Formation of Stable BOBO-3 H-Aggregate Complexes Hinders DNA

J. Phys. Chem. B 2010, 114, 9063–9071

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Formation of Stable BOBO-3 H-Aggregate Complexes Hinders DNA Hybridization Maria J. Ruedas-Rama, Jose M. Alvarez-Pez, and Angel Orte* Department of Physical Chemistry, Faculty of Pharmacy, UniVersity of Granada, Campus Cartuja, 18071 Granada, Spain ReceiVed: April 7, 2010; ReVised Manuscript ReceiVed: June 1, 2010

In recent works, we have been studying the photophysics and binding properties of the trimethine cyanine homodimer dye BOBO-3, a DNA intercalative fluorophore that shows an important fluorescence enhancement upon binding to double-stranded DNA. During the course of studying the interactions of the dye with singlestranded homo-oligonucleotides we detected the apparition of an additional absorption band centered on 466 nm. The large hypsochromic effect and the fact that direct excitation of this band resulted in negligible fluorescence emission are characteristic properties of an H-type molecular aggregate. In this work we study the properties of this H-aggregate, and obtain by means of Principal Component Analysis the spectral shape and association constant of the complex. The H-aggregate complex shows very unique features. On one hand, the nucleotide bases cytosine or adenine are crucially involved in the formation of the aggregate. We describe here that at least six consecutive cytidine nucleosides are required to properly form the BOBO-3 H-aggregate complex. On the other hand, we demonstrate that the formation of such a stable complex prevents hybridization of the bases involved with their complementary strands. This phenomenon draws important conclusions on the anomalously high stability of the BOBO-3 H-aggregate complex. To the best of our knowledge, this is the first time such a stable H-aggregate of a dimeric cyanine dye facilitated by specific nucleotide bases in single strands has been reported. Introduction Cyanine dyes are a large family of fluorescent compounds used in a broad field of application. The common structural feature of these organic dyes consists of two heterocyclic rings linked by several methine groups. Cyanine fluorophores are used in laser technology,1,2 photography industry,3 as sensitizers in solar cells,4,5 or for optical data storage.6 In addition, it is in biological and biophysical applications where the cyanine dyes have undergone a recent boost. Variants of these fluorophores are employed as fluorescent tags for DNA or proteins in many biophysical studies,7 allowing even the detection of fluorescence from individual molecules. The cyanine dyes Cy3, Cy5, or Alexa Fluor 647, among many others, are widely used in single molecule fluorescence and single pair Fluorescence Resonance Energy Transfer (FRET) approaches.8 Several members of the cyanine dyes present the important characteristic of undergoing a large enhancement of the fluorescence emission upon binding to DNA. The dimeric cyanine dyes, composed of two linked cyanine moieties, show the largest fluorescence enhancements when bound to DNA. This feature makes dimeric cyanine dyes very useful for DNA staining in gel or capillary electrophoresis, or microchip-based sensing.7 Staining DNA with cyanine dyes have allowed imaging DNA by fluorescence microscopy9 to follow several properties such as size,10 relaxation,11 adsorption,12 or the effect of individual DNA repairing enzymes over double-stranded DNA (dsDNA).13,14 Furthermore, these dyes are also being used to determine chromosomes fine topology by using super-resolution fluorescence microscopy techniques.15 There are several proposed reasons underlying the fluorescence enhancement upon binding to DNA. Cyanine dyes in solution do not show a particularly high fluorescence quantum yield. * Corresponding author. E-mail: [email protected]. Tel: +34 958 243825. Fax: +34 958 244090.

There is experimental and theoretical evidence showing that the low quantum yield is caused by fast deactivation processes facilitated by the angular motion of the methine bridge.16-19 However, binding into dsDNA hinders the rotation of the heterocyclic rings along the central bridge, which is the main cause for the fluorescence enhancement.20-22 There are other additional processes that reduce the fluorescence quantum yield of the dyes in solution. For instance, the presence of nonfluorescent molecular aggregates, which are disrupted upon binding to DNA, has been suggested to importantly contribute to the low quantum yield of YOYO-1 in solution and the strong fluorescence enhancement in the dsDNA.16 As mentioned above, cyanine dyes have the capability to selfassemble into aggregates with a modified electronic configuration, the so-called J- and H-aggregates. These nanosized molecular aggregates have been extensively studied, especially J-aggregates, because of their technological potential as sensitizers of photographic films or in photoelectric cells.23,24 The difference between the two types of aggregates lies on the structural arrangement resulting in opposite effects over the electronic configuration. J-aggregates present a staggered selfassembly, causing a red-shift of the absorption band, and they are highly fluorescent. On the contrary, H-aggregates selfassemble in a face-to-face conformation with extensive π-packing that causes a hypsochromic shift of the absorption band,25,26 and a reduced fluorescence emission, because of strong excitonic coupling.16,22 Dye aggregation is commonly undergone in aqueous solution because of strong van der Waals attractive forces. This process is normally concentration-dependent, favored at high concentrations of the dyes. However, in dimeric cyanines, such as YOYO-1, the presence of H-aggregates in solution shows little dye concentration dependence,16,22 but a higher dependence on salt concentration.27 This is consistent

