Photoisomerizable arylstilbazolium ligands recognize parallel and antiparallel structures of G-quadruplexes

International Journal of Biological Macromolecules 51 (2012) 576–582

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Photoisomerizable arylstilbazolium ligands recognize parallel and antiparallel structures of G-quadruplexes Izabella Czerwinska 1 , Bernard Juskowiak ∗ Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland

a r t i c l e

i n f o

Article history: Received 28 May 2012 Received in revised form 20 June 2012 Accepted 21 June 2012 Available online 29 June 2012 Keywords: Arylstilbazolium ligands Binding selectivity Equilibrium dialysis G-quadruplex Trans-cis photoisomerization

a b s t r a c t The photoisomerization and DNA interaction studies of three arylstilbazolium derivatives with various samples of nucleic acids (duplexes, triplexes and tetraplexes) are reported. The equilibrium dialysis study revealed high binding affinities of ligands to tetraplex structures. The quadruplex-binding affinity could be switched by light, e.g., the E,E and E,Z isomers of 1,4-bis(vinylquinolinium)benzene (1) interacted with parallel and antiparallel tetraplexes exhibiting different binding selectivity. The E,Z-1 showed higher binding preference for c-myc DNA (a propeller-type quadruplex), whereas the E,E-1 favorably interacted with telomeric DNA (a basket-type quadruplex). The presence of quadruplex DNA hampered photoisomerization of quadruplex-bound ligand. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The DNA structure that attracted a particular research interest in the past decade was the four-stranded DNA alternatively termed G-quadruplex, tetraplex or G4 DNA structure [1]. Interest in the more general therapeutic significance of G-quadruplexes has increased mainly due to their presence in human genome. It has been shown that tetraplexes play an important role inside living cells [2–4]. In G-quadruplex, the stacked guanine-tetrads (G-quartets) formed by the coplanar arrangement of four guanines interconnected by Hoogsteen-type bonds constitute the core of the structure. In general, G-rich strand orientation and variations in loop size and their arrangement, determine tetraplex topology [5,6]. For instance, different G4 structures have been identified in vitro for the sequence of human telomeric repeats d(TTAGGG)n . The NMR, crystallographic, and other studies revealed a baskettype [7,8], a chair-type [9,10], a propeller-type and a mixed-type G4 DNA structures [11–13] depending on the experimental conditions. Moreover, tetraplex-forming sequences have been found in promoter regions of genes involved in carcinogenesis (e.g., c-myc) [14–17].

∗ Corresponding author. Tel.: +48 61 829 1467. E-mail address: [email protected] (B. Juskowiak). 1 Present address: Department of Applied Chemistry, Kyushu Institute of Technology, Kitakyushu-shi, Fukuoka 804-8550, Japan. 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.06.027

Taking advantages of the ability of some ligands to bind G-rich sequences and to stabilize G-quadruplex structures, new strategies in the anticancer therapy have been developed. One is based on the inhibition of telomerase, a reverse transcriptase that is active in most cancer cells. Ligand that induces folding of telomeric DNA (primer for telomerase) into G-quadruplex conformation may inhibit telomerase. As a consequence, telomerase cannot effectively elongate telomeric DNA that leads to telomere shortening and death of tumor cells [18,19]. In accordance with a second strategy that concerns stabilization of G-quadruplexes in gene promoter regions, small ligand molecules may stop the growth of cancer cells by interacting with promoter G4 structures and blocking gene expression [20,21]. Both strategies are driving forces in the anticancer drug discovery research and provide motivation for design of new selective G-quadruplex ligands [1,22,23]. The selectivity and structural binding preferences of DNA-interacting ligands are the most important factors that influence their activity. The extended aromatic ring system and/or positively charged atoms, have a remarkable impact on specific ligand–quadruplex interactions. The commonly observed ligand/G4 DNA binding mode consists in stacking of ligand on the plane of exposed guanine tetrad (end-stacking) and the resulting complex is stabilized by a combination of ␲–␲ stacking, hydrogen bonding, coulombic attractions etc. [1]. However, not only the presence of some structural constituents determines the strength and the mode of interaction, but also the spatial arrangement of these groups may affect binding affinity and preferences of the ligand. For example, the cis-trans photoisomerization is a suitable tool that allows controlled manipulation of the ligand conformation.

