 4, 1–10, 2008

Interaction of Calf Thymus DNA with a CationicTetrandrine Derivative at the Air–Water Interface

Amparo Wong-Molina1, Karen O. Lara1, Mario Sanchez1, Marıa G. Burboa1�2,Luis E. Gutierrez-Millan1�2, Jose L. Marın3, and Miguel A. Valdez1�4�∗

1Departamento de Investigacion en Polímeros y Materiales, 2Departamento de Investigaciones Científicas y Tecnologicas,3Departamento de Investigacion en Física, and 4Departamento de Física,

Universidad de Sonora, Blvd. Luis Encinas y Rosales, Col. Centro, C.P. 83000, Hermosillo, Sonora, Mexico

The interaction of the semi-synthetic dicationic cyclophane-type macrocycle (TC) with calf thymusDNA was investigated at the air–water interface. The macrocycle has been prepared by chemi-cal modification of S,S-(+)-tetrandrine with acridinylmethyl groups. The macrocycle was mixed witharachidic acid in a chloroform solution (1:1, 3:1, 9:1 arachidic acid:macrocycle weight proportions)and spread on a DNA buffer solution in a Langmuir Balance. Results indicate isotherms shifts andphase modification of the arachidic acid/macrocycle mixture monolayers due to the DNA adsorp-tion from the subphase after different times. The presence of the arachidic acid at the interfacegives support to the macrocycle at the air–water interface, otherwise macrocycle molecules do notremain at the interface. Atomic force microscopy images of Langmuir-Blodgett monolayers obtainedon mica demonstrate the DNA adhesion on the macrocycle domains. The DNA adsorption pro-cess was also monitored by Brewster Angle Microscopy images. Circular Dichroism spectra ofdifferent DNA:macrocycle proportions were performed and show secondary structure changes ofDNA. UV spectrometric measurements indicated a strong DNA-macrocycle interaction and molec-ular mechanics simulations corroborate that the macrocycle interaction with DNA occurs mainly inthe major groove of the DNA molecule.

Keywords: DNA, Arachidic Acid, Tetrandrine, Langmuir-Blodgett Films, AFM, CD, BAM.

1. INTRODUCTION

The immobilization of DNA on different substrates hasbecome a subject of a great biological importance becauseof, among others, its relevance on the fabrication ofbiosensors1 and gene delivery.2 It is an emerging field withimportant medical and pharmaceutical applications.3�4 Dif-ferent supports for the immobilization have been used5–7

and recently the investigations on Langmuir monolayers8�9

have achieved great relevance. Due to the importance ofcationic lipids as vehicles for delivery of DNA,10 stud-ies on DNA immobilization at the air–water interfacehave strongly concentrated on electrostatic interactionswith cationic lipid monolayers.11�12 Other surfactantsand lipids13–16 have also used to support DNA at theair–water interface. Monolayers of binary phospholipidsand/or cationic lipids mixtures at the air water interfacehave also been considered to immobilize DNA from thesubphase.17–20 These binary lipid mixtures play an impor-tant role in the DNA cell transfection.19 Some authors have

∗Author to whom correspondence should be addressed.

found that immobilized polynucleotides on a thin film atthe air–water interface preserve the geometry and integrityof DNA molecules as demonstrated by X ray and neu-tron reflectometry techniques applied to liquid systems.21

The aggregation and kinetics of Immobilized DNA at theair–water interface have currently been visualized by fluo-rescence microscopy9 and by Brewster Angle Microscopy(BAM)12�13�22 and the corresponding Langmuir Blodgettfilms have been observed and measured by atomic forcemicroscopy (AFM).9�23–25

On the other hand, the interaction of different moleculeswith DNA produces important changes in the DNA sec-ondary structure which can affect the functionality26 andthe DNA helix structure.27–29

In this paper, we are interested to investigate the interac-tion of DNA with a recently reported dicationic tetrandrinederivative bearing two acridine groups at the air–waterinterface.

Tetrandrine (TET) is a bis(benzylisoquinoline) alkaloidisolated from the root of Stephania tetrandra S. Moore.This natural compound has several biochemical and

J. Biomed. Nanotechnol. 2008, Vol. 4, No. 1 1550-7033/2008/4/001/010 doi:10.1166/jbn.2008.007 1

RESEARCHARTICLE

Interaction of Calf Thymus DNA with a Cationic Tetrandrine Derivative at the Air–Water Interface Wong-Molina et al.

pharmacological properties such as antipyretic, analgesic,antituberculostatic and anti-tumoral.30�31 On the otherhand, the chemical, physical and spectroscopic charac-teristics of TET are widely described in the literature.32

Its crystal structure was also elucidated and according tothis, TET has the conformation of a triangle with a smallcavity.33

Moreno-Corral and Lara recently reported the synthesisof new dicationic TET derivatives bearing anthraquinoneand acridine groups and their complexation studiesdirected toward nucleotides in aqueous medium.34 In thiswork they demonstrated that quaternization of both nitro-gen atoms of the alkaloid, with the apolar and planarunits above mentioned, transform it into a dicationic recep-tor with affinity for negative charges of anionic com-pounds, and with an extended cavity capable of significanthydrophobic and �–� stacking interactions with aromaticmoieties of hydrophobic guests in aqueous medium.

