h*,6-dloarboxyamide-2-phenyl indole (ocx), a non-lonio struc- tural

26
voium. SNumbeno October 1978 Nucleic Acids Research Fluorescent complexes of DNA with DAPI 4',6-diamidine-2-phenyl indole.2HCl or DCI 4\6-dicarboxyamide-2-pbenyl indole Jan Kapuscinski and Bogna Skoczylas M.Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093 Warsaw, 3 Pasteur Str., Poland Received 8 August 1978 ABSTRACT h*,6-Dloarboxyamide-2-phenyl indole (OCX), a non-lonio struc- tural analogue of 4',6-dlamidlnc-2-phenyl lndole«2HCl (DAPI), was synthesized In order to verify the hypothesis of interoala- tlon of both dyes into the OKA double helix. Hie influenoe of pH, vlsooslty, and different concentrations of SDS (sodliisi dodeoylsulphate) or NaCl on the optioal and fluoresoent properties and the ohanges In thermal transition of both dye ooaplexes vlth OKA oonflra the affinity of the dyes to the doable helix as veil as their stabilizing inflaenoe on the seoondary DNA structure. The results of binding studies, oarrled out by fluorescent methods hare shown that the- dyes are strongly bound to DNA. though the number of binding sites is snail* Aooordlng to the experimental data, the fluoresoent properties of DAPZ and DCI ooaplexes with DNA are oonneoted with the Intercalating binding mechanism of these dyes. On the other hand, the eventual lonlo or hydrogen bonds of dyes outside the DNA helix do not ohange notloeably their fluoresoent properties. INTRODUCTION Since the first reports on DAPI, vhloh forming fluoresoent ooaplexes with DNA, this ooapound has qulokly beoome an objeot of great Interest (1,2). It seems to be a very valuable instru- ment for researoh work, having a broad application in bioohe- mistry and oytoohemistry as veil (i-13). The meohanlsm of formation of fluoresoent DAPI-DNA eomplexee has up till now not been fully eluoldated. Some authors postula- te a non-intercalating (9,11,12), others an intercalating mode of binding of this dye to DNA (3,5,13)* The publishing data in- dloate that DAPI may be seleotlve towards DNA sequenoes rich in A-T (1,2,3-7). However, the relation between A-T selectivity and © Information Retrieval Limited 1 Falconberg Court London W1V5FG England 3775 Downloaded from https://academic.oup.com/nar/article-abstract/5/10/3775/1014476 by guest on 08 February 2018

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Page 1: h*,6-Dloarboxyamide-2-phenyl indole (OCX), a non-lonio struc- tural

voium. SNumbeno October 1978 Nucleic Acids Research

Fluorescent complexes of DNA with DAPI 4',6-diamidine-2-phenyl indole.2HCl or DCI4\6-dicarboxyamide-2-pbenyl indole

Jan Kapuscinski and Bogna Skoczylas

M.Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093 Warsaw,3 Pasteur Str., Poland

Received 8 August 1978

ABSTRACT

h*,6-Dloarboxyamide-2-phenyl indole (OCX), a non-lonio struc-tural analogue of 4',6-dlamidlnc-2-phenyl lndole«2HCl (DAPI),was synthesized In order to verify the hypothesis of interoala-tlon of both dyes into the OKA double helix.

Hie influenoe of pH, vlsooslty, and different concentrationsof SDS (sodliisi dodeoylsulphate) or NaCl on the optioal andfluoresoent properties and the ohanges In thermal transition ofboth dye ooaplexes vlth OKA oonflra the affinity of the dyes tothe doable helix as veil as their stabilizing inf laenoe on theseoondary DNA structure.

The results of binding studies, oarrled out by fluorescentmethods hare shown that the- dyes are strongly bound to DNA.though the number of binding sites is snail* Aooordlng to theexperimental data, the fluoresoent properties of DAPZ and DCIooaplexes with DNA are oonneoted with the Intercalating bindingmechanism of these dyes. On the other hand, the eventual lonloor hydrogen bonds of dyes outside the DNA helix do not ohangenotloeably their fluoresoent properties.

INTRODUCTION

Since the first reports on DAPI, vhloh forming fluoresoent

ooaplexes with DNA, this ooapound has qulokly beoome an objeot

of great Interest (1,2). It seems to be a very valuable instru-

ment for researoh work, having a broad application in bioohe-

mistry and oytoohemistry as veil (i-13).

The meohanlsm of formation of fluoresoent DAPI-DNA eomplexee

has up till now not been fully eluoldated. Some authors postula-

te a non-intercalating (9,11,12), others an intercalating mode

of binding of this dye to DNA (3,5,13)* The publishing data in-

dloate that DAPI may be seleotlve towards DNA sequenoes rich in

A-T (1,2,3-7). However, the relation between A-T selectivity and

© Information Retrieval Limited 1 Falconberg Court London W1V5FG England 3775Downloaded from https://academic.oup.com/nar/article-abstract/5/10/3775/1014476by gueston 08 February 2018

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fluoreseenee of the complex is not sufficiently prored.