I. Czerwinska, B. Juskowiak / International Journal of Biological Macromolecules 51 (2012) 576–582 Table 1 Nucleic acid samples used in competition dialysis assay.

2. Experimental

DNA index

Sequence

Monomeric unit

#1

Poly[dA]

Nucleotide

#2

Poly[dT]

Nucleotide

#3

Poly[dA]*Poly[dT]

Base pair

#4

Poly[dG]*Poly[dC]

Base pair

#5

Poly[dA-dT]*Poly[dA-dT]

Base pair

#6

Poly[dG-dC]*Poly[dG-dC]

Base pair

#7

5’-CAATCGGATCGAATTCGATCCGATTG-3’

Base pair

#8

TT-CTTTCTCTCCTCC-3’ 5’-GAAAGAGAGGAGG-3’ CC-CTTTCTCTCCTCC-5’

577

Triplet

#9

Poly[dT]*Poly[dA]*Poly[dT]

Triplet

#10

5’-AGGGTGGGGAGGGTGGGG-3’

G-quartet

#11

5’-AGGGTTAGGGTTAGGGTTAGGG-3’

G-quartet

#12

5’-TTGGGGGGGGGGGGGGGGGGGGTT-3’

G-quartet

2.1. Materials Arylstilbazolium derivatives 1–3 were obtained as trans isomers by the conventional aldol condensation [28]. Cis isomer of 1,4-bis(vinylquinolinium)benzene derivative (E,Z-1) was obtained in the photoisomerization process from trans isomer. Irradiation of E,E-1 and subsequent HPLC separation of the isomeric mixture yielded E,Z-1 isomer. An isocratic elution was applied with a flow rate of 1 ml/min and eluent that contained 70 mM ammonium acetate in 33% acetonitrile. LaChrom HPLC system (Merck/Hitachi, Japan) was composed of dual L-7100 pump system, L-7455 diode array detector and D-7000 interface. Separations were carried out with an Inertsil ODS-3 column, dp = 5 ␮m, 250 mm × 4.6 mm i.d. (GL Sciences Inc., Japan). The nucleic acid samples used in equilibrium dialysis experiments are listed in Table 1. Polynucleic acids were purchased from Sigma Chemical Co. (St. Louis, USA) and were used as received. Oligonucleotides were synthesized and HPLC-purified by Metabion Int. AG (Martinsried, Germany). Other reagents were of analytical grade purity and were used as received. High-purity water (Polwater, Poland) was used throughout this study. 2.2. Measurements and methods

Binding preferences and photoisomerization of some arylstilbazolium derivatives have been investigated previously against double-stranded DNA [24–27]. Two intercalators: E-1-(vinylpyridinium)naphthalene and E-2(vinylpyridinium)naphthalene exhibited similar spectral changes upon binding to calf-thymus DNA. Observed hypochromicity of the absorption bands of ligands (300–400 nm) was consistent with their similar binding affinity to DNA, whereas the binding constants for Z isomers of both ligands were two times lower. The reason for different binding affinities of E and Z isomers was associated with planarity of the ligand structure. The cis isomers exist usually in the non-planar conformation; therefore, an insertion of aromatic rings of intercalator between DNA base pairs was hindered [24]. We have also demonstrated that the size of aromatic part of ligand had an impact on the stoichiometry of DNA–ligand complex formation. The MALDI-TOF mass spectra showed that trans isomers of some arylstilbazolium derivatives exhibited different relative abundance and stoichiometry of complexes formed with both hairpin and bulged duplex structures of sT3 oligonucleotide (5 -CGCTTTGCG-3 ) [25]. In addition, we reported that replacement of pyridinium rings with larger quinolinium groups influenced the DNA binding preference of ligand [26]. These results encouraged us to undertake the photoisomerization and DNA interaction studies with selected arylstilbazolium derivatives (Fig. 1) and various samples of nucleic acids including triplex helices and tetraplex DNA structures (Table 1). We assumed that ligands shown in Fig. 1 are good candidates to serve as G-quadruplex stabilizing agents mainly because of the presence of extended ␲-electron systems and positively charged nitrogen atoms. Moreover, upon light illumination, the trans isomers can undergo controlled isomerization into cis isomers and this property can be useful in controlling the stabilization of G-quadruplex structures. In this paper we present the photoisomerization of arylstilbazolium ligands 1–3, mediated by the complexation with tetraplex DNA. The effect of ligand isomer structure on the DNA binding preferences is also reported on the example of 1,4-bis(vinylquinolinium)benzene (ligand 1) using competition dialysis experiments.