On the other hand, due to the well known ability ofanthraquinone and acridine of intercalating into base pairsin DNA and RNA, there are many reports in literatureof molecules that have incorporated those units in anattempt to find systems capable of showing high affinity toDNA sequences at physiological pH, in order to developnew artificial nucleases, DNA sensors, and intercalatingdrugs.35�36

Due to the acridine pKa of 5.6, its possibilities to beused as a cationic molecule are limited at low pHs. Onthe other hand, because both of the nitrogen atoms of theTET derivative (TC) are formally charged, this moleculeis a dication at any pH. Considering the latter, only TChas a charge complementarity with anionic DNA at phys-iological pH. Besides, the incorporated unit of acridinein TC can help to form a more stable DNA-TC complexas a consequence of the intercalating ability of this poly-cyclic arene. Another advantage of TC over acridine is thepresence of hydrophobic surface regions. In conclusion,TC has several structural advantages when is comparedwith the simple acridine unit and as a consequence a morestable DNA complex is expected for this semi-syntheticmolecule in aqueous medium at physiological pH.

In this paper we report the interaction of calf thymusDNA (DNA) with the TET derivative with two acridinyl-methyl groups (TC). Our interest is to get evidences ofthe DNA-TC interaction in solution and at the air–waterinterface. Due to the solubility of TC in water, we haveachieved holding TC at the air water surface by usingan arachidic acid/TC mixture. This mixture, spread on aDNA buffered solution, interact with DNA and is capableto attach it at the air–water interface. By using BAM andAFM we observed evidences of the DNA-TC interaction.Complimentary UV and CD spectroscopic measurementswere performed to investigate the DNA/TC interaction inaqueous solution. Finally molecular simulations were per-formed in order to visualize the DNA-TC interaction.

2. EXPERIMENTAL DETAILS

Calf thymus deoxyribonucleic acid (DNA) was purchasedfrom Life Technologies (Cat. No. 15633-019, Carlsbad,CA, USA). The average size obtained by gel elec-trophoresis, using a 1.2% agarose gel, was 150 bp.Arachidic acid (AA) (99%) was purchased from Aldrich(St. Louis, MO, USA). Chloroform (grade HPLC, 99.9%)and ethanol (reactive grade) were obtained from Sigma-Aldrich (Mexico). TC was synthesized as described in arecent work.34 Deionized Millipore-Q water (18.3 M�-cm) was used in all cases and it was obtained from an Easypure/Barnstead instrument (E pure, Barnstead, Dubuque,IA, USA).

A Langmuir balance (Nima Technologies, Ltd, Coven-try, England, Langmuir Blodgett through, model type 611),whose surface tension precision is 0.1 mN/m, was usedto obtain isotherms. The surface pressure � = �0 −�, i.e.,the surface tension difference of the clean surface and thesurface tension of the covered surface was measured byusing a Wilhelmy plate made of a chromatography paper.The surface pressure and molecular area data were fedinto a computer and recorded, using a barrier speed of25 cm2/min.

AA and TC were dissolved in chloroform at differentweight proportions 1:1, 3:2, 3:1 and 9:1, respectively. Dif-ferent samples of 50 �l from a 1 mg/ml final concentrationwere deposited on a clean buffered solution (TRIS EDTA,pH 7.4) and on a DNA buffered solution (0.25 �g/ml) witha Hamilton microsyringe. This DNA concentration waschosen according the results obtained by some researchers9

and tested after several experiments performed in orderto obtain smaller DNA aggregates in the AFM images.When the buffer solution was used as subphase, isothermsfor AA and AA/TC mixtures were performed 15 min and20 h after chloroform was evaporated. For the case of AAalone, isotherms showed differences in area smaller than1%. On the other hand, differences of AA/TC isothermswere around 5% when they were compared at differenttimes. For the case of DNA aqueous buffered subphase,isotherms were performed 4, 6 and 20 h after the AA/TCsolution was spread. Temperature was kept at 20 C withthe aid of a water circulator bath (Cole Palmer, model1268-24, Chicago IL, USA). All experiments were carriedout inside a dust free glass box.

In-situ BAM images of AA/TC monolayers wereobtained with and without a DNA solution subphase byusing a MiniBAM Plus instrument (model 03027MBA045,Nanofilm, Menlo Park, CA, USA). Images were obtainedat different pressures and for different AA:TC proportions.

Langmuir-Blodgett monolayers were obtained on freshcleaved mica from different AA:TC proportions at the air–water interface and at the surface pressure of 20 mN/m.Monolayers were dipped at speed 1 mm/min. These mono-layers were analyzed by AFM.

2 J. Biomed. Nanotechnol. 4, 1–10, 2008

RESEARCHARTICLE

Wong-Molina et al. Interaction of Calf Thymus DNA with a Cationic Tetrandrine Derivative at the Air–Water Interface

AFM images were obtained from AA/TC monolayersobtained directly from the air water interface. AdsorbedDNA on AA/TC monolayers were also investigated. TheDNA adsorption was analyzed by submerging AA/TCmonolayers (deposited on mica) in a DNA buffered solu-tion of 10 �g/ml. After 30 min, samples were gentlywashed with milli-Q water and analyzed. The measure-ments were performed in a JEOL instrument (model JSPM4210, Akishima, Tokyo, Japan) in the non-contact mode,using silicon nitride cantilevers NSC15 from MicroMasch(Wilsonsville, Oregon, USA).