10.1021/jp103131r  2010 American Chemical Society Published on Web 06/24/2010

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with the formation of an intramolecular aggregate involving the two moieties of the dimeric dye. If the cyanine dye concentration is not high enough, selfassembly usually needs some type of templating. As these dyes are usually positively charged, their accumulation and aggregation can be typically facilitated by materials with large negatively charged surfaces. For instance, cyanine dye Haggregates have been described to form in polyelectrolytes,28-30 silica films31 or nanoparticles,32 Langmuir-Blodgett (LB) films,33 surfactants under micellar conditions,34 or calix-arenes.35 Similarly, J-aggregate self-assembly can be templated in LB monolayers,36 gold nanoparticles,37 polyelectrolyes,29 or bilayer lipid vesicles.38 Both single-stranded and double-stranded DNA molecules also fulfill this condition along the extensively charged phosphate backbone. Therefore, DNA molecules also have the potential to foster the formation of cyanine dye aggregates. Indeed, several examples of cyanine dye aggregates formed upon binding to dsDNA have been described in the literature including both J- and H-type aggregates.24,39-44 However, all of these previous reports describe templated aggregation along dsDNA but not on single-stranded DNA (ssDNA). Our recent research has been focused on the dimeric cyanine dye BOBO-3 (1,1′-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro(benzo-1,3-thiazole)-2-methylidene]pyridinium tetraiodide) and its potential use as an energy acceptor in fluorescence resonance energy transfer applications. In previous works we described the intercalating behavior and photophysics of BOBO-3 into ss- and dsDNA,45 exploring the base content dependence with DNA homo-oligonucleotides.46 We found the striking appearance of an absorption band around 466 nm when BOBO-3 interacted with some ssDNA, especially with a single strand composed exclusively of deoxycytidine nucleotides. In this work, we further investigate the nature, spectral characteristics, and relevance of this absorption band, which presents typical features of an H-aggregate, for instance, a remarkable hypsochromic effect. Although the formation of H-aggregates of cyanine dyes is well-known, we have found some unique properties reported here for the first time. Experimental Methods Reagents. All experiments were performed with analyticalreagent grade chemicals (obtained from Sigma-Aldrich, Spain, unless otherwise specified) and Milli-Q water. BOBO-3 iodide stock solution (Invitrogen, Carlsbad, CA) was freshly diluted in a pH 7.5 buffer solution that contained 10 mM Tris, 1 mM EDTA, and 100 mM NaCl (TEN buffer). BOBO-3 was added to the DNA solutions and incubated for 10 min in the dark at 25 °C. The pH of the solutions and buffers was adjusted with diluted NaOH and HCl dissolved in Milli-Q water. All of the chemicals were used as received without further purification, and the stock solutions were protected from sunlight and kept at about 4 °C in a refrigerator. The dilution effect in titration experiments was minimized by using a high concentration of titrant stock solutions. The oligonucleotides used in this work were obtained from IBA Technologies (Germany) and their sequences shown in Table 1. The synthesis of long poly inosine oligonucleotides is complicated, and the number of inosine residues left after purification ranged between 30 and 50. The DNA was purified by double HPLC, and received lyophilized. The stock concentration of each ssDNA oligonucleotide was verified by absorption measurements at 260 nm. Potassium salt of high molecular

Ruedas-Rama et al. TABLE 1: Sequences of the Tested DNA

weight polymeric poly cytidylic acid (Poly(C)) was obtained from Sigma-Aldrich (Spain). Instruments. Absorption spectra were recorded at 25 °C (unless otherwise specified), using 5 × 10-mm cuvettes with a Perkin-Elmer Lambda 650 UV/vis spectrophotometer equipped with a Peltier temperature controlled cell holder. Steady-state fluorescence emission spectra were collected at 25 °C on a JASCO FP-6500 spectrofluorometer equipped with a 450-W xenon lamp as the excitation source and an ETC-273T temperature controller. Principal Component Analysis. Principal Component Analysis (PCA) is a mathematical formulation used in multivariate analysis. It has been shown to be especially useful in absorption spectral analysis because it is possible to obtain simultaneously the number of components, their molar absorption coefficients, and the equilibrium constant between them. As the PCA method is well documented,47-49 here we only describe a brief summary of the procedure without going through the details of the method. A set of ns overlapping spectra, with varying concentrations of the optically absorbent components in solution, can be expressed together in an ns × nw data matrix A, where nw is the number of wavelengths recorded in each spectrum. If there are nc optically active components the matrix A can be written according to Beer’s law as:

A)c×ε

(1)

in which ε is the nc × nw matrix containing the molar absorption coefficients of each component at the different wavelengths, and c is the ns × nc matrix constructed with the concentrations of each component in every spectrum. By applying the extended PCA method one can first identify the number of orthogonal absorbing components, and then determine c and ε simultaneously. In our case, the different absorbers come exclusively from different states of BOBO-3. We define a matrix Ar in which the elements of each row have been divided by the total BOBO-3 concentration. The matrix Ar can be considered an apparent molar absorption coefficient. The problem of identifying the number of components that explain the variations in the absorption spectra can be merely reduced to the diagonalization of the product (ArT × Ar), where the superindex T indicates matrix transpose. The product (ArT × Ar) gives nw eigenvalues and the corresponding eigenvectors. The eigenvalues are decreasingly sorted, and a plot of the eigenvalues versus the corresponding index is used to identify a large gap between the ncth and the (nc + 1)th eigenvalues, where nc is the number of components that describe the system. Once the largest nc eigenvalues are chosen, the associated eigenvectors are gathered in the nc × nw matrix E. To find the molar absorption coefficients ε and the concentrations c, an unknown transformation matrix T, of the order nc

Formation of Stable BOBO-3 H-Aggregate Complexes

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× nc, must be obtained. Once the matrix T is known, ε and c are calculated from the equations:

ε)T×E

(2)

c ) A × ET × (E × ET)-1 × T-1

(3)

The estimation of the matrix T is performed taking advantage of the particular feature of an ns × nc matrix L given by:48

L ) Ar × ET × (E × ET)-1

(4)

The row elements of L satisfy a common equation. This equation is obtained by plotting the elements of each column of L against the first column. Importantly, the row elements of T must also fulfill the same equation. Therefore, T is estimated iteratively by using the following procedure. Only restricted values for the elements of T complying with the equation given by L are tried. Additional restrictions to the elements of T are added in order to force nonnegative values for ε and c, and take into account the mass balance. A possible equilibrium between the components is set, and the elements of c are used to calculate an equilibrium constant value Ki at each given condition. If the equilibrium is right, K must be constant over the ns different conditions. Therefore, the iterations to estimate T are those that minimize the parameter R given by: ns

R)

∑

1 (K - 〈K〉)2 ns · 〈K〉 i)1 i

(5)

where 〈K〉 is the average of the ns different Ki values. The minimization of R for elements of T that satisfy the restrictions has a 2-fold advantage: on one hand, different possible equilibria can be tried and those that give best results tested; and on the other hand, an equilibrium constant value is obtained along with the molar extinction coefficient and concentration matrices. Only for the correct T matrix and proper equilibrium scheme is the value of R minimized as well as all the elements of the matrices ε and c being exclusively nonnegative. The total procedure for the diagonalization, to obtain eigenvalues and eigenvectors, minimization of R, and T estimation, was coded in MathCad 14.0 (PTC, Needham, MA) using the eigenVals, eigenVec, Nipals, and Minerr fuctions. Results and Discussion Absorption and Steady-State Fluorescence Spectroscopy. We collected the absorption spectra of 1.5 × 10-6 M BOBO-3 in TEN buffer at pH 7.5 and increasing concentrations of either (dA)30, (dT)30, (dC)30, or (dI)30-50 ssDNA (Figure 1A and Figure SI 1 in the Supporting Information). The interactions between BOBO-3 and single strands of homonucleotides composed of cytosine or adenine bases gives rise to the formation of a complex with absorption around 466 nm. The spectra showed a gradual decrease in the absorption of free BOBO-3 and the appearance of the complex band at 466 nm for (dC)30 and to a less extent for (dA)30. Furthermore, this complex was detected to be nonfluorescent, because the excitation spectra (λem ) 600 nm) of BOBO-3 in the presence of (dC)30 or (dA)30 ssDNA did not show any band around 466 nm, nor was any emission detected when the samples were directly excited at 466 nm (data not shown). The large hypsochromic effect is characteristic of

Figure 1. (A) Absorption spectra of titration of free BOBO-3 (1.5 × 10-6 M) with (dC)30 ssDNA (concentration range: 5 × 10-9 to 6 × 10-8 M). (B) Absorption and (C) emission spectra (excitation wavelength ) 573 nm) of free BOBO-3 (1.5 × 10-6 M) (black), BOBO-3 + (dC)30 ssDNA (3 × 10-8 M) (red), BOBO-3 + (dC)30 ssDNA (3 × 10-8 M) + (dI)30-50 ssDNA (3 × 10-8 M) (blue), and BOBO-3 + (dCdI)30-50 dsDNA (3 × 10-8 M) (green). Note that the black and red lines are overlapped in panel C.