To gain more information about the meohanlsm of fomatlon of

the DAPI-DXA fluoreaoent oomplex, we oarried out the synthesis

of a new DNA fluorophore (4',6-dloarboxyamide-2-phenyl indole) -

- OCX which is a structural analogue of DAPI but, unlike it, is

a nonionio ooapound and therefore oannot fora ionio bonds with

DHA. Ve also tried to explain the influence of some physioo-

ohemloal agents on the fluoresoenoe of both the unbound dyes,

DAPI and DCX, and their oomplexes with DHA, in order to eluoir

date the meohanlsm of fomatlon of these oomplexes.

MATERIALS AMP METHODS

DCX aay be prepared by way of hydrolysis of both DAPI and

4',6-dloyano-2-phenyl indole (DCPI)(Fig. 1)(i4,15)t a) DAPI,

0.5 g (1.43 mM), was boiled under reflux for 3 h with 50 ml of

10jt NcuCO^. After cooling the preoipitate was filtered, washed

with water, dried and crystallized several times from absolute

ethanoli 0.28 g (7i£ of yield) of yellow orystals, a.p. 3<X>°C,

were obtained. The result of elementary analysis corresponds to

the formula C^H^NjOgf b) DCPI, 0.5 6 (2.34 mM) was heated for

30 mln under stirring at 95°C with 10 ml of 95> gSOfc* After

oooling the mixture was poured onioe, and the preoipitate was

filtered and purified as above. Thus obtained DCI was soluble

in both DMSO and DMP, but unlike DAPI it was hardly soluble In

oold water.

Sucrose and SDS (sodium dodeoylsulphate) were purohased from

Serva Fainbioohemioa, Hsidelberg, cetavlon (oetyltrlmethylammo-

nlua bromide) from Kboh-Light Lab., Colnbrook and rhodamine RB

200 from Gurr Ltd., London. All the other reagents and materials

used in this work were desoribed In the previous paper (6).

All reaotlons were oarriad out In buffer H (0.005 M Hepea

with 0.01 M NaCl), pH 7, unless stated otherwise. The oonoentra-

tions of DAPI and DCI stook solutions were 5 ;ug/ml and 2 Ag/al,

respectively. DCX was dissolved on a 100°C water bath. Degrada-

tion of DHA (Sigma I) stook solution (250 ng/ml) to yield a

sheared DNA preparation was oarried out with an MSB ultrasoni-

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Tig. 1. Seham* of OCX preparation

oator (30 • sauloationa repeated 4 times, at 30 • intervals),

or by expelling the OKA, solution through a syringe needle Ho 27

(16). DMA denaturatlon vas oarrled out with a 5 times dilated

DBA stook solution. The samples were heated for 10 min at 100°C

and immediately ohilled In ioe.

. OV absorption measurements and DHA thermal transition stu-

dies vere performed vlth the use of a Cbloam SP-500 speotropho-

toawter. Fluoreaoenoe speotrophotoaietry - the outfit and oourse

of measurements vere described previously (6). Some differenses

are marked In the description of experiments, the measurements

of fluoreaoenoe intensity (I) (referred to as relative fluores-

oenoe intensity in the mentioned paper), vere performed vith

excitation at 372 nm (OAFI and DAPX-DHA, complex) and at 354 am

(DCT and Dd-DHA oomplex). Bnission vas measured at k$k and

450 na, respectively. To eliminate quenohing, solutions of 0D<

0.05 vere used both in exoitement and emission bands, lhe pola-

rization ooeffioient of fluoresoenoe (P) vas measured by means

of a MPF-3 spectrofluorimeter vlth polarizers belonffin^ to the

equipment of this apparatus. The components of the polarized

emitted light Ivv> Ivh, Ihv.yand 1 ^ (17) vere measured, and the

ooeffioient of polarization vas assumed as equal to P s Ivv-

Ivn»t/Ivv.+Ivn»t vhere t = InT'

/^hh t n o *ran8"itt*lloa» whioh vas

found to be 0.88. Quantum efficiency of fluoresoenoe (q) of DAPI

and DCI vas determined aooording to Parker and Rees (18) using

rhodamine B (q s 0,69) as standard. Quantum effloienoies of com-

plexes of these dyes vlth USA vere oaloulated aooording to Pao-

letti and Le Peoq (i9)t employing as reference the values of q

of free DAPI and DCI and talcing into consideration changes of

OD and P.

Fluoresoenoe studies of the binding dyes. Titration of the

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DBA solution (o « 5 jug/vl, 3 «1) va* peifotaed directly in a

quartz oell, dosing the DAPI solution (o s 2.5 xig/al) in 5>25,ul

portions. The fluoresoenoe intensity (I) and polarization ooef-

fleient (P) were measured about 3 ain after addition of DAPI.

Prior to the experiment, we determined the fluoresoenoe inten-

sity of nonbound DAPX (X~) and that (L) of the saae oonoentra-

tlan of dye bound to a large excess of DNA (aolar ratio, r+ a

o a-./Onjn • 0.00013)* the ratio of these two fluoresoenoe in-

tensities, V a Xw/ttf a 31.7 was used to determine the concen-

tration of bound (o^) and free (o.) dye for each point of the

binding plot (19). Proa these data, we oould plot, the Soat-

ohard equation (20) r/of a K(n-r), where the aolar ratio, r a

°b/cDNA' K A* *n* *" i n i*T oonstant for the binding and n the

ntasber of dye aoleoules bound per nuoleotide at saturation of

dye. Measurements were performed within bonds of r. a 0.00^5 f

O.O67. On aeoount of the slow establishment of the state of

equilibrium, the DCI binding oonstant was found in another way.