Circular dichroism (CD) measurements were performedin a JASCO J-810 spectropolarimeter (Easton, MD, USA)at room temperature (around 23 �C). DNA concentrationwas kept fixed at 0.1 mg/ml and was mixed with differ-ent TC aqueous solutions (0.01, 0.025, 0.05, 0.1, 0.2, and0.3 mg/ml) in the same buffer and pH as the DNA solu-tions. Both sample and reference were run three times andaveraged. The wavelength range used was 180–300 nmand the cell path length was 0.1 cm.

UV spectrometric measurements were performed ina spectrophotometer lambda 2 (Waltham, Massachusetts,USA). A one centimetre quartz cuvette cell holder wasused. Spectrophotometric titrations were performed on1�75×10−5 M TC solution in a phosphate buffer (0.032 Mand 0.05 M NaCl). Typically aliquots of fresh DNA stan-dard solutions were added to TC solutions. The pH waschecked at the beginning and at the end of the titrationand was found constant in all cases. The TC concentra-tion was kept constant and the DNA concentration variedin the range 1�25× 10−6–5�0× 10−5 M. The absorbancemeasurements were taken at the 368 nm wavelength. Theassociation constant, K, was obtained fitting the experi-mental data to the Eq. (1),37 using a nonlinear least-squaresregression:

Aobs = A0 +A�KG�T /1+KG�T (1)

Where Aobs is the observed absorbance of the complex,A0 is the absorbance of free TC alone, A� is the maximumabsorbance of TC induced by the presence of DNA andG�T is the total DNA concentration. The Origin 7.5 soft-ware was used to get the better fit with the experimentaldata.

Molecular modeling. Hypercube’s hyperchem 7.52package38 was used to perform molecular mechanics sim-ulations. The MM+ force field was employed as imple-mented in the 6.03 version and the likely structure of TCwas found following the method explained by Moreno-Corral and Lara.34 Various TC-DNA complexes wereexplored electronically using the MM+ force field with thepurpose of establishing the factors that govern the molec-ular recognition of the TC with the double-stranded DNAfragment (GTAAGATGATTC), obtained from the salmonDNA sequence.39 The procedure was the following: thedouble-stranded DNA fragment was constructed from the

nucleic acid databases implemented in Hyperchem 7.52.38

The TC molecule was positioned manually to the DNAminor and major grooves. All complexes were optimizedusing the MM+ force field followed by molecular mechan-ics simulations and finally re-optimized with the MM+method.

3. RESULTS AND DISCUSSION

In Figure 1 we show the isotherms of AA and the mix-ture AA/TC in the weight proportion 1:1 spread on buffersolution. We also show the effect of the DNA adsorptionon the mixture monolayer 4 and 20 h after the AA/TCwas spread on the DNA solution. It can be observedan isotherm shift due to the DNA adsorption at the airwater interface, similar to the one observed by severalresearchers.13�16�19�22 After 20 h DNA molecules have pen-etrated the AA/TC monolayer and the isotherm reaches alimiting area around 77 A2, in comparison with the limit-ing area of the AA/TC mixture spread on the buffer solu-tion (around 37 A2). On the other hand, in the condensedzone, the extrapolated area at zero pressure gives 36 A2

for the isotherm, 20 h after the AA/TC spreading on theDNA solution, in comparison with the extrapolated areaof the pure AA/TC mixture (22 A2). This last value isalmost the same value obtained for pure AA. This wouldmean that for this AA:TC weight proportion, correspond-ing to 3 AA molecules per one TC molecule in the mono-layer, both species occupy the same average moleculararea as AA alone. This is could be an evidence of anAA-TC interaction at the air–water interface. Other inter-esting feature is that the kink produced in the AA isothermat 27 mN/m is changed by a smoother curve, indicating

Fig. 1. Isotherms of arachidic acid (AA) (1) and the mixture 1:1 w/wAA: tetrandrine derivative (TC) on buffer TRIS EDTA (2). (3) and (4)represent isotherms of the 1:1 AA/TC monolayer 4 h and 20 h after themixture AA/TC was spread on a DNA buffered subphase, respectively.DNA concentration used in the subphase was 0.25 �g/ml /buffer TRISEDTA and pH 7.4. Temperature was kept at 20±0�1 �C.

J. Biomed. Nanotechnol. 4, 1–10, 2008 3

RESEARCHARTICLE


Fig. 2. Isotherms of the mixture 3:1 w/w AA:TC on buffer TRIS EDTA(1) and isotherms of the 3:1 AA/TC monolayer 4 h (2) and 20 h (3) afterthe mixture AA/TC was spread on a DNA buffered subphase, respec-tively. DNA concentration used in the subphase was 0.25 �g/ml /bufferTRIS EDTA and pH 7.4. Temperature was kept at 20±0�1 �C.

a longer Liquid Expanded phase as a consequence ofthe AA-TC interaction. This change of phase disappearswith the DNA adsorption, producing a more flexible andexpanded monolayer.

