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Spectrochimica Acta Part A 74 (2009) 421–426

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

journa l homepage: www.e lsev ier .com/ locate /saa

ultiplex binding modes of toluidine blue with calf thymus DNA andonformational transition of DNA revealed by spectroscopic studies

uan Wang a,b, Xiurong Yang a,∗

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,625 Renmin Street, Changchun 130022, PR ChinaGraduate School of the Chinese Academy of Sciences, Beijing 100039, PR China

r t i c l e i n f o

rticle history:eceived 13 April 2008eceived in revised form 30 April 2009ccepted 12 June 2009

eywords:

a b s t r a c t

It is noteworthy to understand the details of interactions between antitumor drugs and DNA becausethe binding modes and affinities affect their antitumor activities. Here, The interaction of toluidine blue(TB), a potential antitumor drug for photodynamic therapy of tumor, with calf thymus DNA (ctDNA) wasexplored by UV–vis, fluorescence, circular dichroism (CD) spectroscopy, UV-melting method and surface-enhance Raman spectroscopy (SERS). The experimental results suggest that TB could bind to ctDNA via

oluidine blue (TB)NAartial intercalationlectrostatic interactiononformational transition

both electrostatic interaction and partial intercalation. The fluorescence quenching of TB by ctDNA wasstatic and due to electron transfer from bases to the excited singlet state of TB. At low [TB]/[DNA] ratio,TB mainly partially intercalated into ctDNA resulting in the slight increase of base stacking degree; athigh [TB]/[DNA] ratio, excessive TB externally stacked along the helix surface via coupling with partiallyintercalated ones, thereby inducing B-A transition of ctDNA. The conformational transition of DNA wasconfirmed by the obvious improvement of the thermal stability of ctDNA. The SERS spectra suggest thatTB could partially intercalate into DNA basepairs with its ring C1NC1′ side buried.

. Introduction

Nucleic acids as the carriers of genetic information pro-ide important targets for many biomacromolecules and smallolecules. The interactions between DNA and small molecules

re of special importance in chemotherapeutic applications andutagenesis studies [1–3]. Many small molecular anticancer drugs

re known to exert their biological activity through interactingith DNA [1–4], while lots of carcinogens and potent mutagens

ttack nucleic acids thereby changing DNA replication or repara-ion and lead to uncontrolled growth of tumor cells [2]. Although

lot of important DNA-binding compounds have been exploredn phenotypic, cell-based screens, in vitro studies are needed foromprehensive understanding of the interaction mechanism [1].he investigations on the nature of interactions between smallolecules and nucleic acids, not only shed light on understanding

he chemical basis for the antitumor mechanism of drugs and the

arcinogenicity of pollutants, but also give chemists and pharma-ologists opportunities to discover or design new drug candidates.

Phenothiazinium dyes are photocytotoxic in living systems,nd even cause photoinduced mutagenic effects when some cells

∗ Corresponding author.E-mail address: xryang@ciac.jl.cn (X. Yang).

386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.saa.2009.06.038

© 2009 Elsevier B.V. All rights reserved.

survive from damages [5]. DNA is an important target in the phe-nothiazinium dye-photosensitized biological damage, and guanineresidues are particularly accessible [6–8]. Moreover, some phe-nothiazinium photosensitizers, such as toluidine blue (TB) andmethylene blue (MB), have the promising applications in photody-namic therapy of tumor [9–11]. These potential anti-tumor drugscan kill cancer cells through indirect or direct reactions with DNA[5]. TB has been widely used for detection and delineation of somecancers and displays good safety in humans. Furthermore it appearssome selectivity for cancer cells in vivo, therefore TB has beenexplored as a potential anti-tumor drug [11]. It is important toobtain comprehensive understanding of the DNA-binding proper-ties of TB.

To our knowledge, there are only a few investigations on theinteraction between TB and DNA. Resonance light scattering spec-tra of TB with the presence of DNA indicated that TB could aggregatealong DNA surface [12]. Jiao et al. [13] have studied the interactionof TB with DNA by electrochemistry and proposed the electrostaticbinding of TB to DNA. Since TB has the planar ring structure, it isquite possible that TB would bind to DNA via intercalation. However

details on the binding mode of TB to DNA and the conformationalchanges of DNA are still unrevealed, which has great relevanceto the bioactivity of TB. In this study, the interaction between TBand ctDNA was investigated by UV–vis, fluorescence, CD, surface-enhanced Raman spectroscopy (SERS) and UV-melting method.