Thus, 5 ml of the mixture of DBA (o a 10 or 25 «g/ml) and DCI

(rt a 0.0011 f 0.12) was inoubated for 2k h at 37°C and then I

and P were aeasured. The ratio T a 2,72 was determined for r+ a

O.0005. Further oaloulations were done as in the oase of DAPI.

RESULTS AMD DISCUSSION

Some physioo-ohemical properties of the dyes DAPI and DCI

V,6-Diamldlne-2-phenyl Indole is a strong base. The results

of tltration of its hydroohloride (DAPI) with NaOH solution in-

dloate that it exists in neutral solutions In the fora of a bl-

oat ion (pKa>11)(Fig. 2A). Unlike DAPI, k*,6-diearboxyaalde-2-

-phenyl indole (DCI), as an aaide of an aromatic acid, does not

show baslo properties. Both these oompounds bare, however,

nearly identical dimensions and the saae indole skeleton oonjo-

gated with the phwnyl group. The resonsnee energy of oenju-

gatlon in such systems reaches its —-•«»"- when the rings, In

this ease the indole and pnenyl rings, are oo-planar. The

absorption spectra in or (Table 1) oonfirm the effeot of

conjugation (the appearance of a third, longwave aaxiaua whloh

is not present in the Indole speetrua). The resonance energy of

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10

Fig. 2. A - Tltration of DAPI, 19.8 ag in 5 ml of 60% ethanol, by Meansof 1 M ifaOB so In. in 60}t ethanol, left scale. B - fluorescence

intensity I of DAPI, eoncn. 0*25 yng/nl, as a function of pB, left scale.C - Fluorescence intensity I and D - Polarization coefficient P, rightscale, of DAPI-OHA complex, DAPI conen. = 2.5 ng/ml, DRA concn. =25 Ag/mlas a function of pH, 1 - Alkaline transition of OKA -o- and D HA-DAP Icomplex -A - , right scale, expressed as an increase of log I/I , as thefunction of pfl. 50 ml of DHA or DAPX-DNA complex was titrated withRaOB, DNA concn. = 50 jig/ml, DAPI concn. in the conplex = 1.1 ng/ml

suoh systems i s of the order of 5 fcoal/uol (21) . I t means thattheir oo-planarity la not so stable aa that of aromatlo conden-sed aysteas ( e . g . oarbasol) and the relaxation of their exoitedstate may be due to the internal rotational diffusion (22) . I tla v e i l known that the relaxations of the exoited. s tate throughnon-radiative passages, i . e . internal vibrational and r o t a t i o -nal prooesses are very rapid, of the order of 10" s . On theoontrary radiative relaxation, for example fluoresoenoe, i s aprooess slower by several orders.

lhe low quantum y ie ld ot fluoresoenoe (q) of DAPI and DCIaqueous solutions (Table 2) indioates the quioker nonradlatlverelaxation to be dominating, unless some additional agentsooour whloh might s t a b i l i z e the oo-planar structure of dyes andprovoke the elimination of internal rotational di f fusion, e .g .around the axis of the 1'-2 bond (Fig. 1 ) .

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fable 1. Speetrophotometrlo properties of dyes and their,mixtures with UNA and SDS

Dye

DAPI

DCI

Amax

3*0259223

332258221

270001830022200

195001*50019000

n»Aa)

Amax

3*7

338

£max

23600

16600

SDS^

Amax

35626*225

Emax

259001580022300

a*

"(molar r a t i o , r tJ DAPX-DSA = 0.0O*, DCI-DHA a 0.0075'molar r a t i o DAPI/SDS s 0.0O1

Table 2. Comparison of fluorescent properties of dyesand their mixtures with UNA and SDS

DAPI *DAPI-DNA?<DAPI-SDS^

DCI ..Dd-DHA?<DCX-SDS0'

exoltation

AB

355372368

353360359

emission«m^< IMWI ) Tim

453458*5545O457451

1.031.7*0.0

1.02.7*.5

quantumyields

4

0.050.900.970.220.590.98

polarizationooefflolent

P'>

0.090.330.11

0.0*0.270.08

' Rjttio (V) of fluoresoenoe Intensity emitted by a bound dyeto the fluoresoenoe intensity emitted by a free dye and po-larization ooeffiolent (P), measured for samples containingDAPI, at AT. = 372 and An> « *5* nm and for samples containingDCI, at A B * 35* andA p = *50 nm.