In Figure 2 we show the isotherms of the AA/TC mix-ture in the weight proportion 3:1, which corresponds to10 AA molecules per one TC molecule at the interface.The isotherms obtained 4 and 20 h after the AA/TCspreading on the DNA aqueous subphase are also shown.For the isotherm obtained 20 h after the AA/TC spread-ing, the limiting and the extrapolated areas were smaller(48 and 25 A2, respectively) in comparison to the onesobtained for the 1:1 AA/TC monolayer, probably due tothe decreasing number of TC molecules at the air–waterinterface. In this case, the kink observed in the AAisotherm at 27 mN/m is also observed for this AA:TCproportion, probably due to the higher AA proportion.We also see that the solid like region characteristic ofthe AA isotherm does not completely disappears. On thecontrary, after 20 h the isotherm shows two solid likestructures at higher pressures. Notice the plateau observedaround 47 mN/m as a consequence of the equilibriumbetween the two solid like phases. For the case of theisotherms obtained with the 9:1 AA:TC weight propor-tion (not shown), the limiting area, obtained 20 h after theAA/TC spreading, was around 39 A2, larger also in com-parison with the corresponding area of the AA/TC mixturedeposited in buffer solution (32 A2). Therefore, we canobserve a clear correlation between the TC proportion inthe monolayer and the area increase of isotherm shift pro-duced by the DNA-TC interaction.

It is difficult to explain in detail the mechanism of DNAadsorption at the interfacial film. However, considering thechemical structural characteristics of TC (and the previ-ously reported studies of TC directed toward nucleotides),

we can expect that the major forces stabilizing its DNAcomplex result from ion pairing between the chargedgroups of DNA and TC and from stacking or hydropho-bic interactions. As we will see later, both results of themolecular modeling and the complexation studies in solu-tion of this work prove, in several ways, participation ofelectrostatic interactions. On the other hand, a possibleexperimental evidence of the �-stacking interaction in thecomplex, also provided by the complexation studies insolution, is the hypocromic effect induced in the DNAUV-vis absorption spectra in the presence of TC.

The compressibility of monolayers, defined as Cs =1/A dA/d� , where A and � are the area and the sur-face pressure of the monolayer, respectively, is a moreprecise tool to analyze the equilibrium phases and the flex-ibility of monolayers along isotherms.40�41

In Figure 3 we show the behavior of the compressibilityat different pressures of three different AA/TC complexes,when DNA is adsorbed after 20 h at the monolayer inthe air–water interface. As a reference we show also thecompressibility behavior of the AA monolayer. For AA iseasy to observe the Liquid Condensed-Solid (LC-S) equi-librium phases represented by the peak at 26.1 mN/m witha Cs value of 0.014 m/mN. Another peak is shown at51.2 mN/m with a Cs value of 0.0049 m/mN, correspond-ing to the solid-collapse equilibrium. The presence of TCmixed with AA causes significant changes in the mono-layer rigidity. When the AA:TC 3:1 proportion is usedonly on buffer solution, the transition between the Liq-uid condensed and liquid expanded phases is observed at28 mN/m. For the proportion 1:1 this equilibrium appearsat 27 mN/m (not shown). This phase equilibrium can benoticed also in Figures 1 and 2. As long as DNA is being

Fig. 3. Compressibility of AA/TC mixtures with different AA:TC w/wproportions, 20 h after mixtures were spread on a DNA buffered solution.DNA concentration used in the subphase was 0.25 �g/ml /buffer TRISEDTA, pH 7.4. Temperature was kept at 20±0�1 �C. The compressibilityof arachidic acid (AA) is plot as reference (solid line).


RESEARCHARTICLE


adsorbed, this phase equilibrium is shifted to higher pres-sures as can be observed in Figure 3.

The presence of DNA in the monolayer, 20 h afterthe AA/TC spreading, modifies the compressibility for thethree AA:TC mixtures analyzed as can be observed inFigure 3. We observe that the values of Cs , correspondingto a LC-S equilibrium, are higher in comparison with thevalues without the presence of DNA for the three mix-tures analyzed: 0,030, 0.020 and 0.016 m/mN, respectivelyfor the 9:1, 3:1 and 1:1 AA:TC monolayers adsorbed byDNA molecules 20 h after spreading. This indicates thatthe DNA adsorbed at the AA/TC monolayer produces amore flexible film. Notice in Figure 1 that the pressurefor this transition is shifted to around 42 mN/m for the1:1 proportion as a consequence of the DNA adsorptionand the higher TC concentration in the monolayer. Thecalculation of Cs for the monolayer corresponding to the3:1 AA:TC proportion, performed after 20 h DNA wasadsorbed, shows two peaks corresponding to the equilib-rium of two solid phases, occurring at 43.8 and 50 mN/m

(a)

(c) (d)

(b)

Fig. 4. BAM images of different AA/TC monolayers obtained at a pressure around 4 mN/m: (a) and (c) were obtained from 3:1 and 1:1 AA:TC w/wproportions, respectively, spread directly on water. (b) and (d) were obtained from 3:1 and 1:1 AA:TC w/w proportions, respectively, spread on a DNAbuffered solution. Images were obtained 4 h after the AA/TC mixtures were spread on the DNA subphase. DNA concentration used in the subphasewas 0.25 �g/ml /buffer TRIS EDTA, pH 7.4. Temperature was kept at 20±0�1 �C.

approximately. These transition points, showing the tran-sition, can better be observed in Figure 2.

