Interaction of cyanine dyes with nucleic acids . 5 . Towards model of « half intercalation » of monomethyne cyanine dyes into double-stranded nucleic acids

Recently we have proposed a model of «half intercalation» of monomethyne cyanine dye into the double-stranded helix. Benzothiazole terminal heterocycle «classically» intercalates, nestled between the adjacent base pairs just when second heterocycle is spatially fixed by nucleic acid groove. We consider that heterocycle with high basicity hits in the more nucteophilic groove whereas heterocycle with low basicity insert in more electrophilic interbase space. The purpose of this study is to determine the possible fixation mode of second heterocycle in nucleic acid groove. We synthesized and investigated the stries of structure-tike cyanine dyes which have different charge electronic distribution. We suppose that binding of second heterocycle of high basicity is caused by both spatial fixation and electrostatic interaction, with phosphate group of nucleic acid. Mommethyne pyridinium cyanine dye (Cyan 40; 4-[( 1-m.ethylbenzothiazoleilidene-2)methyIJ J,2,6-trimethylpyridiniurn p-toluenesulfonate interacted with native DNA and RNA with strong fluorescence enhancement. It seems to be perspective for the development of new nucleic acid binding cyanine dyes.

Introduction.The qualitative and quantitative determination of nucleic acids is of great significance for detection of pathogenic organisms (bacteria, viruses) [1 j, determination of mutated genes (hereditary diseases, such as diabetes mellitus, phenylketonuria, and sickle cell anemia) [2], and solving of sexual crimes in forensic medicine [3].Radioisotopes are widely used in the most approaches for the detection of nucleic acids.The numerous disadvantages of radioactive detection such as the handling licenses required, disposal costs, precautions necessary to prevent risks to health, short shelf-life have contributed to shifting the emphasis towards development of nonradioactive methods.
Fluorescent cyanine dyes are very promising for the development of new nonradioactive approaches of labeling and detection of nucleic acids.They are increasingly used for fluorescent microscopy, DNA sequencing [4], staining nucleic acids in gel electrophoresis [5].
Monomethyne cyanines are weakly fluorescent in free state but show strong fluorescence upon binding to DNA or RNA, and some of these dyes are the most sensitive nucleic acid stains currently available [6,7 ].Today commercial availability of highly sensitive spectroscopic apparatus promotes development of new homogeneous approach of nucleic acids determination.Capillary electrophoresis represents an additional improvement in assays of nucleic acids compared with conventional gel electrophoresis systems [8].
In homogeneous analysis there is no separation of fluorescence probe excess and there is record of fluorescence signal changing upon forming of probenucleic acids complex.The strong enhancement of fluorescence of dye molecules is the result of decreased rotational mobility around the internuclear bridge between the two aromatic ring systems of the dye chromofore [9 ].
The possible fixation mode is main question of any model of cyanine dyes interaction with nucleic acids.Jacobsen at al. [10] proposed «interaction model» of binding of TOTO to dsDNA involving the insertion of dye into the interior of (he helix with a conjugated ring system nestled between the adjacent base pairs of the DNA.It was shown that the binding of TOTO to larger oligonucleotides is site selective with CTAG : CTAG as preferred binding site [10].In Ihis model dye fixation is caused only by intercalation mechanism.
Recently Rye and Glazer have proposed a «partial intercalation» model of TOTO binding to DNA to explain stability and spectroscopic properties of ТО-ТО -ssDNA complexes.In their model the nucleic acid serves as a scaffold upon which highly cooperative dye-base-dye stack aggregates are formed.In their model dye fixation is caused by both stacking and intercalation mechanisms [10].
In our previous study we developed the possible «half intercalation» model of monomethyne benzthiazole cyanine dyes binding with double-stranded nucleic acids.We proposed that dye fixation is the result of both intercalation mechanisms and steric fitting of dye in nucleic acid groove [11 ].In this work we show that electrostatic interaction of high basicity hetorocycle with phosphate groups of nucleic acids may be possible mode of dye fixation too.
Materials and Methods.Spectroscopic measurements.The absorption spectra were obtained on Specord M-40 (Germany).Absorption of free dyes was measured immediately after dissolving dimethylsulfoxide (DMSO) dye stock solution in appropriate solvents: 50 mM Tris-HCl, pH 6 or DMFA.Fluorescence spectra were recorded with a serial fluorescence spectrophotometer Hitachi Model 850 (Japan).Fluorescence measurements were: carried out in thermostable quartz cell (0.5 χ 0.5 crn).Fluorescence was excited with a 150 Watt Xe-Iamp emission.