Extrapolated values (rt -»»0).b'

A rutine method of inhibition of molecular motions in solu-

tions as veil as of their internal rotation, is the inorease of

solution vlsoosity (22). It has been found that both DAPI and

DCI shov a remarkable inorease in fluoresoenoe intensity I in

suorose solutions (Fig. 3A, 4A). A considerable Inorease of the

fluoresoenoe of DAPI solution* 5 ng/ml in the solid phase

(0°C) as compared with that of the solution in the liquid phase

at the same temperature, has also been observed. These results

indicate a relationship between Inhibit ion of the internal

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700

500

300

100

J•

/

B _

BP

y

ai

Sucrose40 % */»

Tig. 3* Influence of sucrose concentration on the fluorescence intensityI, oft A (DAPI), B (DAPI-DHA) complex, left scale and on the polarizationcoefficient of fluorescence, P oft C (DAPI), and D (DAPI-DHA) complex, rightscale* Dye conch, in ali samples - 0.25 ag/al, DHA conea. in the complex -25 Ag/ml. Identical results were obtained without reference tosuccessiveness of addition of components and also after 24 h of Incubation•t 37°C

rotation in the moleoulea of dyes and the inorease of I. On the

other hand, the deorease In the molecular notions In solutions

of elevated viscosity manifests itself in the inorease of the

polarization ooeffloient, P (Fig. 3C, kc) (17).

The observed SDS effeot (Klg. 5A) is another argument indl-

oating that inhibition of internal rotation in the DAPI aoleou-

le is followed by an inorease of I. SDS is an anionio detergent

oapable of formation of mleellar systems (for rev. see 23).

There is no doubt that in 'solvent oases' (2k) torating under

the influence of SDS, a strong spatial blookade ooours vhioh

makes the motions more diffioult. Ve also have observed ohanges

of P under the influenoe of SDS (Pig. 5C). In the oourse of ti-

tratlon of DAPI vith the detergent solution there is initially

a qulok inorease of P, with a n&xinua at °SDS/ODAPI'V/'*00*

Afterwards, with further addition of SDS, the P value decreases.

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100

Fig. 4. Influence of sucrose concentration on the fluorescence intensity I, of: A (DCI), B (DCI-DNA)complex, left scale and on the polarization coefficient of fluorescence, P of: C (DCI) and D (DCI-DNA) complex, right scale. Dye concn. in all samples - 0.15 jig/ml, DNA concn. in the complex- 78jjg/ml. The complex, prepared by incubation of DCI and DNA at 37°C, was mixed with thesucrose solution and incubated again. Identical results were obtained when DNA was added to themixture of DCI and sucrose and then incubated.

Ve have not been able to find a satisfactory explanation of

this deoline. It seems however, that this effect, though Inte-

resting, has no essential importance for the problems disoussed

in this paper* As In the oaae of a series of other DNA Uganda

and anlonlo detergents (23-25), ve hare observed a red shift

(16 tun) and a hypoohromlc effeot (-6%) In the DAPI speotrum at

A 3^0 nm after addition of SDS (Table 1).

The sudden increase in I of DAPI under the influence of SDS,

almost linear in the initial phase of titration (Fig. 5A), may

beoome a basis for a seleotive and very sensitive fluoriaetrlo

method for determination of anionlo detergents, for example in

pollution. Ve do not intend, however, to deal with this problem.

Ibis observation may explain the negative opinion of Williamson

and Pexmell oonoerning the application of DAPI as a reagent for

quantitative determination of DNA (2). These authors, in the

described mettxod of isolation of DNA used the anlonio detergent,

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3 g/l SDS

Fig. 5. Influence of SDS concentration on the fluorescence intensity I, of: A (DAPI), B (DAPI-DNA)complex, left scale, and on the polarization coefficient of fluorescence P, of: C (DAPI), D (DAPI-DNA) complex, right scale. Dye concn. in all samples 0.25,ug/ml, DNA concn. in the complex25 jig/ml. Continuous line, B represents the results obtained directly after mixing of SDS with thesolution of the complex and also after 24 h of incubation of this mixture at 37°C. The results ob-tained after incubation of the solution prepared by addition of DNA to the mixture of SDS-DAPIare presented as open circles -o-

Saroosyl. Ibis oould be followed by a remarkable Increase In the

Intensity ot fluoresoenoe of the dye not bound with UNA.

The titration ot DAPI solution with oetavion, which is a

oationio detergent, was not followed by any significant increa-

se of I and P. This result oould be foreseen, because binding

interactions between ions of detergent and DAPI, oharged iden-

tically, are hardly imaginable. It shows, however, that the I

and P values of dyes are not influenced by ohangea in the super-

fioial tensions.

The influence of oetavlon and SDS on DCI is similar, but the

changes in I and P values are greater in the oase of SDS action

(Fig. 6A, C).

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5 fi/l SDS

F i g . 6 . Influence of SDS concentration on the fluorescence intens i ty I ,and on the polarization coeff ic ient P of OCI and DCI-DHA com-

p l e x e s . Symbols and solutions concentration as in Fig. 5 . Solutions ofthe dye or of the complex after the addition of SOS were incubated 24 h*at 37°C. Open c i r c l e s - o - , as in Fig . 6 .

The I and P ohangee of DAPI s o l u t i o n s , depending on NaClconcentration (Fig. 7A., C), suggest that a relatively small chlo-ride anions are not able to inhibit successfully the rotationaldiffusions. A small P Increase is perhaps due also to theinorease of the visoosity of the solution.

Summarizing the above results ve are of the opinion that theIncrease in the fluoresoenoe intensity, the red shift and the de-crease in OS in the uv spectrum of DAPI and DCZ solutions relateto the stabilization of the co-planar structure of dyes and tothe inhibition of relaxation processes of moleoular rotation.