In Figure 4 we show the BAM images obtained at lowpressure (around 4 mN/m) for the different AA/TC mono-layers and also the images obtained 4 h after the sameAA:TC proportions were spread on the DNA subphase.We notice that images of the AA/TC monolayers on purewater show bright aggregates probably rich in TC sur-rounded by a dark zone which corresponds to the LiquidExpanded (LE) phase of AA. These aggregates becomemore dense and interconnected when the TC concentra-tion in the monolayer is higher. The presence of DNAin the subphase produces aggregates even brighter andmore dense at the interface. Specially interesting is theFigure 4(d) for the 1:1 AA:TC proportion. In this casewe notice a more fibrillated structure due to the DNA-TC interaction at the interface. We also notice the holedareas, probably corresponding to the AA in the LE phase.The appearance of the condensed domains with the surfacepressure increasing, confirms the existence of different


RESEARCHARTICLE


Fig. 5. CD spectra of the DNA/TC complexes. DNA concentration waskept fixed at 0.1 mg/ml and was mixed with different TC aqueous solu-tions (0.01, 0.025, 0.05, 0.1, 0.2, and 0.3 mg/ml) in the same buffer andpH as the DNA solutions.

phases.13 The domains grow larger and stack up to form atightly aggregated morphology at high surface pressure.

Results of the CD spectroscopic measurements of dif-ferent DNA/TC proportions are shown in Figure 5. Thestructure of DNA solution corresponds to a B form, sim-ilar to the structure reported by other researchers at lowsalt concentration.13�24�28�42�43 The absence of a negativeband around 295 nm, as demonstrated by Cowman andFasman,44 is due to the long size of DNA molecules. Theinfluence of the TC interaction on the DNA structure pro-duces changes similar to the ones due to a C form in theDNA structure, similar to the C DNA structure observedby Hanlon et al.,42 due to a high salt concentration and tothe influence of some solvents on the shift to longer wave-lengths and to the decreasing of the ellipticity values.13�45

A similar decrease of the intensity was observed by Prokopet al.,27 around the positive band of 280 nm due to theDNA-anti-tumor platinum complexes. Notice that the peakaround 278 nm is shifted to shorter wavelengths when theTC proportion is increased, probably caused by the lowsolubility of the DNA/TC complex.

In order to study the TC-DNA complex formation, weperformed titration experiments by the UV-vis spectropho-tometric technique. Studies were carried out in aqueoussolution and pH and ionic force were also controlled toguarantee having predominantly the DNA in its fully ion-ized form. A DNA stock buffered solution was added stepby step to the TC dissolved in the same solvent. Dur-ing the titration, the UV spectra of TC showed a sig-nificant hypochromism at its maximum absorption as anindication of the TC-DNA interaction. This hypochromiceffect is frequently observed with similar complexes andcan be attributed to the intercalation of the acridine unitsinto base pairs in DNA helices driven by �-stackinginteraction.46–48

Fig. 6. Plot of the absorbance at 368 nm of TC (1�75 × 10−5 M)with respect to the concentration of added DNA in phosphatebuffer. Solid line is the fitting curve in accordance with Eq. (1) in thetext.

The absorptions recorded at 368 nm were plotted ver-sus the DNA concentration. A typical hyperbolic behaviorwas found corresponding for a complex of 1:1 stoichiom-etry and therefore data could be fitted by Eq. (1) (seeexperimental section). Figure 6 shows the UV titrationplot. A binding constant of 1�6× 106 M−1 was obtainedwhich indicates formation of a very high stable TC-DNAcomplex in solution. It is important to mention that theorder of the association constant found by this study isin agreement with those reported in literature for similarsystems.35�49�50

On the other hand, in Figure 7 we show the AFMimages of monolayers using two different AA:TC propor-tions. Figures 7(a) and (b) show the images for mono-layers of 9:1 and 1:1 AA:TC proportions, respectively.We notice that both images show separated regions bya thin strip, probably rich in TC molecules. For the 9:1AA:TC monolayer, the large areas have an average heightof 1.5 nm, probably constituted mainly of AA moleculesin a liquid condensed phase and the thin darker strips havean average height of 0.5 nm. In the case of 1:1 AA:TCmonolayers, the large areas have an average height of1.0 nm. This different average height is probably caused bya higher TC proportion in these regions, interacting withAA molecules. The dark strips are wider in comparisonwith the ones observed for the 9:1 monolayer and have thesame average height.

The effect of DNA on AA/TC monolayers observedafter they were submerged on a DNA solution and gen-tly washed, as described in the experimental part, can beobserved in Figures 7(c) and (d), where a 9:1 and 1:1AA:TC proportions, respectively were used. In Figure 7(c)we can observe two different domains, as observed inFigure 7(a). We observe that DNA aggregates are attachedat zones where TC molecules are found probably withoutAA molecules. These aggregates have an average height


RESEARCHARTICLE


(a) (b)

(c) (d)

Fig. 7. AFM images of AA/TC monolayers on freshly cleaved mica. (a) and (b) correspond to the 9:1 and 1:1 AA:TC proportions, obtained from theair–water interface. (c) and (d) are images obtained after the 9:1 and 1:1 AA:TC monolayers, respectively were submerged in a DNA solution. DNAconcentration used was 10 �g/ml /buffer TRIS EDTA, pH 7.4. The size of the corresponding images are: 7(a), 5 �m; 7(b), 12 �m; 7(c), 9 �m; and7(d), 1 �m.