Equilibrium dialysis experiments were performed as described elsewhere [29]. Briefly, a dozen of dialysis units (Pierce; Molecular weight cut off of 3500) containing nucleic acid samples DNA #1–DNA #12 listed in Table 1 (100 ␮L; 75 ␮M in terms of the monomeric units) and one dialysis unit with BPES buffer as a reagent blank (100 ␮L) were placed in a beaker with ligand solution (250 mL of 1 ␮M) in BPES buffer (6 mM Na2 HPO4 , 2 mM NaH2 PO4 , 1 mM Na2 EDTA, 185 mM NaCl, pH 7) and equilibrated with continuous stirring for 24 h at room temperature. Next, the DNA samples were transferred to a microplate and the ligand/DNA complexes were dissociated by adding 1% SDS (sodium dodecylsulfate). Finally, absorption and emission spectra of all samples were recorded using a microplate reader (Infinite M200, Tecan, Austria). Two main parameters were calculated using absorbance or fluorescence intensity measurements. Firstly, concentration of bound ligand (Cb ) was obtained from the relationship Cb = Ct − Cf (Ct – total concentration of ligand and Cf – concentration of free ligand). Total and free ligand concentrations were determined using calibration graphs that were obtained for each compound. Free ligand concentration (blank sample) did not vary significantly from the initial value of 1 ␮M. Secondly, the apparent binding constants (Kapp ) were calculated using the simple relation Kapp = [Cb ]/[Cf ] [NAt − Cb ], where NAt is the total nucleic acid concentration expressed in monomer units. Photoisomerization experiments were carried out by irradiation of sample solution at selected wavelength in the thermostated (25 ◦ C) cell compartment of spectrofluorimeter (Shimadzu RF 5000, Japan) equipped with a 150 W Xe lamp. Buffer BPES solution (1 mL) containing ligand (10 ␮M) and DNA (20 ␮M) was placed in a quartz cell, deaerated with argon and irradiated. The trans → cis photoisomerization was observed by recording chromatograms of samples (30 ␮L) collected at appropriate time intervals. Separation of photoproducts was carried out with LaChrom HPLC system (Merck/Hitachi, Japan) using XTerra MS C18 column (dp = 3.5 ␮m, 150 mm × 4.6 mm i.d., Waters, USA), with gradient elution method. The solvent A with 100 mM triethylammonium acetate (TEAA) in water and the solvent B containing the same concentration of TEAA in 80% acetonitrile were used for gradient elution. Linear gradient was run at a flow rate of 1 mL/min): 0–20% B increased in 20 min and 20% of B was maintained for another 5 min, next 20–70% B

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Fig. 1. Structures of arylstilbazolium ligands: E,E-1,4-bis(vinylquinolinium)benzene (1), E,E,E-1,3,5-tris(vinylpyridinium)benzene (2), E-9-(vinylpyridinium)phenanthrene (3).

increased in 10 min and 70% B was maintained for another 5 min. Chromatograms were monitored at the isosbestic point of ligand, and the conversion percentage of trans isomer was determined from peak areas of trans and cis isomers. Irradiation and monitoring wavelengths for particular ligands were as follows: 450 and 394 nm (ligand l), 390 and 285 nm (ligand 2), and 390 and 302 nm (ligand 3). The trans → cis photoisomerization was also monitored by recording fluorescence traces against the time of irradiation. The same photoisomerization experiments were performed for free ligands (10 ␮M) and in the presence of DNA (20 ␮M).