DAPI and PCI complexes with DWAOptioal properties. In spite of examination of solutions

within a large range of concentrations, i t vas not possible tofind visible changes in the nv spectrum of DMA, measured at

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700

500

300

,2

0.1

Tier1:-

rig, 7. Influence of 5aCl concentration on th« fluorescence intensity I,and on the polarization coefficient P of DAPI and DAPI-OHA

complex. Symbols and solution concentrations as in Fig* 5»

200-300 an, under the Influence of both dyes. The measurements

were oarried oat using DNA solutions of final oonon. of 5> ug/al

and ohanging rt = °(tysa/0oK4 from O.O8 to O.OO4 (all the men-

tioned solutions were prepared under standard conditions, see

Mat. and meth.). However, ohanges in the speotra of both dyes,

ooourring in the presence of DNA (Flff. 8» 9)» were observed.

The red shift of absorption maxima» observed in this oaset ln-

oreases with the decrease of r. value. The shift of some «— »<•«

extrapolated for r. -•O is presented in Table 1. The bypoohro-

mio effect was also visible on the presented diagrams. Similar

effects were observed for BthBr (27,28) as well as for a series

of other ligands and also for those, for whioh the intercalating

mechanism of binding had been assuaed (26,29-3*0.

Ve also observed both, the red shift and a hypoohroalo effect

under the influence of SDS, in spite of the fact that this com-

pound, being an aliphatio derivative, had no 7T -electrons and

in oonsequenoe, oould not fora 3t -ooaq>lexes with aromatic ooo-

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pound*, Thus, the changes in the uv spectrum of dyes were

probably due to the stabilization of the oo-planar atruoture and

gave evi denoe for a rigid binding with INL

The excitation and emission speotrua of DAPI-DNA oomplex has

been published In the previous paper (6). Fig. 10 represents

the excitation and emission speotrum of OCX and of the DCT-DNA

oomplex whloh, like the DAPI-DNA oomplex, though to a lesser

degree, shows the red shift and the increase of emission as ooa-

pared with the solutions of free dye. Slmilarily to the observed

ohanges in the absorption speotrua, the red shift value inorea- ,

a»a vlth the decrease of the r^ value. The nuaerioal data oon-

oernlng with these effeots are listed In Table 1.

Quantum yield of fluorescence (q) of DAPX and OCX oomplexes

with DNA increases markedly as oompared with q of free dyes.

This is particularly distinct in the oase of the former

(Table 2). It should be noted that the speotrofluorlmetrioally

observed ratio V of f luoresoenoe intensity emitted by bound dye

I b as compared with fluoresoenoe Intensity emitted by free dye

Xf, may be higher than the ratio of quantum yields, beoause V =

X^/E- = qb/q* *£b/fff» *•* means that It depends also on the ex-

tinction coefficient of bound (eb) and free (£f) dye (35).

Fluoresoenoe of emission anlsotropy. Polarization ooeffiolsnt

P of a ohromophore (see Hat. and Meth.) refleots the rotation

this ohromophore undergoes between exoitation and emission. The

emissions are maximally polarized, or partially polarized, de-

pending upon how rigidly the dye is bound to OKA and so, P mea-

sures the rigidity of the dye binding (for rev. see 36). Aqueous

solutions of OAPX and DCI show strong polarization of emission,

0.09 and 0.04, respectively. This may be due to solvatation, or

to a very short lifetime of fluorescence (17). The DAPI-DNA

complex is oharaoterized by a high P value, close to the values

observed In EthBr oomplexes (37)(Table 2). It is interesting to

note that this high P coefficient Is found for binding sites of

UNA of various affinity to DAPI (see below, 'Binding Studies'

and Pig. 3). The results oonflrm a very rigid DAPI-DNA binding.

The P value for DCT-DNA oomplex is lower than that observed

for the DAPI-DNA oomplex (Table 2). In this oase, however, the

binding is also rigid* A ooaparlson of P of the oomplex with P

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JOO

211

JOO

220 200 300 340 3 0 0 W O W

Fig. 8. DAFI absorption spectrum in DAPI-DNA complex measured versus DNA. DAPI concn. = 1.1ug/ml, DNA concn. was A - 0, B - 5 jjg/ml, C - 240 jig/ml. All solutions in standard condition.Light path length = 4 cm.

220 400 Qi

Fig. 9. DQ absorption spectrum in DCI-DNA complex measured versus DNA. DCI concn. = 1 Jig/ml,DNA concn. was: A -0, B - 5.ug/ml, C- 150 jjg/ml. All solutions in standard conditions. Light pathlength = 4 cm.

of the free dye in a very visooua morose solution is a proof

for it (Fig. k).