H-aggregates, which are well described for cyanine dyes interacting with negative polyelectrolytes,30,50 including dsDNA.40,43 Indeed, several examples of cyanine dye aggregates formed upon binding to dsDNA have been described in the literature.24 These aggregates usually present hypsochromic shifts in the absorption bands, suggesting stacking interactions, for instance in thiazole orange (TO),39 but in other cases, such as in Cyan βiPr, the aggregates present a staggered J configuration.42 Most of these aggregates are formed in the minor grooves of the double helix, because they provide the required fit and negatively charged environment to promote self-assembly of the dyes.40,42 Formation of dye aggregates in the minor grooves results in an interesting

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consequence: usually AT-rich regions are preferred opposite to GC grooves because of steric hindrance caused by guanine bases.40-44 There are several models of H-aggregates formed in DNA, such as dimers of 3,3′-diethylthiadicarbocyanine that grow cooperatively by adding additional dimers in adjacent sites,40,41,43 half-intercalation models proposed for TO and Cyan 13,39 or high order arrangements described for unsymmetrical cyanine dyes.44 However, all of these models template along dsDNA. This makes the BOBO-3 H-aggregate complex formed in single-stranded DNA rather unique. Another unique feature of the BOBO-3 H-aggregate that we have found is that certain specific bases, but not the others, seem to be crucially involved in the formation of the aggregate. More importantly, we investigated whether the interactions to form the H-aggregate complex might affect the hybridization of such bases with their complementary strands. When the dye interacted first with (dC)30 ssDNA the peak of the complex (maximum 466 nm) was observed, and it remained even after hybridization with its complementary (dI)30-50 ssDNA (Figure 1B). The spectral shape is completely different when the two strands are hybridized to form (dCdI)30-50 dsDNA prior to the addition of BOBO-3. Under such circumstances, there is no remaining absorbance of the complex at 466 nm. These results were also supported by fluorescence experiments. The enhancement of emission of BOBO-3 intercalated into (dCdI)30-50 dsDNA was higher when the dye was added after hybridization in opposition to a lower fluorescence intensity when the complex is preformed with (dC)30 ssDNA (Figure 1C). A similar behavior was observed in the case of the BOBO-3 complex with (dA)30, although the spectral differences between BOBO-3 intercalated into previously hybridized dsDNA and when the dye interacted first with (dA)30 ssDNA were smaller than those shown in Figure 1 for the cytosine-BOBO-3 system (see Figure SI 2, Supporting Information). We therefore focused our work on the interactions between BOBO-3 and cytosine-rich ssDNA. These experiments allowed drawing very important conclusions. The formation of the H-aggregate complex between BOBO-3 with the DNA single strands prevents total hybridization to the complementary strand, i.e., the interactions are rather stable and not all the bases are totally available to form hydrogen bonding with their complementary ones. This complex should therefore present a very high stability and certain structural hindrance for the formation of the duplex. However, when the duplex is previously formed, the BOBO-3 behaves as it normally does, showing typical spectral features in dsDNA: red shift of the maxima absorption and emission, and a large enhancement of the intrinsic fluorescence of the dye after binding without any new absorption band (Figure 1B,C). These results point out that when cytosine bases are interacting with inosines in dsDNA, they are not available for the formation of the H-aggregate complex with BOBO-3. This indicates that the cytosine moiety implicated in the formation of the hydrogen bond with inosine is directly involved in the formation mechanism of the aggregate complex with BOBO-3. Furthermore, the stabilities of the double helix with intercalated BOBO-3 and the BOBO-3 H-aggregate complex with cytosine are rather similar because the effects are not reversible and neither situation is capable of shifting the equilibrium of the other case. The BOBO-3 H-aggregate complex presents very striking and unique features when compared to similar cases found in the literature. First, the H-aggregate complex is specifically formed with cytosine and (to a less extent) adenine bases. This strongly suggests an important involvement of the bases themselves in the complex. By looking at the base structures, cytosine and

Ruedas-Rama et al. adenine have a primary amine group in positions 4 and 6, respectively, in a very similar environment. On the contrary, in all thymine, guanine, and hypoxanthine this primary amine is substituted by a carboxylic group. Therefore, the primary amine group may be involved in the formation of the complex of the bases forming a BOBO-3 H-aggregate. The second unique characteristic is the high stability of the BOBO-3 H-aggregate complex with cytosine bases. The few examples reported in literature of dimeric cyanine dye H-aggregates describe the formation of the aggregates in solution. However, the presence of dsDNA disrupts these aggregates, and the dye binds dsDNA in a monomeric form.16,22,27,51 Therefore, the H-aggregates of related homodimers, such as YOYO-1,16,22,27 or the bichromophoric Cyan 4051 are not as stable as the normal intercalation mechanism. On the other hand, we have detected an H-aggregate so stable that it is able to prevent hybridization of the DNA strand with its complementary. This clearly suggests that the mechanism of formation of the cytosine-induced H-aggregate must be different from that of the aggregates found in solution. In the next section we further explore the stability of the complex. Finally, we tested whether the cytosine-templated H-aggregate is a characteristic exclusive of BOBO-3, or a general feature of homodimeric cyanine dyes. We performed similar experiments but using YOYO-3 (quinolinium, 1,1′-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl]]bis[4-[3-(3-methyl-2(3H)-benzoxazolylidene)-1-propenyl]]-, tetraiodide) instead of BOBO-3. Figure SI 3 (Supporting Information) shows that a similar blueshifted band appears when YOYO-3 interacts with (dC)30 ssDNA. This suggests a common general mechanism underlying the formation of cytosine-induced H-aggregates of dimeric cyanine dyes, which we are currently investigating with other cyanine dyes. Melting Experiments. We support the conclusions on the stability of the complex with melting experiments followed by absorption measurements. Absorption spectra of (dC)30 ssDNA (2.5 × 10-7 M) in the presence of BOBO-3 (5 × 10-7 M) were recorded at different temperatures. The cuvette holders were thermostatically controlled between 20 and 70 °C. Neither the absorption intensity at 260 nm changed nor was the absorption maximum shifted for (dC)30 ssDNA in all range of temperatures tested (Figure 2A). This indicates the absence of self-hybridized double stranded structure in the presence of the dye. The absorption at 466 nm, corresponding to the H-aggregate complex, gradually decreased by increasing the temperature. This decrease indicates the partial decomposition of the complex at high temperatures (inset in Figure 2A). However, still at 70 °C there is a large contribution of the H-aggregate complex that remains stable. Absorbance spectra of previously hybridized (dCdI)30-50 dsDNA (2.5 × 10-7 M) mixed with BOBO-3 (5 × 10-7 M) and mixtures BOBO-3/(dC)30 ssDNA (with formation of the aggregate) and the subsequent addition of (dI)30-50 ssDNA were performed at the same concentration ratio for each component. When (dCdI)30-50 dsDNA was hybridized prior to the addition of the dye the absorbance in the DNA region, at 260 nm, was slightly higher than that in the case of the preformed complex. This effect indicates the existence of more base pair matches in the case of the prehybridized stands (dCdI)30-50 dsDNA. Hyperchromicity at 260 nm was observed over 30 °C as the DNA duplexes melted in both cases (Figure 2B,C). The increase of the absorbance at 260 nm was 25% when (dCdI)30-50 were hybridized prior to the addition of BOBO-3. In contrast, the absorbance increase was 13% when the complex was preformed.