higher than 12 nm and an average diameter of 400 nm.Similar size of DNA aggregates were found by Mitraand Imae.25

In the case of monolayers with a larger TC proportion,we should observe a higher DNA concentration attachedto the monolayer. In Figure 7(d), where a 1:1 AA:TC pro-portion was used, in fact we notice at the bottom of thepicture the AA domains surrounding TC molecules (smalldark zones). It seems that DNA aggregates are attachedto planar zones constituted mostly by TC molecules. Theaverage diameter of the DNA strips are 50 nm and theaverage height is 1.8 nm, similar to the one found by Mitraand Imae.25 We notice in this case that DNA aggregatesare longer than the ones observed on the monolayer builtwith 9:1 AA:TC proportion. It is possible that as longas many TC molecules are present, more DNA moleculescan remain more elongated, interacting with TC along theDNA chains and showing a more fibrillated structure.

Finally in Figure 8 we show the image of the DNA-TC interaction resulted with the lowest energy found bymolecular dynamics. The force field, MM+, suggests thatinteractions between TC and DNA are mainly into themajor groove. These electrostatic interactions are formedbetween quaternary nitrogen atoms of the TC charged pos-itively and oxygen atoms charged negatively of the phos-phate groups of the DNA fragment. Figure 8(a) shows theTC molecule localized in the major groove of the DNAfragment. In Figure 8(b), atoms participating in these inter-actions are labeled with their respective symbols. As wecan see, there are two main interactions. One of these inter-actions is between O1 and N1 and separated by a distanceof 4.30 Å. The second electrostatic interaction has a dis-tance of 4.44 Å between O2 and N2.

The TC prefers the phosphate groups of the DNA frag-ment GTAAGATGATTC. This is due to the steric hin-drance generated by the acridine groups. These bulky


RESEARCHARTICLE


(a)

(b)

Fig. 8. Molecular structure of the TC-DNA complex. (a) The greenmolecule is the TC molecule localized in the major groove of theDNA fragment. (b) Main interactions sites are formed between quater-nary nitrogens (positively charged) and the phosphate groups (negativelycharged) of the DNA.

groups prevent the approach of the quaternary nitrogenatoms to other functional groups localized in the minor orthe major groove.

Other calculated configurations show interactionsmainly between oxygen atoms of phosphates of the DNAnucleotides and the quaternary nitrogen atoms of the TC.However, these structures have higher energies, usuallygreater than 24 kcal/mol.

Additional simulations were performed in order tounderstand the possible AA-TC molecular interactions.

We found that both of the oxygen atoms of the COO−

group in the AA molecule interact electrostatically withthe hydrogen atoms on the carbon atom adjacent to thequaternary nitrogen atoms of the TC molecule shown inFigure 8(b) (results not shown).

In addition to the possible hydrophobic interactionsbetween the tail of the AA and the hydrophobic regionof the TC molecule, the previously mentioned interactionscould explain the stability of the AA/TC monolayers. Itis also possible to conclude from the results, that one TCmolecule remaining at the air–water interface could besupported by one AA molecule and simultaneously inter-acting with a DNA chain.

4. CONCLUSIONS

The overall results of the present study corroborate theDNA adhesion on the TC domains. It seems to be possi-ble that, as long as many TC molecules are present, moreDNA molecules can become enlarged, interacting with TCalong the DNA chains, showing a more fibrillated struc-ture. The latter feature would constitute a peculiar behaviorof this system and make it an attractive candidate for thedirect detection of DNA. Work is in progress along thisline and will be published elsewhere.

Acknowledgments: This work was supported by Con-sejo Nacional de Ciencia y Tecnología CONACyT(Mexico) under grant Sep-2004-C01-46749 and a scholar-ship to A. Wong.

References and Notes

1. D. J. Caruana and A. Heller, Enzyme-amplified amperometric detec-tion of hybridization and of a single base pair mutation in an 18-baseoligonucleotide on a 7-�m-diameter microelectrode. J. Am. Chem.Soc. 121, 769 (1999).

2. J. O. Radler, I. Koltover, T. Saldit, and C. R. Safinya, Structureof DNA-cationic liposome complexes: DNA intercalation in multi-lamellar membranes in distinct interhelical packing regimes, Science275, 810 (1997).

3. E. Southern, K. Mir, and M. Schepinov, Molecular interactions onmicroarrays. Nat. Genet. 21, 5 (1999).

4. S. P. A. Fodor, DNA SEQUENCING: Massively parallel genomics.Science 277, 393 (1997).

5. R. Arshady, Microspheres for biomedical applications: Prepara-tion of reactive and labelled microspheres. Biomaterials 14, 5(1993).

6. M. Sasou, S. Sugiyama, T. Yoshino, and T. Ohtani, Molecular flatmica surface silanized with methyltrimethoxysilane for fixing andstraightening DNA. Langmuir 19, 9845 (2003).

7. A. Kumar, M. Pattarkine, M. Bhadbade, B. A. Mandale, N. K.Ganesh, S. S. Datar, V. C. Dharmadhikari, and M. Sastry, Linearsuperclusters of colloidal gold particles by electrostatic assembly onDNA templates. Adv. Mater. 13, 341 (2001).

8. O. Okahata, T. Kobayashi, and K. Tanaka, Orientation of DNAdouble strands in a Langmuir-Blodgett film. Langmuir 12, 1326(1996).