Influenoe of pH on the stability of DAPI-DHA oowplex. This

influence was examined by tltratlon of the complex solution

vith diluted NaOH or HC1 to the required pH and by Measuring

the I and P and also OD values in separate experiment. Ihe ob-

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Fig. 10. Excitation and fluorescence spectrum of DCI and DCI-DNA complex. The excitation .spectrum: la (DNA-DCI) complex, lb (DCI) solution. The fluorescence emission spectrum: 2a(DNA-DCI) complex, 2b (DCI) solution. DNA concn. in samples la and 2a was lOOjig/ml. DCIconcn. in all samples was 0.1^g/ml. The excitation spectrum measurements were made at emissionwavelength 450 nm. The emission spectrum (normalized to excitation spectrum) • at excitationwavelength 354 nm.

talned results (Fig. 2) indicate the stability of the complex

within a large range of pH (6) . A sudden fall of I end P in the

Interval of pH 11-12 may be due rather to alkaline DNA melting

(38) than to the change of the oharge of the DAPI ion. This is

confirmed by OD changes, kO% between pH 11.5-12. Previous

data relative to a close relationship between the fluoresoenoe

of the DAPI-DNA oomplex and the secondary DNA structure (6,8)

are oonfirmed by the experiment.

Thermal denaturation of the complexes. Ve observed an excep-

tionally high increase of the melting temperature, Tm, of the

DAPI-DNA oomplex in comparison with the Tm of DNA (Fig. 11).

Such a great difference (&T« = 21°C) was observed solely in the

case of some intercalating ligands (19,32,39) and the ohange of

transition profiles is a proof of the strong stabilization of

the structure of the DNA double helix by the dye. It should be

added that the low moleoular weight ligands for which the

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intercalating mechanism is exoluded, e.g. aliphatio diamines

(40,^1), or tetraalkyl-amcnlu salts (4a) shov a AIM several

tines lower, though the amount of bound llgand Is many times

higher than in the oase of DAPI (aee 'Binding studies', belov).

A small inorease of Tm = 1°C was also found for the DCI-DHA

oomplez (experimental oonditioms vere identioal to those des-

cribed in Fig. 11).

Influenoe of ONA structure on the ability ot formation of

oosiplexes with PAP1. A comparison of f luoresoenoe intensity of

DMA native, sheared, sonifioated and heat denatureted oomplexes

with DAPI administered in exoess, is presented in Fig. 12, Ao-

oording to these results the amount of forming oomplex depends

on mol. wt. of SNA. though DAPI does not form fluoresoent oom-

plexes with one-stranded DNA (8), the thermally denature ted DNA

shows a oertaln ability for oomplex formation. This is probably

due to denaturation, as reannealing prooeeds In the solution.

Influenoe ot visooelty of solution upon the stability of oom-

iplexes. Aooording to the experiment presented in Fig. 3B, D, the

Increase of solution vlsooslty oaused by the Increase of suorose

oonoentration has no significant influenoe on the stability of

DAPI-DNA oomplexes. The small increase of I and P is a proof that

there is but a small quantity of free dye, and that DAPI exists

there in an exceptionally rigid binding with DMA.

On the oontrary, the DCI-DNA oomplex undergoes almost oom-

plete decomposition in suorose concentrated solutions (Fig.

kB, D). As a strong inhibiting influenoe of ethanol (several %)

on the formation of the oomplex was also observed, we suppose

that the above desoribed effeot of solvent oages ooours.

Effect of salt oonoentration on DAPI-DNA oomplex is repre-

sented In Fig. 7. The effeot of *inrtTt*+ltArte I under the Influ-

enoe of MaCl was desoribed previously under slightly different

experimental conditions (6). The laok of diminution of P, as in

the oaae of the BthBr-DNA oomplex (for rev. see k3), is a proof

that the nature of the oomplex is not drastioally modified.

Influenoe of SDS on the dissooiation and on the formation of

complexes. Anionlo detergents, e.g. SDS, decompose oomplexes of

DNA with various dyes (26), without ohanging the DNA structure.

Probably the moleoules of a tv»9 dye are trapped in solvent

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It

linn'JT 1U

40 BO 100°G

Fig. 11. Comparison of melting temperatures of thymus DNA and its complex with DAPI. A (DNA),B (DAPI-DNA) complex. DNA concn. = 50 ug/ml, DAPI concn. = 5 pg/mL All solutions in standardBconditions.

5.0ngDIA/nl

Fig. 12. Fluorescence intensity I of the mixture. DAPI concn. = 20ijg/ml and DNA concn. = 50jug/ml—o— native, rnpl.wt. ~20.10 , —A — native sheared, mol.wt.~5.10 , — •— native sonificated,mol.wt.~4.10 , —•—denatured.

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oages made up of detergent moleoules. In binding prooesses in

the state of equilibrium the SDS owleoolea sequester the free

dye, thereby preventing the reverse raaotion to re-form the

oomplex (24,25). The interaction of SDS with DCI and DAPI mani-

fests itself, especially in the latter oase, in an enormous

increase of flnoresoenoe intensity I (this effect vas dlsoassed

previously) vhioh reaohes a value close to that of I of the

DAPI-DNA oomplex (Fig. 5A, 6A). The addition of SDS to the so-

lution of this oootplex is followed by an increase of I, for

vhioh the free dye is responsible. This level of I remains oon-

stant even after 2k h incubation at ,37°C (Fig. 5B). The reaar-

kable inhibiting influence of SDS on the formation of the DAPI-

DHA oomplex is oonfimed by the fact that, after addition of DMA

to DAPI-SDS solution,the increase of the intensity I is very

alov, after 2k h at 37° C only 1/3 of this quantity of the com-

plex is formed, vhioh fonts vithout the addition of SDS

(Fig. 5). It is difficult to interpret ohanges of P ooourring

under the influence of SDS in the oaae ot the complex and of

DAPI (this problem has been dlsoussed above).