Figure 3. (A) Titration of free BOBO-3 (5 × 10-7 M) at 20 °C with high molecular weight Poly(C) (1 × 10-7 - 2 × 10-6 M in bases). Titration of free BOBO-3 (1.5 × 10-6 M) at 60 °C with different sequences of ssDNA (5 × 10-9 to 5 × 10-8 M): (B) (Olig3C)21; (C) (Olig4C)26; (D) (Olig5C)28; and (E) (Olig6C)30.

Figure 2. Absorption spectra of (A) (dC)30 ssDNA (2.5 × 10-7 M) + BOBO-3 (5 × 10-7 M); (B) (dCdI)30-50 dsDNA (2.5 × 10-7 M) + BOBO-3 (5 × 10-7 M), and (C) (dC)30 ssDNA (2.5 × 10-7 M) + BOBO-3 (5 × 10-7 M) + (dI)30-50 ssDNA (2.5 × 10-7 M) at different temperatures: 20 (black), 30 (red), 40 (blue), 50 (green), 60 (pink), and 70 °C (yellow). Insets: Corresponding absorption spectra of BOBO-3 in each system.

The absorption spectrum of BOBO-3 concomitantly changed as a result of the separation of the strands as the temperature increased. The maximum at 573 nm, characteristic of BOBO-3 interacting with dsDNA, underwent a spectral shift and an increase of the shoulder corresponding to free BOBO-3 (inset in Figure 2B). After heating above the duplex melting temperature and slow cooling, the characteristic absorption peak of the H-aggregate complex appears. In this case, the separation of the strands allows BOBO-3 to form the complex with the cytosine-rich strand, preventing hybridization with its complementary upon cooling. On the contrary, when the complex with (dC)30 ssDNA was preformed, a small decrease in the complex

peak occurred with heating, but it remained even after the partial hybridization (inset in Figure 2C). After slow cooling, the absorption intensity always returned to the original value and the shape of the absorption spectrum of BOBO-3 was shifted to the initial form, meaning that the complex is stable to thermal cycling as happens for other cyanine dye H-aggregate forms.44 The H-aggregate complex is still very visible at 60 °C, which confirms its high stability compared to other cases. For instance, H-aggregates of TO-PRO-3 disappear at temperatures above 48 °C.44 Stoichiometry of the Complex BOBO-3/Cytosine. We investigated the minimal sequence of consecutive dC required to effectively form the complex with BOBO-3 in order to gain more insight into its stoichiometry and 3D arrangement. First, BOBO-3 was titrated with high molecular weight poly cytidylic acid (Poly(C)) (Figure 3A). The BOBO-3 H-aggregate complex is formed with Poly(C) as demonstrated by the clear absorption band at 466 nm. Next, similar titrations were carried out with single stranded oligonucleotides with a different number of consecutive cytosines from 3 to 6 cytosines at each end (see Table 1 for sequences). The experiments were performed at 60 °C to favor the separation of the strands, because there was certain possibility of self-hybridization of the strands promoted by the presence of the dye,45 which may prevent the formation of the complex of interest. The absorption spectra of the BOBO-3 during the titration with (Olig3C)21 (with regions of three consecutive cytosines) did show the reduction of the absorbance by increasing ssDNA concentration, but the characteristic peak of the H-aggregate complex was not observed (Figure 3B). In addition, there was negligible red shift of the absorption maximum. The same behavior was observed for mixtures of BOBO-3 with oligonucleotides containing either four or five consecutive cytosines ((Olig4C)26 and (Olig5C)28)