Preparation of DNA, RNA and dyes stock solutions.Stock solution of dyes (2-Ю' 1 M) were prepared by dissolving dyes in DMSO.All dyes were stable in this solvent for several months, whereas in aqueous solutions some dyes gradually lost their fluorescence properties.Working solutions were prepared immediately prior to use.For spectral studies total calf thymus DNA («Sigma», USA) and yeast RNA («Sigma») were used.Nucleic acids stock solutions were prepared in TE buffer (50 mM Tris-HCl, 10 mM EDTA, pH 8.0) in concentration 6.1 IO" 3 b.p. for DNA and 1.2 IO" 2 b. p. for RNA.
Absorbance and fluorescent emission spectra.For spectral measurements the complexes of dyes with nucleic acids were obtained by mixing dye stock solution with DNA or RNA solution in Tris-HCl (50 mM, pH 6) buffer.The final concentration of DNA an RNA was 1.2 IO" 4 and 2.4 IO" 4 M respectively.Final dyes concentrations were 0.02 mM.Dye-nucleic acid complexes were prepared with approximate ratio of 1 dye per 12 base of RNA and 6 b. p. of DNA.Fcr measurement of optical properties of free cyanines the same dyes concentrations were used.
Preparation of cyanine dyes.Standard procedures were used for the synthesis of unsymmetrical monomethyne cyanines [13].The purity of synthesized dyes was controlled by UV-spectroscopy and elemental analysis.
pH Investigation.Two procedures were used to study pH effect on spectral properties of dye/DNA complexes.In the first procedure dye/DN A complexes were prepared by adding dye stock solution to DNA working solution at pH 6. Ther pH of solution was changed to pH 5 by adding the corresponding amount of 0.05 M CH 3 COOH or to pH 9 by adding 0.1 M NaOH.In second procedure pH of DNA solution (50 mM Tris-HCl, pH 6) was previously changed to pH 5 or pH 9 and then dye stock solution was added.
Results.Dye structures and spectroscopic proper ties of free dyes.Physical data for three synthesized cyanine dyes (Cyan 39, Cyan 40 and Cyan 37), dye base (Cyan 42), salt of dye base (Cyan 41) and TO are presented in Table 1.The fluorescent proper ;ies of these dyes and their complexes with nucleic acids have not been previously studied.Dye TO with well investigated spectral properties was included for the comparison [14].The wavelengths of absorption maximum ( шо '^га«) of benzothiazole cyanines slightly depend on solvent.Absorption maxima of the dyes showed the red shift of 2-8 nm after going from aqueous buffer to less polar DMFA.The fluorescence intensity (I 0 ) of free dyes is very low and have no clear maximum ( em A max ) and Stokes shift (AS).Cyan 39, 40, 41 and TO have a strong tendency to form Η-aggregates in water buffer ( agr A raax ) in contrast to unpolar DMFA.Cyan 37 with polar carboxyl group aggregates weakly.
NA-Interaction properties of dyes.The data on the absorbance and fluorescence emission spectra oi the nucleic acid-bound form of dyes are presented in Table 2.All dyes had low Stokes shifts (AS, 28.0-78.0nm).Nucleic acid presence does not influence on absorption maximum ( abs A max ) of dye ( abs A max (complex) -mol A max (free); 0-4 nm).
The Cyan 40 with 1,2,6-trimethylpyridinium heterocycle has the most promising characteristics for the nucleic acids analysis.Its fluorescence intensity (J) is equal for DNA and 2.5-fold greater for the RNA than that of TO.Unfortunately, dye has high fluorescence in free state (0.8) in contrast to TO (0.09).It is not efficient for the homogeneous system detection (AQ dna = 21.8,AQ rna = 61.25) in contrast to TO (AQ dna = 186.0,AQ rna = 217.1).Titration of Cyan 40 with DNA and RNA.Fluorescence titration of Cyan 40 was carried out in 0.05 M Tris buffer (pH 6) with total calf thymus DNA and yeast RNA at 495 and 490 nm respectively (Fig. 1).The fixed dye concentration was 2 •1(Γ 5 M. The DNA and RNA concentration was changed from 2.9 IO" 6 to 4.1-10" 4 M and from 1.1-10" 5 to 9.1-IO" 4 respectively.For the Cyan 40-DNA complexes fluorescence plateau is reached at 6 base pairs per dye (A).Titration of RNA showed that the fluorescence emission of bound Cyan 40 reaches a maximum at 10 base/dye (B).
pH -Influence on the spectroscopic properties of cyanine dyes complexes with nucleic acids.Spectroscopic properties of complexes and dye aggregation sharply depend on pH changes (Table 3).In the case of pH 5 and pH 6 -» pH 5 there is strong aggregation C oi Df ir D < 1) in absorption spectra of dye-nucleic acid complexes.Consequently, fluorescence intensities at pH .5 are 3-10 times lower than at pH 6.