4 ica Acta Part A 74 (2009) 421–426

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his research is helpful for evaluating the antitumor activityf TB.

. Experimental

.1. Equipments

The absorption spectra were obtained by Cary 50 UV–vispectrophotometer (Varian, USA). All fluorescence spectra wereeasured on a PerkinElmer LS-55 luminescence spectrometer.

he CD spectra were recorded using a 62A DS CD spectrometerAVIV, USA). UV-melting profiles of ctDNA were obtained by Cary00 UV–vis-NIR spectrophotometer (Varian, USA) equipped withquartz cell of 1 cm path length and a thermoelectrically con-

rolled cell holder. The SERS spectra were measured on a Renishaw000 model confocal microscopy Raman spectrometer (Renishawtd., Gloucestershire, U.K.) using radiation of 514.5 nm for the SERSxcitation.

.2. Reagents

TB was obtained from Acros Organic Co. (USA) without furtherurification. Deoxyribonucleic acid sodium salt from calf thymustype I) was purchased from Sigma Chemical Co. (USA). All reagentssed were of analytical grade and aqueous solutions were preparedsing doubly distilled deionized water. The TB stock solution ofmM was kept away from light to avoid photochemical decom-osition and was diluted just before use. The DNA concentrationbasepairs) was spectroscopically determined using molar absorp-ion coefficient of 13,200 cm−1 M−1 at 260 nm. The ratio of thebsorbance at 260 and 280 nm was larger than 1.8, indicating thattDNA was sufficiently free of protein. All the experiments involvingnteraction of TB with ctDNA were carried out in buffer containing0 mM NaAc-HAc and 50 mM NaCl (pH 5) unless otherwise statednd the ionic strength was modulated by adding an appropriate vol-me of stock solution of NaCl (5.0 M in doubly distilled deionizedater).

.3. Procedures

.3.1. Absorption spectroscopy studyAbsorption spectra were recorded in the range of 500–800 nm

sing a quartz cell of 1 cm path length at the ambient temperature,nless otherwise stated. The absorbance titrations were performedy keeping the concentration of TB fixed at 0.01 mM while gradually

ncreasing the concentration of ctDNA from 0.002 to 0.03 mM. Fromhe absorption titration data, the binding constant was determinedccording to [14].

0/(A0 − A) = εf/(εf − εa) + εf/(εf − εa) × (1/K[DNA])

here A0 and A are the absorbances of drug in the absence andresence of DNA, εf and εa are the absorption coefficients of drugnd its complex with DNA, K is the binding constant betweenrug and DNA, respectively. The plot of A0/(A − A0) versus 1/[DNA]as constructed using the data from the absorbance titrations

nd a linear fitting of the data yielded the binding constant (i.e.= intercept/slope). To investigate the influence of temperature on

he binding of TB with ctDNA, absorption titrations were carriedut at 20, 30 and 40 ◦C, respectively.

.3.2. Fluorescence spectroscopy studyFluorescence spectra were obtained in the range of 640–800 nm

pon excitation at 634 nm at the ambient temperature, unless oth-rwise stated. A 1 cm path length quartz cell was used for all theeasurements. The fluorescence quenching experiments were car-

Fig. 1. TB molecule structure.

ried out by the similar operation as absorbance titrations. Thefluorescence intensity at the maximum emission of TB (672 nm)was determined for the calculation of quenching constant KSV. Thedata were plotted according to the Stern–Volmer equation [15]:

I0/I = 1 + KSV[DNA],

where I0 and I are the fluorescence intensities in the absence andpresence of DNA, respectively, KSV is the Stern–Volmer quenchingconstant, which is a measure of the efficiency of quenching by DNA.The influence of temperature on quenching was investigated at 20,30 and 40 ◦C, respectively.