3. Results and discussion 3.1. Equilibrium dialysis assay: nucleic acid binding preferences In order to investigate binding selectivity of ligands to different DNA samples, the equilibrium (competition) dialysis method was used [29–33]. In the competition dialysis experiment, the different nucleic acid samples are placed in separate dialysis units and dialyzed against a common solution of free ligand. After equilibration, the amount of ligand bound to each DNA sample is determined using proper analytical technique. Upon equilibration, the amount of bound ligand is directly proportional to the binding constant of the particular DNA/ligand complex and represents the ligandbinding preferences. Concluding, the DNA/ligand complexes are simultaneously investigated in this competition assay, hence the equilibrium dialysis method is a powerful tool for studying the ligand-DNA binding selectivity [30–33]. DNA binding preferences of ligands 2 and 3 have been reported previously [34]. Therefore, equilibrium dialysis experiments were carried out for ligand 1 only. Irradiation of the solution of 1 and subsequent semi-preparative HPLC separation yielded two photoproducts of 1, the E,E-1 (trans) and the E,Z-1 (cis) isomers. These compounds were dialyzed against a dozen of different nucleic acids: oligo- and polynucleotides presented in Table 1. It is worth mentioning that oligonucleotide #10 represents sequence of human c-myc oncogene promoter and oligonucleotide #11 possesses human telomere sequence. Dialysis experiments were performed according to procedure described in Section 2.2 and after the SDS treatment to dissociate the complexes, the concentrations of bound E,E-1 or E,Z-1 were calculated from calibration graphs. Fluorescence measurements were carried out with excitation/emission wavelengths of 442/521 nm for E,E-1 and 410/512 nm for E,Z-1, respectively. Calculations based on the absorption spectra were done for abs = 410 nm and 442 nm for cis and trans isomer, respectively. In order to show the

competition dialysis results in a more distinguishable way, the amounts of bound ligands to each nucleic acid are represented as a 3D bar graph (Fig. 2). The nucleic acids can be grouped into categories according to different DNA forms: single-stranded DNA (ssDNA: #1, #2), double-stranded DNA (dsDNA: #3–7), triplestranded DNA (triplex: #8, #9) and four-stranded DNA (tetraplex: #10–12). From the results shown in Fig. 2, it is clear that E,E-1 and E,Z-1 exhibit binding preferences for AT base pairs (#3 and #5) if one considers double-stranded DNA and only a faint binding to single-stranded DNA is observed. Both isomers display appreciable binding affinity to triplestranded oligonucleotides, but E,Z-1 binds triplex #8 with higher selectivity. The highest binding affinity is observed for all fourstranded DNA samples included in the assay. Finally, a striking result that emerges from the competition experiments is a strong preference of E,E-1 for tetraplex #11 possessing human telomere sequence, for which CD spectrum (Fig. S1) confirmed an antiparallel quadruplex structure stable in room temperature (Fig. S2) in agreement with literature reports [7–10]. The amount of E,E1 bound to quadruplex #11 is almost two times higher than that for tetraplex #10 representing c-myc sequence that formed stable parallel quadruplex (Figs. S1 and S2) [14–17]. On the contrary, isomer E,Z-1 shows higher binding selectivity to c-myc quadruplex (#10 DNA) comparing with telomeric quadruplex (#11). Although

Fig. 2. Relative affinities of compounds E,E-1 and E,Z-1 to different DNA polymorphs (#1–12) determined using competition dialysis method. The vertical columns indicate the relative binding of each ligand to the nucleic acid structure. The values were normalized for both compounds with maximum concentration of bound ligand corresponding to 8.69 ␮M.

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Scheme 1. The proposed mechanism of interactions of E,E and E,Z isomers of ligand 1 with tetraplexes: (A) c-myc G-quadruplex with propeller structure prefers E,Z-1 isomer; (B) telomeric G-quadruplex with basket-type structure binds planar E,E-1 isomer preferentially.