For the pH 9 and pH 6 -» pH 9, fluorescence intensities of complexes slightly decrease as compared to pH 6, but in absorption spectra molecular peaks increase C [ Dr ir D >1).
Discussion.The spectral characteristics of ionic cyanine dyes can depend on their concentration or counterion nature.In this case their disobey of Beer's law and change of their absorption characteristics were caused by the aggregation process [15], i. е., the formation of dimers, trimers, and n-iners.The aggregates of polymethyne dyes divided into H-aggregates (Η-band) with a blue shift and J-aggregates (J-band) with red shift from molecular absorption band (Mband) of unaggregated dye.It is considered that hydrophobic interaction causes the aggregation process preferably in aqueous solution [16].
Monomethyne cyanine dyes have a tendency to aggregate in aqueous solution [17].Their interaction with nucleic acids seems to be accompanied by aggregation process too, i. е., the formation of Haggregates with consequent increase of И-band intensity in absorption spectra and quenching of fluorescence intensity.Fig. 2 represents absorption spectra of Cyan 40 in nonpolar DMFA, aqueous buffer and in presence of nucleic acid.The additional absorption peak at shorter wavelength may arise from dime•' formation (Η-band, ig D).Intensity of Η-band decreases in aqueous solution of nucleic acid.Obviously, nucleic acid decreases hydrophobic or dispersion forces which cause dye aggregation process.Data presented in Table 3 show that increase of Η-band intensity relatively to that of M-band causes decrease of fluorescence intensity.All well interacted with nucleic acids monomethyne cyanine dyes seem to possess a high aggregation tendency in water solution and aggregation decreases after their interaction with nucleic acids [18].

Table 3 Dependence of the spectroscopic properties of cyanine dyes-nucleic acids complexes on pH of working solutions and procedure of their preparation
However, little is known about influence of aggregation interaction mechanism and possible model of cyanine dyes binding with nucleic acids.Recently proposed models of interaction of cyanine dyes with nucleic acids do not take into account aggregation processes [10].Besides, existence of Η-band of Η-aggregate at high dye : DNA ratios was the basis for the development of new binding mode of YOYO with double-stranded DNA [19].We assume that diitner formation and interaction with any substrate surface can explain a blue shift in absorption spectra of YOYO-nucleic acid complexes [20 [.Recently Rye and Glazer proposed «a partial intercalation-like» model of interaction of cyanine dyes with single-stranded nucleic acids.In their model, single-stranded polymer serves as a scaffolds upon which highly cooperative dye-base-dye stack aggregates are formed.This model is in gcod agreement with possible dye aggregation, i. е., it explains blue shifts in absorption spectra of the bounded dyes and quenching in fluorescence spectra [11].Obviously, stacking of cyanine dyes causes formation of aggregate structures like dye-dye, dye-dyebase and dye-base-dye under interaction of monomethyne cyanines dyes with double-stranded nucleic acids too.Titration of fixed amount of Cyan 13 with dsDNA (A) and titration of fixed amount of the same dsDNA with the same dye (B) were represented on Fig. 3.
Absorption and fluorescence spectra are not identical in the cases A and B. Titration of fixed amount of Cyan 13 with dsDNA changes only absorbance and lluorescense intensities, whereas reverse titration leads to spectral patterns changes.New H-aggregate structures at ratio 2 : 1 dye/b.p. are formed in the case B. In absorbance spectrum peaks at 420-440 nm decrease and new peak at 400 nm appears.Aggregation process is accompanied by quenching of main fluorescence peak (495 nm) and appearance new red shifted peak (540 nm) with low intensity.Double-stranded nucleic acids seem to serve as scaffold for the formation dye-dye-base and dye-dyedye-base aggregates like in Rye's model [11] for single-stranded nucleic acids.We suppose the process of formation of Η-aggregates with n> 2 begins at some excess of dye with participation of some structure elements of nucleic acid.
Recently we have developed the «half intercalation» model of interaction of monomethyne cyanine dyes with double-stranded nucleic acids.We proposed that one heterocycle of cyanine dye intercalates into the interior of the helix whereas second heterocycle is fixed in nucleic acid groove.According to this model, less basic hetercycle intercalates and fixation of more basic one appears to be the result of coulombic interaction with negative phosphate groups of nucleic acid [12].