2.3.3. CD spectroscopy studyThe induced CD spectra of 0.02 mM TB in presence and absence

of 0.005 or 0.05 mM ctDNA were recorded in the range of500–700 nm. The ionic strength was changed by increasing con-centration of NaCl in buffer. The CD spectra of 0.05 mM ctDNA inabsence and presence of 0.005 and 0.02 mM TB were recorded inthe range of 220–320 nm. All CD measurements were carried outat the ambient temperature with a 1 cm path length rectangularquartz cell.

2.3.4. UV-melting profiles measurementsAbsorbance changes at 260 nm versus temperature were deter-

mined with a heating rate of 0.5 ◦C min−1 for ctDNA meltingexperiments. The UV-melting profiles of 0.03 mM ctDNA in absenceand presence of 0.003, 0.005 and 0.01 mM TB were measured.

2.3.5. SERS studyThe sodium citrate protected silver colloid for SERS measure-

ments was prepared as follow [16]: 50 mL of 1 mM AgNO3 aqueoussolution was boiled, then 1 mL of a 1% trisodium citrate solutionwas added with vigorous stirring, and the mixture was boiled withrefluxing for 1 h. The silver colloid obtained showed a turbid grayaspect and had surface plasmon band around 429 nm indicatingspheroidal shape with a diameter from 30 to 60 nm [17]. The solu-tion for SERS measurements was prepared by diluting the stocksolution of TB and ctDNA to suitable concentration with silver col-loid solution (a mixture of one volume of silver colloid and ninevolumes of NaAc-HAc buffer described in Section 2.2). The SERSspectra of 0.001 mM TB in the absence and presence of 0.01 mMctDNA were recorded in the range of 200–1800 cm−1 (Fig. 1).

3. Results and discussion

3.1. Multiplex binding modes revealed by absorption spectra

Fig. 2a shows the absorption spectra of TB after addition of differ-ent amounts of ctDNA. With the [DNA]/[TB] ratio shifting from 0.0 to3.0, The absorption spectra of TB showed moderate hypochromicity(about 16%) at 634 nm and bathochromic shift (about 10 nm). The

hypochromicity is a feature common to stacked chromophores. Atransitory dipole formed in one chromophore (A) absorbing lightinduces a dipole in the opposite direction of a neighboring stackedchromophore (B), which partly counteracts the dipole formed in B.Thus the stacked chromophores have less absorbance than the sum

J. Wang, X. Yang / Spectrochimica Acta Part A 74 (2009) 421–426 423

Fig. 2. (a) Absorption spectra of 0.01 mM TB in the presence of 0, 5, 10, 15, 20,25, 30 �M ctDNA. Arrow shows the absorbance changes with increasing amountof ctDNA. Inset: enlarged view of the area inside the circle. (b) Absorption spectraoN2t

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tmsbiastNwselwh

f 0.01 mM TB in the presence of 0.03 mM ctDNA with increasing concentrations ofaCl. Dash line: only TB in buffer; solid lines: TB and ctDNA with 50, 100, 150, 200,50, 300 mM NaCl. Arrow shows the absorbance changes with increasing concen-ration of NaCl. Inset: enlarged view of the area inside the circle.

f each chromophore, resulting in hypochromicity [18]. The sub-tantial hypochromicity (about 30%) and large bathochromic shiftabout 15 nm) have been widely reported for the DNA intercala-ors [19–21]. Therefore TB should not be a classical intercalator.t has been reported that porphyrins with varying substituentsould interact with DNA in different binding modes (intercalation22–24], outside binding [23] or partial intercalation [24–25]). Theresence of bulky non-coplanar groups in the planar core of por-hyrin might hinder the classical intercalation [24]. Therefore theubstituents of TB molecule might also result in some hinderanceo its intercalation into DNA.

There was no distinct isosbestic point during the absorptionitration of TB with ctDNA (Fig. 2a), indicating multiplex binding

odes [20,26,27]. Since TB has a cationic charge, the electro-tatic attraction of TB to the negatively charged sugar-phosphateackbone of ctDNA is inevitable [13]. Therefore, both electrostatic

nteraction and partial intercalation should contribute to the inter-ction between TB and ctDNA. Fig. 2b displays the absorptionpectra of TB in the presence of ctDNA and after five equal addi-ions of NaCl in succession. At low ionic strength, the addition ofaCl induced an obvious reverse of the hypochromicity, however,ith increasing ionic strength the addition of NaCl resulted in much

maller reverse of the hypochromicity. This might be because thelectrostatic interaction between TB and ctDNA was favourable atow ionic strength, and weakened with increasing ionic strength,

hile intercalation was less affected and became predominant atigh ionic strength [28].

