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