In order to explain the possible fixation mode of basic heterocycle in the groove space we synthesized a series of structure-like cyanine dyes (Table 1).Unsymmetrical monomethyne pyrylium (Cyan 39) and pyridinium (Cyan 40) cyanine dyes have verysimilar chemical structures.The pyrylium heterocycle of Cyan 39 has however low basicity as compared to pyridinium moiety of Cyan 40.As a resit't, interaction of Cyan 40 with nucleic acids leads to enhanced fluorescence increase.Due to the presence of COOH group in Cyan 37 its interaction with nucleic acids and therefore fluorescence intencity of dye-nucleic acid complexes are lower than those of Cyan 40.
TO, its base (Cyan 42 ) and salt of this base (Cyan 41) have analogous chemical structures (Table 1).In this series, TO and Cyan 41 have similar electronic charge distribution.Acidification of Cyan 42 (base) at pH 5 protonates benzthiazole heterocycle to yield Cyan 41 (salt).In basic environment (pH 9) Cyan 41 (salt) is converted into Cyan 42 (base).The pH-depended absorption spectra of cyanine base and their salt clearly show large red or blue shifts caused by base-into-salt or salt-into-base conversion (Fig. 4).
Two procedures were used to prepare working solution of dye/DNA complexes.In the first procedure, dye/DNA complexes were prepared by adding dye stock solution to nucleic acid solution at pH 6 The pH of working solution was then changed to • 5 and pH 9 by acidification or adding of base consequently (Table 2, pH 6 -> pH 5, pH 6 -pH 9).In this case spectroscopic properties of dye/DNA complexes ( em A max , abs A max and S) were very alike.Probably all cyanines Cyan 41, Cyan 42 and TO interact with nucleic acids by similar mode.However, all dyes have low aggregation and high fluorescence intensity in basic environment in contrast to acidic environment C [ DZ itr D, I).In the second procedure, DNA solutions were prepared previously at pH 6, pH 9 and pH 5 (Table 3).Working solutions were prepared by adding stock dye solution to nucleic acid solutions with various pH.It was very surprising to observe change of colour of Cyan 42 (base) in basic environment (pH 9) after its interaction with nucleic acid.Perhaps formation of dye-nucleic acid electrostatic complex is the first stage of cyanine dye-nucleic acid binding.Spectroscopic properties of formed complexes are very similar to those of complexes prepared according to first procedure.
Recently we proposed possible mechanism of cyanine dyes-nucleic acids interaction [12].As a rule asymmetric cyanines in aqueous solutions exists in the form of aggregation structures (dimers, trimers).We suppose cyanine dyes in aqueous solution to interact with nucleic acids in aggregated form.«Nucleic acid medium» disperses dye aggregates that resulted in the fixation of monomelic dyes.
As a rule asymmetric cyanine dyes are'to represent dipoles because of their asymmetric arrangement of a charge.DNA also can be approximately considered as asymmetrically charged environment with negative charge located at sugar-phosphate backbone and positive charge in the interbase space.So we considered dipole-dipole interaction to play an important role in dye fixation.
«Nucleic acid medium» disperses dye aggregates resulting in the fixation of monomelic dyes.Intercalation of the dye into the double-stranded helix possibly takes place according to model of «half intercalation.One heterocyclic part of cyanine intercalates, nestled between the adjacent base pairs, just when another heterocycle is spatially fixed by the groove of nucleic acid.We suppose that binding of heterocycle of higher basicity is caused by electrostatic interaction with phosphate group of nucleic acid.The first stage of interaction is electrostatic external binding of the dyes aggregates.After that the heterocycle with low basicity intercalates into interbase space.
The increase of mixing ratio dye/nucleic acid results in the increase of H-aggregates cor сен !ration in working solution with rise of aggregate bands («blue shift»).At the same time, nucleic acids can serve as scaffolds for the formation of H-aggregates of another type for some dyes, with formation of aggregation structures which are not observed for free dye.In this case new aggregate bands are observed in the spectrum.Η-aggregates of this type (n > 2) were found for Cyan 13 (Fig. 3).Probably aggregates of stacking type could be formed on the accessible part of partly intercalated dye, i. e. heterocycle that was not placed between base pairs of nucleic acid.
This model accounts well also for the pHdepended experiments with cyanine dyes (Tabl.3).Acidic medium decreases coulombic interaction between helerocyclic bases of the dye and phosphate groups in the nucleic acid groove.In this case we observe an increase of dye aggregation and decrease -of fluorescence intensity of dye complexes with nucleic acids.On the other hand, basic medium increases the nucleophilicity of nucleic acid phosphate groups in contrast to acidification.11: causes strengthen ing of dye-nucleic acid complexes and increase of their fluorescence intensity.
So, model of partial intercalation of cyanine dyes can explain spectroscopic properties of cyanine complexes with nucleic acids.
Acknowledgements.We are grateful to Dr. I. Dubey for valuable discussions.