Fig. 3. Stern–Volmer quenching plots of TB with increasing concentrations of ctDNAat 20 ◦C (�), 30 ◦C (�), 40 ◦C (�). Inset: fluorescence spectra of 0.01 mM TB after addi-tion of ctDNA at 20 ◦C. Arrow shows the intensity changes upon increasing ctDNAconcentration.

The influence of temperature on the binding of TB to ctDNA wasalso studied. The binding constants of TB to ctDNA at 20, 30 and40 ◦C were 1.63 × 105, 5.38 × 104 and 3.95 × 104 M−1, respectively.Increasing temperature is likely to result in decreasing stability ofTB-ctDNA complex. At the same [DNA]/[TB] ratio, the higher thetemperature was, the smaller the hypochromicity would be, indi-cating that higher temperature facilitated the decomposition ofTB-ctDNA complex. The binding constant of TB to DNA reported byJiao et al. [13] is 7.16 × 104 M−1, which is slightly smaller than thatobtained from the absorption titration experiment at room tem-perature. The difference might come from the different methodsadopted and different buffer conditions.

3.2. Fluorescence quenching study

The fluorescence emission of TB decreased with the additionof ctDNA, meanwhile the emission maximum shifted from 672 to678 nm (inset of Fig. 3). It indicates that the binding of TB to ctDNAwould change the average local environment of TB chromophores[29]. Since the electronic absorption of ctDNA is at much lowerwavelength with respect to the emission band of TB, the fluores-cence quenching of TB by ctDNA through energy transfer could beruled out. It has been reported that the quenching constants of thephenazine and phenothiazine family of dyes by four different DNAbases decreased with increasing oxidation potential of the DNAbases, indicating that the fluorescence quenching was due to elec-tron transfer from the DNA bases to the excited dye [7,30]. Kelly etal. [8] have studied the laser flash spectroscopy of MB with nucleicacids and proposed the fluorescence quenching by electron trans-fer from the guanine base to the excited singlet state of MB [8,31].Another phenothiazinium dye thionine has also been suggested thesame fluorescence quenching process as MB by DNA [6,7,32]. So it isreasonable to suggest that the phenothiazinium dye TB also under-went fluorescence quenching by electron transfer from the DNAbases, especially guanine, to the excited singlet state of TB.

The linear Stern–Volmer quenching plots obtained from the flu-orescence titrations of TB by ctDNA indicated merely one type ofquenching process, namely static or dynamic quenching, under theexperimental condition [33] (Fig. 3). The slope of Stern–Volmerquenching plot, namely Ksv, decreased with the increase of tem-

perature indicating that the fluorescence quenching of TB by ctDNAwas static [33]. Non-fluorescence complexes formed upon TB bind-ing to ctDNA resulted in the fluorescence quenching of TB. The Ksv

of TB by ctDNA at 20, 30 and 40 ◦C are 4.18 × 104, 3.23 × 104 and2.57 × 104 M−1, respectively. The increase of the incubation tem-

424 J. Wang, X. Yang / Spectrochimica Acta Part A 74 (2009) 421–426

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Fig. 5. (a) CD spectra of 0.05 mM ctDNA in the presence of 0, 5, 20 �M TB. Inset:

ig. 4. (a) Induced CD spectra of 0.02 mM TB (dash line) in the presence of 0.05 mMtDNA (dash dot line) with the addition of 50 mM NaCl (solid line). (b) Induced CDpectra of 0.02 mM TB (dash line) in the presence of 0.005 mM ctDNA (dash dot line)ith the addition of 50 mM NaCl (solid line).

erature resulted in the destabilization of the TB-ctDNA complexeading to smaller quenching constant of TB by ctDNA. It was agreed

ith absorption spectroscopic results.