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(Figure 3C,D), in which the peak of the H-aggregate was not detected. When the number of consecutive cytosines in the sequence is low, the H-aggregate complex is not formed, probably due to an unsuitable three-dimensional conformation of the bases. However, when the titration is performed with a DNA strand containing six consecutive cytosine bases ((Olig6C)30) at 60 °C, the absorption peak at 466 nm was evident (Figure 3E). This suggested that the BOBO-3 H-aggregate complex with cytosine is not formed with less than six consecutive cytosine bases in the sequence, possibly related to its 3D structural arrangement. The fact that the formation of an H-aggregate complex of BOBO-3 with cytosines requires at least six dC in a row is in good agreement with other stoichiometries found in literature for templated H-aggregates of cyanine dyes. Gadde et al. found polystyrenesulfonate-templated H-aggregates of pseudoisocyanine and pinacyanol with a sulfonate/dye molar ratio about 3:1.30 Similarly, Armitage and colleagues reported a stoichiometry of 2.5 base pairs per dye in 3,3′-diethylthiadicarbocyanine aggregates formed in the minor grooves of poly(dAdT).40 If one considers an H-aggregate dimer, involving two cyanine dye units, these two examples showed a stoichiometry of six sulfonate groups and five base pairs per dimer, respectively. These values are similar to the requirement of six cytosine bases to form the BOBO-3 H-aggregate complex. In contrast, there are other examples of templated H-aggregates showing a different stoichiometry. For instance, Lau and Heyne reported recently a thiazole orange H-aggregate templated in calix[4]arene with a molar stoichiometry of 3:1 (TO:calix[4]arene). This involves three TO molecules in the aggregate and four sulfonate groups in the calix[4]arene.35 All our results put together and the previous literature provided the basis to set a model for the complex. Given BOBO-3 is a homodimeric cyanine with a flexible linker, we propose the H-aggregate is formed intramolecularly between the two cyanine units stacked in a parallel manner (see Figure SI 4, Supporting Information). Similar intramolecular H-stacking has been proposed as the feasible structure of other homodimeric cyanine dyes aggregates in solution.16,22,27 However, in the BOBO-3 aggregate found by us the primary amine groups of six cytosine bases (or adenine bases) must be essentially involved in a tridimensional arrangement by hydrogen bonding with the H-aggregate. We are currently testing structural and computational models to prove the validity of this model. Computational works have successfully shown the capability of nucleotide bases’ primary amine groups of forming Hbonding adducts with certain fluorophores.52 Moreover, cytosine bases have several groups available for hydrogen bonding and protonation that can lead to the formation of different structures as happens for instance in i-motifs. The i-motif is formed by DNA stretches of two or more cytidines: two cytidine stretches form a parallel-stranded duplex with C · CH+ pairs, with two of such duplexes associate head-to-tail by base pair intercalation into a quadruplex.53,54 This structure is favored at acidic pH, sometimes close to neutrality, but its formation depends on the total concentration and the specific sequence.55-57 We investigated whether the formation of the i-motif structure could be related to the observed H-aggregate of BOBO-3. Nevertheless, the absorption peak of the H-aggregate complex was still observed at basic pH (∼8.5) (Figure SI 5, Supporting Information). At these conditions the presence of the C · CH+ pairing needed for the formation of the i-motif structure is unlikely, which indicates that the cytidine stretches are not involved in the formation of the BOBO-3 H-aggregates. Therefore, other

Ruedas-Rama et al. processes such as electron withdrawing, protonation, and hydrogen bonding should play a crucial role in the formation of the H-aggregate complex described in this work. Principal Component Analysis To Resolve the Absorption Spectra. Principal Component Analysis (PCA) is a wellestablished mathematical tool, very useful in spectroscopy analysis for the identification of the minimum number of orthogonal species that originate variations in spectra of any kind. This is a technique-independent mathematical method that provides the number of species responsible for the main variations in a series of spectra. We applied PCA to the absorption spectra of BOBO-3 in the presence of either (dC)30 or (Olig6C)30 ssDNA. In both cases the H-aggregate complex is detectable (Figures 1A and 3E). The PCA method described in the Experimental Methods section has been successfully applied to obtain the spectral profiles of stacking aggregates of a monomeric cyanine dye, 3,3′-diethylthiacyanine iodide, formed in the presence of anionic surfactant aerosol-OT.34,48 We first run PCA to the absorption spectra of BOBO-3 in the presence of (dC)30. We globally analyzed the spectra of a titration of a fixed amount of BOBO-3 and increasing concentrations of (dC)30 (Figure 1A), as well as a titration of (dC)30 with additions of different amounts of BOBO-3 (Figure SI 6, Supporting Information). In total, the spectral matrix Ar was composed of 21 spectra with 251 different wavelengths each. The eigenvalues of the matrix (ArT × Ar) suggested the presence of solely two principal components, which explained 99.95% of the spectral variation. The first four eigenvalues were 4.1 × 1012, 1.5 × 1011, 1.5 × 108, and 4.3 × 107. The difference between the second and the third eigenvalue was high enough to consider only two main absorbers. We then applied the extended PCA method to obtain the 2 × 2 transformation matrix T. Because the number of principal absorbers was two, we posed an equilibrium involving free BOBO-3 and the H-aggregate complex with DNA. We established a tentative equilibrium constant K given by:

K)

[complex] [BOBO-3]m · [DNA]n

(6)

where [BOBO-3] and [complex] are given by the elements of the first and second column of the matrix c, and [DNA] is the amount of free DNA. The exponents m and n are related to the order of the complex formation. We minimized the value of R (eq 5) by calculating the Ki values for exponents n ) 0 and 1 and m ) 1, 2, 3, and 4. The exponent values in eq 6 that satisfied the set of restrictions (nonnegative elements for matrices c and ε, mass balance, and minimum R) were m ) 1 and n ) 1, with an average value for K of (2.3 ( 0.8) × 107 M-1. Once the matrix T is determined, the 21 × 2 matrix c of concentrations of each species and the individual molar extinction coefficient 2 × 251 matrix ε are obtained through eqs 2 and 3. The spectra of the two species are depicted in Figure 4A. One of the species shows the spectral profile of free BOBO-3 in solution, whereas the second form shows a single band peaking at 463 nm, with an extinction coefficient ε463 of 54 840 M-1 cm-1. The concentrations of each absorber in both experiments are shown in Figure 4B. We focus first on the titration of BOBO-3 with (dC)30 (Figure 4B, open symbols). At low ssDNA concentrations the free dye is the major component. The free BOBO-3 disappears and more H-aggregate complex is formed as ssDNA is being added. We globally fitted both traces to a Hill equation58 and obtained an apparent formation constant of (3.9 ( 0.3) × 107 M-1, with a Hill coefficient of 1.4 ( 0.2 that indicates a certain



Figure 4. PCA results of the interaction of BOBO-3 with (dC)30 (A and B) and (Olig6C)30 (C and D). (A) Molar extinction coefficients of the absorber 1 (free dye, black solid line) and absorber 2 (H-aggregate complex, red solid line) obtained for BOBO-3 in the presence of (dC)30. (B) Obtained concentrations of free BOBO-3 (black symbols) and H-aggregate complex (red symbols) in the titration of 1.5 × 10-6 M BOBO-3 with (dC)30 ssDNA (open symbols) or the titration of 6 × 10-8 M (dC)30 ssDNA with BOBO-3 (closed symbols). The x axis indicates the titrant concentration in each experiment, either (dC)30 ssDNA or BOBO-3. Solid lines represent the global fit of the data to a Hill equation. (C) Molar extinction coefficients of the absorber 1 (free dye, black solid line) and absorber 2 (interaction with ssDNA, red solid line) obtained for BOBO-3 in the presence of (Olig6C)30. The decomposition of the absorber 2 in two components, H-aggregate complex (dashed green line) and electrostatic interaction with ssDNA (dashed orange line), are also shown. (D) Mole fraction of free dye (black), H-aggregate complex (red), and electrostatic interaction with ssDNA (blue) obtained for the titration of 1.5 × 10-6 M BOBO-3 with (Olig6C)30 ssDNA.

degree of heterogeneity in the binding sites. This formation equilibrium constant represents the midpoint of the transition in the formation of the complex and should only be considered approximate for the given conditions. However, it is in good agreement with that obtained in the recovery of T. The titration of (dC)30 with BOBO-3 showed increasing concentrations of both free and complexed dye (Figure 4B, closed symbols) as the total BOBO-3 concentration increased. The ratio free dye: complex, however, changes from values as high as 1.5 to values around 0.4 with high contributions of the complex. At low total dye concentrations the free BOBO-3 is the major species. As the total concentration increases, the complex contribution becomes higher. Finally, the free dye proportion increases again when the complex is close to saturate the available binding sites in the ssDNA, and further addition of dye just stays as free chromophore. These trends can be seen in Figure SI 7 (Supporting Information) and they are in agreement with the equilibrium theory and saturation of the binding sites. Finally, the obtained molar extinction coefficients ε and concentrations c are able to simulate the experimental absorption spectra through eq 1 as can be seen in Figures SI 6 and SI 8 in the Supporting Information. An interesting result is the value of m ) 1. This supports the picture described above, that the H-aggregate complex is formed with a single molecule of BOBO-3. The structure therefore should be the intramolecular stacking of the two cyanine groups of the dimer facilitated by the bending of the flexible linker group (Figure SI 4, Supporting Information), similar to the intramolecular H-stacking suggested for molecular aggregates of homodimeric cyanines in solution.16,22,27 We also applied PCA to the titration of a constant amount of BOBO-3 with (Olig6C)30 ssDNA shown in Figure 3E. We used 7 different spectra so the spectral matrix A dimensions were 7 × 251. The nonzero eigenvalues of the matrix (ArT × Ar) were 3.2 × 1012, 1.2 × 1010, 2.5 × 107, and 1.6 × 107, supporting