TlhUiro the DAPI-DNA oomplex, the DCI-DNA oomplex undergoes

almost oomplete decomposition under the influence of SDS (Fig.

6). SDS Inhibits completely the formation ot the oomplex vhen

DMA Is added to the DCI-SDS mixture. The decomposition of the

oomplex is oonfiroed by the observed ohanges of P.

Binding studies. Villiamson and Fennell (2) found that vhen

using ion exohangers the DAPI-DNA oomplex may be separated

vithout changing the UNA structure. The reversibility of the

reaction of formation of this oomplex vith sonlfloated DNA vas

confirmed by preliminary observations on the eleotrophoretio

separation ot DAPI-DNA (plate eleotrophoresis, 1% agarose in

buffer (H), pH 7, ourrent density aoo. to Espejo)(44). Vhen

submitted to eleotrophoresis DAPI and DNA migrate in opposite

directions.

The dissociation oonstant of the DAPI-DNA oomplex vas esti-

mated by Kania et al. (3) as exceedingly lov (3.3* 10~12 M).

LdJcevise, our observations relative to the influence of SDS on

the stability of the oomplex (see above) confirmed a very

strong DAPI-DNA binding. This fact Imposed the necessity of

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examination of the states of equilibria of very dilated solu-

tions and in this situation the applloatlon of uv measurements

and dialysis m a Impossible. A. speotrofluorimeter of very high

quality vaa used, but in spite of that it was indispensable to

oarry out measurements at the highest degrees of amplification.

In oonsequenoef the exactitude of measurements vaa diminished

considerably and it was possible to estimate only the approxi-

mative values of the binding constant. The application of DNA

solutions of a concentration > 5 yug/al brought about a oonsi-

derable deorease of K and n values. This fact was not fully

dear to us. In the oase of DCI, the low value of the ratio V

of fluoresoenoe intensities emitted by bound and free dye and

the poor solubility of DCI in water, made the exact measure-

ments diffloult.

the shape of the Soatohard plot (k5,k6) for DAPI (Fig. 13)

indioatea the exist enoe of at least two kinds of DNA binding

sites differing In the affinity to DAPI. If we analyse the

ohanges of P in the f unotion of r and know the meohanlsm of

DAPI fluoresoeneejit may be supposed that DAPI is equally

rigidly bound to both kinds of binding sites and that the

quantum yields of fluoresoenoe of bound DAPI have in both

oases similar value. The numerieal data are presented in Tab-

le 2. It should be added that, in comparison with DAPI, the

results obtained for DCI show a muoh lower affinity of this

dye to BNA (Fig. 14).

The influenoe of hiatones (total fraotlon) on the affinity

constant K and on the number of DAPI-DNA binding sites n is

relatively small (Fig. 13, Table 3). This is a proof that blooking

of binding sites outside the double helix by various hi stone

fractions has no deciding signlfioanoe on DAPI binding in the

fluoresoent DNA oomplex. This is the next argument for the

Intercalating formation mechanism of this oomplex since a similar

phenomenon was observed in the oase of formation of intercal-

ating EthBr oomplexes with UNP

BINDING MECHANISM - CONCLUSIONS

DAPI and DCI are dyes the fluorescence efficiency of whloh

depends on the degree of inhibition of Internal rotational dif-

fusion prooesses. DAPI shows a pronounced affinity to the DNA

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Fig. 13. Scatchard function of DAPI-DNA binding —o— and of histone mixture —A—, and the changesof polarization coefficient of these complexes, P as a function of r —•—, —A— respectively, rightscale. See Methods

double helix. Hie quantity of a bound dye depends on mol. vt.

of DNA and nothing eonfiras the formation of a fluorescent ooo-

plex with one-stranded DNA or UNA (6). Fluoresoent DAPI and DCI

ooaplexea have a hi^x polarization ooeffioient indioating rigid

binding with the double helix. They also produoe a stabilizing

effeot on the aeoondary DMA struoture what is manifested in the

rise ot Da of ooaplexes. The free energy ohsnges (^O), calcula-

ted from the approximative values of K, are equal to about -11

and -8 koal/aol tor DAPI and DCI, respectively. These values

indicate that the oocurring interaction cannot be exclusively

due to the formation of hydrogen or ionlo bonds (50). This oon-

elusion is confirmed by the slow dissociation of the complex in

the presence ot SDS. The.calculated ohanges of free energy do

not indioate the formation of stable oovalent bonds. The disso-

ciation of the oomplex under the influence of a oationite (2)

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ourFig. 14. Scatchard function of DCI-DNA binding —o—, left scale and the change of polarizationcoefficient of fluorescence P, — #—, right scale as a function of r. See Methods.

and of an eleotrlo field has bean observed and thla ia also

evidence against the formation of theae.

The only explanation of the observed effect vould be the

supposition that stacking Interactions ooour aooompaning the

Intercalation of dyes (50). The oharaoterlstio changes of the

av absorption apeotrum do not exolade this hypothesis.