.3. Binding modes at different [TB]/[DNA] ratios confirmed byhe induced CD of TB

The induced CD (ICD) spectrum of TB in the visible region canrovide the direct information for identifying the binding modef TB with ctDNA. In the solution, TB was free to rotate having no

ntrinsic CD signal; however, its orientation was restricted after itsinding to ctDNA. This resulted in the ICD signal of TB as shown

n Fig. 4a and b. Since ctDNA has no absorption in the visibleegion, the observed CD signal is merely the ICD signal of TB. TheTB]/[DNA] ratio had obvious effects on the ICD spectra of TB sug-esting different binding modes at low and high [TB]/[DNA] ratio.t the [TB]/[DNA] ratio of 0.4 the ICD spectrum consisted of oneajor negative band at the wavelength around 630 nm. It has been

eported that an intercalated chromophore centered near the helixxis of DNA should exhibit the negative ICD for a transition polar-zed perpendicular to the long axis of the basepairs [34]. Thereforehe intercalation is a binding mode in the system. When the ionictrength increased, the ICD spectrum slightly changed, however

he negative band remained with about the same wavelength andmplitude (Fig. 4a). It suggests that the intercalation might beot affected by ionic strength of the solution. At the [TB]/[DNA]atio of 4 the ICD spectrum consisted of a positive band at 537 nmnd a negative band at 590 nm, an exciton-like CD spectrum [35],

enlarged view of the area inside the circle. Arrows show the ellipticity changes uponincreasing TB concentration. (b) UV-melting profiles of 0.03 mM ctDNA in the pres-ence of 0, 3, 5, 10 �M TB. Arrow shows profiles with increasing concentrations ofTB.

which dramatically weakened even vanished with increasing ionicstrength (Fig. 4b). This exciton-like CD signal could be explained asone externally bound TB molecule stacked along the helix surface ofctDNA through coupling with one partially intercalated TB moleculeto form an aggregating complex [35]. The externally bound TB wereattracted to the phosphate backbone of ctDNA through electro-static interaction [36]. With the increase of NaCl concentration,the electrostatic interaction dramatically weakened leading to dis-association of the complexes, therefore the exciton-like CD signalvanished.

3.4. B-A transition revealed by CD spectra of ctDNA andUV-melting profiles

Generally, ctDNA has the characteristic CD signal in the ultravi-olet region, one positive band at 274 nm due to the base stackingand one negative band at 245 nm due to the right-handed B-formhelicity [35]. The negative band remained nearly unchanged whilethe positive band increased remarkably with the addition of TB(Fig. 5a). It suggests that the binding of TB to ctDNA would notapparently disturb the right-handed helicity of ctDNA, howeverdramatically increase the base stacking degree of ctDNA, induc-

ing conformational change of ctDNA. B-form is the most commonconformation of double helix DNA in solution. The molecules thatcompete with the water molecules coating around the grooves andphosphate skeleton of the double helix may dramatically affect theconformation of DNA, which usually induce DNA into more com-

J. Wang, X. Yang / Spectrochimica Acta Part A 74 (2009) 421–426 425

Table 1Raman frequencies in cm−1 of the main bands observed in the SERS spectra of TBwith a tentative assignment and their changes upon the addition of DNA.

Raman peaks (cm−1) Tentative assignment Change uponaddition of DNA

1627sa Ring CC stretch + ring C1′ NC1stretch

Hypochromicity

1499m Ring CC stretch Unchanged1445m Ring C1′ NC1 asymmetric

stretchHypochromicity

1388m Ring CC stretch Unchanged1150w Ring CH bend Unchanged1066w Ring C6SC6′ stretch + NH2 wag Unchanged900w C6SC6′ stretch + HN-C stretch Unchanged74

pcwccc[fctrDr[

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ccchdotc1r

63w Ring CH wag + CH def (CH3) Unchanged95w Ring C6SC6′ bend Unchanged

a Relative intensities as: s, strong; m, medium; w, weak.

act form [27,37,38]. In our experiments, ctDNA was prepared inoncentration of 0.05 mM and the conformational changes of ctDNAere determined under the [TB]/[DNA] ratio of 0.4, because higher

oncentration of TB easily precipitated ctDNA. According to the dis-ussion above, at low [TB]/[DNA] ratio TB partially intercalated intotDNA and moderately increased the base stacking degree. At highTB]/[DNA] ratio excessive TB externally stacked along the helix sur-ace of ctDNA via coupling with the partially intercalated ones toompete with the water molecules on the helix surface. Thereforehe externally bound TB could effectively reduce the electrostaticepulsion of the negatively charged sugar-phosphate backbone ofNA making the conformation of ctDNA more compact, which

esulted in B-A transition of ctDNA even the precipitate of DNA27,37,38].