RESEARCHARTICLE


9. A. Bahaumik, M. Ramakanth, L. K. Brar, A. K. Raychaudhuri,F. Rondelez, and D. Chatteriji, Formation of a DNA layer onLangmuir-Blodgett films and its enzymatic digestion. Langmuir 20,5891 (2004).

10. M. T. Kennedy, E. V. Pozharski, V. A. Rakhmanova, and R. C. Mac-Donald, Factors governing the assembly of cationic phospholipid-DNA complexes. Biophys. J. 78, 1620 (2000).

11. K. Kago, H. Matsuoka, R. Yoshitome, H. Yamaoka, K. Ijiro, andM. Shimomura, Direct in Situ observation of a lipid monolayer-DNAcomplex at the air–water interface by X-ray reflectometry. Langmuir15, 5193 (1999).

12. L. Sun, M. Xu, and X. Hou, In-situ observation of the aggregatedmorphology and interaction of dialkyldimethylammonium bromidewith DNA at air/water interface by Brewster Angle Microscopy.Mater. Lett. 58, 1466 (2004).

13. X. How, L. Sun, M. Xiu, L. Wu, and J. Shen, Electrostatic complexof didodecyldimethylammonium and DNA: Langmuir monolayer,Langmuir-Blodgett film and dye recognition at air/water interface.Colloids and Surfaces B: Biointerfaces 33, 157 (2004).

14. M. Shimomura, R. Mitamura, J. Matsumoto, and K. Ijiro, DNA-mimetics: Towards novel molecular devices having molecular Infor-mation. Synth. Met. 133–134, 473 (2003).

15. D. L. Thomas, L. J. Blum, and A. P. Girard-Eqrot, Effect ofdeoxyribonucleic acid interaction on the interfacial properties ofa fluid functionalized lipidic matrix. Thin Solid Films 483, 319(2005).

16. X. Chen, J. Wang, and M. Liu, Influence of surfactant molecularstructure on two-dimensional surfactant–DNA complexes: Langmuirbalance study. J. Colloid Interface Sci. 287, 185 (2005).

17. V. Matti, J. Saily, J. Alakoskela, S. J. Ryhanen, M. Karttunen, andK. J. Kinnunen, Characterization of sphingosine-phosphatidylcholinemonolayers: Effects of DNA. Langmuir 19, 8956 (2003).

18. A. Frantescu, K. Tonsing, and E. Neumann, Interfacial ternary com-plex DNA/Ca/lipids at anionic vesicle surfaces. Biolectrochemistry68, 158 (2006).

19. D. Mirska, K. Schirmer, S. S. Funari, A. Langner, B. Dobner, andG. Brezesinski, Biophysical and biochemical properties of a binarylipid mixture for DNA transfection. Colloids and Surfaces B: Bioin-terfaces 40, 51 (2005).

20. V. Ramakrishnan, M. D’Costa, K. N. Ganesh, and M. Sastry, Effectof salt on the hybridization of DNA by sequential immobiliza-tion of oligonucleotides at the air–water interface in the presenceof ODA/DOTAP monolayers. J. Colloid Interface Sci. 276, 77(2004).

21. K. Hago, H. Matzukoda, R. Yoshitome, H. Yamakoa, K. Ijiro, andM. Shimoura, Direct in Situ observation of a lipid monolayer-DNAcomplex at the air–water interface by X-ray reflectometry. Langmuir15, 5193 (1999).

22. M. Cardenas, T. Nylander, B. Jonston, and B. Lindman, The interac-tion between DNA and cationic lipid films at the air–water interface.J. Colloid Interface Sci. 286, 166 (2005).

23. S. Dai, X. Zhang, Z. Du, and H. Dang, Fabrication of nanopatternedDNA films by Langmuir-Blodgett technique. Mater. Lett. 59, 423(2005).

24. X. Chen, L. Li, and M. Liu, Assembly and characterization of ternarySV-DNA-TMPyP complex Langmuir-Blodgett films. Langmuir 18,4449 (2002).

25. A. Mitra and T. Imae, Nanogel formation consisting of DNA andpoly(amido amine) dendrimer studied by static light scattering andatomic force microscopy. Biomacromolecules 5, 69 (2004).

26. P. B. Farmer, K. Brown, E. Tompkins, V. L. Emms, D. J. L. Jones,R. Singh, and D. H. Phillips, DNA adducts: Mass spectrometrymethods and future prospects. Toxicol. Appl. Pharmacol. 207, 293(2005).

27. R. Prokop, J. Kasparova, O. Novakova, V. Marini, A. M. Pizarro,C. Navarro-Ranninger, and V. Brabec, DNA interactions of new

antitumor platinum complexes with trans geometry activated by a2-metylbutylamine or sec-butylamine ligand. Biochem. Pharmacol.67, 1097 (2004).

28. S. Mahadevan and M. Palaniandavar, Spectral and electrochemi-cal behavior of copper(II)-phenanthrolines bound to calf thymusDNA. [(5,6-dimethyl-OP)2Cu]2+ (5,6-dimethyl-OP = 5,6-dimethyl-1,10-phenanthroline) induces a conformational transition from B toZ DNA. Inorg. Chem. 37, 3927 (1998).