A slight influence of the presence of histones upon the

affinity oonstant K and the number of DNA binding sites, also

suggest the intercalating •eohanissj of fluorescent binding of

the DAPI-DNA complex.

Exaaination of atootiLo Models oonfinaed a potential possibi-

lity of intercalation of DAPI and DCT. The speolfioity of bin-

ding of those dyes with the DMA double helix, but not with RNA

is probably due to the difference of conformation of the poly-

nuoleotides (51). However, in our opinion, the hydroxide group

at C-2' may fora a spatial (sterio) hindrance for noleoulee of

intercalating ooapounds, the sizes of whloh are ooaparable to

thoae of DAPI or DCI (but greater than BthBr sizes) and the

presence of such a group stay aake incorporation of the dye

molecule into the double helix iapoaslble (Fig. 15a, b).

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Table 3. Approximative binding data obtained from toe inter-pretation of the Soatohard plot, Fig. 13. 14(see text)

Affinity oonstants K.(mole"1) K

*2

Number of bound dye n.•oleoulea/1 nuoleo—tide at saturation n,with dye

DAPI-DNA

3 x 108

3 x 107

0.010

0.026

DAPI-(DNA-HLstone)1:1 w/Sr

3 x 1 0 8

2 x 107

0.007

0.022

DCI-DNA

k x 10 5

0.017

The Intercalating mechanism does not exclude, obviously,

the ionlo binding of DAPI outside the double helix. Neverthe-

less, aooording to our observations, this kind of binding is not

the oause of the increase of fluoresoenoe intensity and there-

fore oatmot be observed speotrofluorlmetrioally. The binding of

QAPI outside the helix is oertainly much veaker than the inter-

calating binding. Probably, it is these interactions that

caused the differentiation of the buoyant density of DHAs ob-

served by Villiaason and Fennell (2). This oonolusion may be

drawn from the faot that to produoe the separation effeot, it

was neoessary to use muoh more OAFX, considering even the CaCl

effeot, than the maximum amount oapable of binding into the

fluoresoent oomplex. The ionio bond may be formed also with

one-stranded DNA. This was actually observed by the above men-

tioned authors.

Up till now, there are no experimental data relative to the

epeoifioity of fluoresoent DAPI complexes in relation to the

determined sequenoe of bases in DNA. It seems that there exist

at least two kinds of binding sites whioh bind the dye in the

same rigid way. Conclusions about the speciflolty of DAPI to

the sequences rioh in AT pairs were drawn from the experiments

in which DAPI was used in excess (2,3,8-10) and, for the reason,

it is difficult to oonolude as to the specifioity of the inter-

calating processes (numerous ionlo Interactions may mask the

stronger, but not numerous, intercalating bindings).

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DNA BASE

DAPI

ETBIDIUM

or DCI

Fig. 15. a and b. Schematic representation of intercalating model of DAPI or DCI binding with theDNA double helix. Dotted lines indicate the eventual ionic (DAPI) or hydrogen (DCI) bonds. Forcomparison, the dimensions of the Ethidium Bromide molecule are given in diagram b. Hatchedareas indicate a supposed steric hindrance in the case of the presence of a hydroxyl group at C—2\i.e. the steric hindrance which probably makes the formation of the intercalating complex withRNA impossible. The DNA base-pairs scheme ace. to W. Fuller 52.

In this eontext the question arises, whether it is possible

to observe ohanges of hydrodynaaio properties of superooiled

oiroular DNA under the influence of DAPI or DCI. If so, it

would be the aost oonvinoing proof of intercalation (for rev.

see 53). Tb±a proof is baaed on the fact that any interoalative

prooess requires looal unwind Ing of the helix at the point of

dye intercalation. At the oritloal level of binding (rQ) the

eirolea are unstrained and have no superoolls at all (5*0 • At

this point S2Q of the closed olroles ooinoides with the S2Q of

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nloked oirolea. lbs unwinding angle ( ) for DAPI and DCI Is

unknown, bat,since the aoleoules of these ooapounds are flat

and have no substltusnts union ooald defora toe helix (wedge

effeot), as It is the ease vith BthBr and Aetinoayoine D (55),

this angle should not exceed the value observed for proflavine,

and it should be olose to 0.7^ of JBthBr. "tiTi e <m the known re-

gularity that for a given DRA the produot (r «^) of various

dyes is a oonstant value and knowing r for SthBr (0.04 - 0.06)

(44,56), it is possible to oaloulate the anticipated rQ value

for DAPI and DCI (0.06 - 0.09). The oaloulated values of »Q are

nearly 3 tiaes higher than the aaxinua amount of the bond dye n

oaloulated froa Soatchard's plot. It does not seea to be easy

to discover the hydrodynaalo differences between nloked and

olosed oiroular DNA for r = 1/3 r by means of the aotually

applied methods (44,56).

ACKNOWLEDGEMENTS

The authors wish to thank Prof. K.L. VLerzehovskl and

Or H. Flkus for useful dlsousslons ooneeming this work. Appre-

tlatlon is expressed to Mrs. D. Kuoharozyk for her teohnioal

assistance and Mr*. J.PloszaJ and O.Krysztofiak for help in

preparing the manuscript for publication.

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