that only two spectral components were required to account for 99.93% of the spectral variation. We obtained the transformation matrix T by applying the minimization method described above, and the concentration 7 × 2 matrix c and molar extinction coefficient 2 × 251 matrix ε. The spectra of the two species are shown in Figure 4C. The spectral profile of free BOBO-3 is in good agreement with that recovered for (dC)30 (Figure 4A). Nevertheless, the absorption of the second species shows a broad profile with contributions from 450 to 600 nm. Two main bands can be inferred: one around 460 nm and another centered around 570 nm. Assuming the absorption at 460 nm is caused by an H-aggregate complex with the cytosine bases at the two ends of the ssDNA strand of the same nature of that formed with (dC)30, we decomposed the spectral profile into two contributions: the pure H-aggregate complex, whose profile is known (Figure 4A), and the rest obtained by subtraction. We applied a factor of 0.45 to the total complex spectral profile and subtracted this from the total spectrum of species 2. We obtained a broad band centered at 562 nm (Figure 4C). Given the sequence of the ssDNA oligo (Olig6C)30, the H-aggregate complex of BOBO-3 induced by cytosine bases can be formed only at the two ends of the ssDNA. However, the BOBO-3 may interact with the central region of the ssDNA just by electrostatic binding as it occurs in randomly sequenced ssDNA. We described in a previous paper that interactions with ssDNA cause shifts in the BOBO-3 absorption spectra.45 Therefore, we can assign the peak of species 2 centered at 562 nm to this kind of interactions with ssDNA. The picture is therefore BOBO-3 forming the H-aggregate complex at the two ends of the single strand (Olig6C)30 and interacting electrostatically at the central part of the ssDNA. It is known that there are cases in which different species may appear as a single one in PCA, for instance, when the concentration of two species are always proportional to each other, so they do not cause orthogonal spectral changes. It is therefore more correct to use the term

9070


“absorber” to allude to species in PCA.47 The fact that both the H-aggregate complex and the electrostatically driven interactions of BOBO-3 behave as a single absorber confirms that their concentration grows proportionally to each other as a function of (Olig6C)30 concentration. This suggests that both types of interactions have comparable affinity constants to each other. However, the H-aggregate complex cannot form in the central region of the ssDNA, so the two types of interactions are not competing in this region. Finally, Figure 4D shows the molar fraction of each species, free dye, H-aggregate complex, and BOBO-3 interacting electrostatically with ssDNA. The molar fractions were obtained from the concentration matrix c and taking into account the spectral decomposition shown in Figure 4C for the two types of dye interaction with ssDNA. Figure SI 9 (Supporting Information) shows the good agreement found between the experimental absorption spectra and those calculated with eq 1 and the obtained spectral profile of the two absorbers, ε, and the concentration matrix c. Conclusions Intercalative binding dyes are widely used in biological applications to stain dsDNA because of their large fluorescence emission enhancement upon binding to the double helix. In a series of papers, we have thoroughly described the photophysics and binding behavior of a trimethine cyanine homodimer, BOBO-3, a potential energy acceptor FRET-based hybridization detection approaches. In previous works we investigated the intercalating and photophysical features of BOBO-3 in DNA with mixed base composition and in homo-oligonucleotides.45,46 The major findings were that BOBO-3 interacts with dsDNA by means of two different binding modes, and that important differences can be found depending on base composition. In the course of this research we found the formation of a complex between the BOBO-3 and certain homonucleotide ssDNA. This complex gives rise to an absorption band at 466 nm. Here we have presented an in-depth study of the behavior of this complex. The BOBO-3 complex shows the main features of an H-aggregate: hypsochromic effect and negligible fluorescence. This H-aggregate is only formed in the presence of cytidine homopolynucleotides, and less remarkably in adenosine polynucleotides, which suggests an important role of the bases themselves through the primary amine groups. We have obtained the spectral profile of the hypsochromatic band by the PCA method, and concluded that at least six consecutive cytosine bases are required for the formation of a stable complex, although further work is ongoing to investigate its structural arrangement. Importantly, we have demonstrated that the formation of such stable complex prevents hybridization of the bases involved with the complementary strands. Therefore, this complex between BOBO-3 and cytosine or adenine bases may be a handicap in the development of methods for the analysis and quantification of dsDNA based on intercalating dyes, such as fluorescence in situ hybridization (FISH) methods.59,60 Under similar conditions, oligonucleotides containing polycytidine sequences might form the H-aggregate complex at physiological pH, preventing their biological activity. In particular, the C-rich strand of telomeres61,62 is a potential example, whose structure is of current interest. Therefore, the consideration of such interactions is an important aspect when BOBO-3 is used as staining dye. Acknowledgment. This work was supported by grants CTQ2007-61619/BQU from the Ministerio Espan˜ol de Educacion y Ciencia (cofinanced by FEDER funds) and P07-FQM-

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JP103131R

Formation of Stable BOBO-3 H-Aggregate Complexes Hinders DNA

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