29. B. Kankia, V. Buckin, and V. A. Bloomfield, Hexamminecobalt(III)-induced condensation of calf thymus DNA: Circular dichroism andhydration measurements. Nucleic Acids Res. 29, 2795 (2001).

30. Y. J. Chen, Potential role of tetrandrine in cancer therapy. Acta Phar-macol. Sin. 23, 1102 (2002).

31. L. H. Meng, H. Zhang, L. Hayward, H. Takemura, R. G. Shao,and Y. Pommier, Tetrandrine induces early G1 arrest in humancolon carcinoma cells by down-regulating the activity and induc-ing the degradation of G1-S–specific cyclin-dependent kinasesand by inducing p53 and p21Cip1, Cancer Research 64, 9086(2004).

32. A. Thevand, I. Stanculescu, C. Mandravel, P. Woisel, andG. Surpateanu, Total assignment and structure in solution of tetran-drine by NMR spectroscopy and molecular modelling. Spectrochim.Acta Part A 60, 1825 (2004).

33. C. J. Gilmore, R. F. Bryan, and S. M. Kupchan, Conformation andreactivity of the macrocyclic tumor-inhibitory alkaloid tetrandrine.J. Am. Chem. Soc. 98, 1947 (1976).

34. R. Moreno-Corral and K. O. Lara, Complexation studies ofnucleotides by tetrandrine derivatives bearing anthraquinone andacridine groups. Supramol. Chem. (2007), in press.

35. B. Armitage, C. Yu, C. Devadoss, and G. B. Schuster, Cationicanthraquinone derivatives as catalytic DNA photonucleases: Mecha-nisms for DNA damage and quinone recycling. J. Am. Chem. Soc.116, 9847 (1994).

36. R. W. Armstrong, T. Kurucsev, and U. P. Strauss, Interaction betweenacridine dyes and deoxyribonucleic acid. J. Am. Chem. Soc. 92, 3174(1970).

37. H. J. Schneider and A. K. Yatsimirski, Principles and Methods inSupramolecular Chemistry, John Wiley and Sons, England (2000),pp. 142–151

38. Hyperchem, Release 7.52, Hypercube Inc. (2002).39. P. Chevaillier, F. Chirat, and P. Sautiere, The amino acid sequence

of the Ram Spermatidal Protein 3. A transition protein TP3 or TP4?Eur. J. Biochem. 258, 460 (1998).

40. P. I. Ihalainen and J. Peltonen, Miscibility of lipids in monolayersinvestigated through adsorption studies of antibodies. Langmuir 19,2226 (2003).

41. J. Minones, Jr., J. Minones, J. M. Rodriguez-Patiño, O. Conde, andE. Iribarnegaray, Miscibility of amphotericin B-dipalmitoyl phos-phatidyl serine mixed monolayers spread on the air/water interface.J. Phys. Chem. B 107, 4189 (2003).

42. S. Hanlon, S. Brudno, T. T. Wu, and B. Wolf, Structural transi-tions of deoxyribonucleic acid in aqueous electrolyte solutions. I.Reference spectra of conformational limits. Biochemistry 14, 1648(1975).

43. J. Kypr and M. Vorlíckova, Circular dichroism spectroscopy revealsinvariant conformation of guanine runs in DNA, biopolimers(Biospectrocopy) 67, 275 (2002).

44. M. K. Cowman and G. D. Fasman, Circular dichroism analysisof mononucleosome DNA conformation. Proc. Natl. Acad. Sci. 75,4759 (1978).

45. J. C. Girod, W. C. Johnson, Jr., S. K. Huntinggton, and M. F.Maestre, Conformation of deoxyribonucleic acid in alcohol solu-tions. Biochemistry 12, 5092 (1973).

46. F. Seyama, K. Akahori, Y. Sakata, S. Misumi, M. Aido, andC. Nagata, Synthesis and properties of purinophanes. Relationshipbetween the magnitude of hypochromism and stacking geometry ofpurine rings. J. Am. Chem. Soc. 110, 2192 (1988).


RESEARCHARTICLE


47. S. Claude, J.-M. Lehn, F. Schmidt, and J. P. Vigneron, Binding of nucle-osides, nucleotides and anionic planar substrates by bis-intercalandreceptor molecules. J. Chem. Soc., Chem. Comm. 1182 (1991).

48. O. Baudoin, F. Gonnet, M.-P. Teulade-Fichou, J.-P. Vigneron, J.-C.Tabet, and J.-M. Lehn, Molecular recognition of nucleotide pairs bya cyclo-bis-intercaland-type receptor molecule: A spectrophotomet-ric and electrospray mass spectrometry study. Chem. Eur. J. 5, 2762(1999).

49. L. T. Ellis, D. F. Perkins, P. Turner, and T. W. Hambley, The prepa-ration and characterization of cyclam/anthraquinone macrocycle/intercalator complexes and their interactions with DNA. DaltonTrans. 2728 (2003).

50. Y. Y. Fang, B. D. Ray, C. A. Claussen, K. B. Lipkowitz, and E. C.Long, Ni(II)-Arg-Gly-His-DNA interactions: Investigation into thebasis for minor-groove binding and recognition. J. Am. Chem. Soc.126, 5403 (2004).

Received: 8 October 2007. Revised/Accepted: 12 November 2007.


interaction of calf thymus dna with a cationic tetrandrine derivative at the air-water interface

Documents