The conformational transition of ctDNA was further confirmedy the change of the thermal stability of ctDNA in the absence orresence of TB (Fig. 5b). It was obvious that DNA became moretable in the presence of TB, which was consistent to other inter-alators [27]. However the improvement of the thermal stabilityf ctDNA with the presence of TB was not as large as that could bechieved with the presence of mitoxantrone (MTX) [27]. This mighte because TB bound to ctDNA through a weaker association thanhe intercalation between MTX and DNA.

.5. Molecular detail of TB partial intercalation into ctDNAevealed by SERS

The SERS bands of TB in silver colloid were in the same posi-ions as those in aqueous solution and all bands were almost equallynhanced (data not shown). Therefore TB probably adsorbed on theurface of silver colloid through physisorption without special ori-ntation. The tentative assignment [39–42] is shown in Table 1.NA had no Raman signal under the experimental condition. It haseen reported that the adsorption of DNA on the surface of silverolloid neither disturbed the construction of DNA [43–45] nor per-urbed the small molecule-DNA interaction [43]. Therefore SERSas used to gain further information on the interaction between

B and ctDNA.The SERS spectra of TB-ctDNA complex displayed nearly identi-

al band positions except the decrease in intensity of certain bandsompared to that of TB (Fig. 6, curve a and b). It suggests that TBould bind to ctDNA via intercalation [43,44,46]. Nabiev et al. [43]ave reported that only particular SERS bands of MTX displayed theecrease in intensity upon intercalation into DNA indicating that

nly those bands related components were involved in intercala-ion. The difference spectrum between the SERS of TB and TB-ctDNAomplex (Fig. 6, curve c) showed that only two bands at 1627 and445 cm−1, which are assigned to the ring CC stretch coupling withing C1,NC1 stretch and the ring C1,NC1 asymmetric stretch, respec-

Fig. 6. Surface enhanced Raman spectra of 1 �M TB (a) in the presence of 10 �MctDNA (b) and their difference spectrum (c).

tively [39–42], exhibited the decrease in the intensity upon TBbinding to ctDNA. This suggests that the ring C1′ NC1 componentmight insert into DNA basepairs. On the other hand, the bands at1499, 1066, 1030, 900, 763, 495, 451 cm−1, which are mainly relatedto the ring C6SC6′ component and the substituents [39–42], werenearly unchangeable upon complexation. So those componentswere not buried into DNA helix. It is reasonable to propose thatTB could partially intercalate into DNA with the C1′ NC1 componentburied into DNA base pairs, while the steric hindrance resultingfrom the substituents blocked the insertion from the C6SC6′ side ofTB.

4. Conclusions

In this paper, the interaction between a potential antitumor durgTB and ctDNA was studied by absorption, fluorescence, CD spec-troscopy, UV-melting method and SERS. The experimental resultssuggest that TB could bind to ctDNA via both electrostatic interac-tion and partial intercalation. At low ionic strength the electrostaticinteraction was favorable; while at high ionic strength the partialintercalation was prominent. The fluorescence quenching of TB byctDNA was static. The binding modes were also affected by the[TB]/[DNA] ratio. At low [TB]/[DNA] ratio TB mainly partially inter-calated into ctDNA with the molecular plane preferably insertinginto DNA basepairs from the ring C1′ NC1 side. At high [TB]/[DNA]ratio some TB molecules partially intercalated into ctDNA andexcessive TB stacked along the helix surface inducing B-A transitionof ctDNA. The conformational transition of ctDNA was confirmed bythe improvement of the thermal stability of ctDNA. The multiplexbinding modes of TB with ctDNA and conformational transition ofDNA upon complexation were first reported. An overall picture ofTB binding to ctDNA is presented, which should be important indeeper insight into the antitumor activity of TB.

Acknowledgements

This work was supported by the National Nature Science Foun-dation of China (No. 20475052) and the Project of Chinese Academyof Sciences (No. KJCX2. Y W. H09).

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