binding preferences of isomers are diverse, the binding affinities are not so different since Kapp values are in the order of 105 M−1 for both compounds. The apparent binding constant for E,Z-1/cmyc (#10) complex is Kapp = 0.9 × 105 M−1 and that for complex of E,E-isomer with telomeric tetraplex (#11) is only slightly higher, Kapp = 1.3 × 105 M−1 . Variation in binding selectivity toward particular G-quadruplex structure should be caused by the planarity of ligand structure and steric factors. It is known that trans isomers of most stilbene derivatives exist in planar conformation, while cis isomers occur in a non-planar conformation. The MM2 geometry optimization with Chem3D package confirmed such a rule for isomers of ligand 1. The optimized structures of E,E-1 and E,Z-1 are presented in Scheme 1. Consequently, the binding interactions in the ligand-quadruplex complex should be different for both isomers, which resulted in specific binding selectivity governed also by topology of the quadruplex structure. C-myc sequence (#10) forms a parallel propeller-type structure, in which TA loops are extended out from the sides of the G-quartets [14]; therefore, ligand molecules may stack externally to the exposed G-tetrads without steric obstacles. Comparable values of binding affinities for both isomers with #10 quadruplex suggest that only a part of ligand 1 structure is responsible for interactions with external tetrad of guanines. This conclusion is consistent with the size of G-tetrad that overlaps with the structural fragment of 1 that contains quinolinium and benzene rings linked by the ethene bond. In such a binding mode, the second vinylquinolinium arm can easily undergo trans-cis isomerization without affecting binding interactions. A slightly higher binding constant for E,Z isomer may even suggest a contribution from additional stabilizing interactions between the

quinolinium arm in cis conformation and negatively charged phosphates of quadruplex groove or loop. On the other hand, telomeric DNA (#11) in the presence of sodium ions can fold intramolecularly to form an antiparallel quadruplex, in which TTA loops are arranged in lateral and diagonal positions (basket-type form) [7–10], hence only sufficiently planar ligand molecule may stack on guanine tetrads that are flanked by TTA loops. This arrangement may suppress binding interactions of guanine tetrads with bulky ligand molecules. Therefore, binding affinity of E,Z-1 with the baskettype structure of telomeric quadruplex (#11) is significantly lower (Kapp = 0.6 × 105 M−1 ) comparing with that for E,E-1/#11 complex (Kapp = 1.3 × 105 M−1 ). The steric hindrance between TTA loops and vinylquinolinium arm in cis configuration is probably responsible for this effect. In contrast, very high binding affinity of E,E-1 suggests that binding of planar E,E-1 molecule is additionally stabilized by the presence of lateral TTA loops, probably because of more negative potential that phosphate groups in loops exert on the bound ligand. Proposed binding models of ligand 1 isomers with parallel and antiparallel quadruplexes are shown in Scheme 1. Undoubtedly, NMR study and/or molecular dynamic simulation calculations are needed to confirm the postulated binding modes of ligand 1 isomers. It is commonly believed that the main mode of interactions between ligands and G-quadruplexes is the end-stacking association. However, one cannot rule out other binding modes since some ligands were suspected to interact with G-quadruplexes, for instance, by a groove binding mode [35,36]. Such groove or loop binders usually possess multiple positive charges that enable coulombic interactions with anionic backbone of tetraplex.

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Table 2 Results of competition dialysis experiments. Binding parameters are shown as mean values of three determinations with uncertainties not exceeding 20%. DNA

Ligand E,E-1

E,Z-1

E,E,E-2a

E-3b

#10

−6

Cb /10 M Kapp /105 M−1

5.8 0.8

6.3 0.9

4.0 0.6

11.2 1.8

#11

Cb /10−6 M Kapp /105 M−1

8.7 1.3

4.0 0.6

3.8 0.5

1.9 0.3

a b

From Ref. [34]. From Ref. [29].

Previously, we have studied binding selectivity of other stilbazolium ligands, including compounds 2 and 3. Binding constants for these two ligands with tetraplexes #10 and #11 are shown in Table 2. Interestingly, ligand E,E,E-2 that possesses three positive charges exhibits low quadruplex-binding selectivity, although the presence of lateral loops in the basket form of DNA #11 should disturb in effective stacking interactions between the guanine tetrad and planar ligand with three extended arms. One can conclude; therefore, that other binding modes, e.g., groove binding or loop interactions play important role in stabilizing the G-4 complex with multi-charged ligands. This conclusion is consistent with binding parameters for ligand E-3 that possesses large planar system (phenanthryl group) and only a single positive charge. In this case external stacking interactions are preferable that explains much higher binding affinity toward the parallel quadruplex of #10 (exposed G-tetrads) comparing with that for basket-type quadruplex of #11. In the later case, TTA loops may impose steric hindrance and disturb in effective external stacking interactions. Unfortunately, our efforts to characterize binding selectivity for cis isomers of ligands 2 and 3 ended in failure. Irradiated E,E,E-2 yielded an isomeric mixture containing more than two products that were difficult to separate. On the other hand, Z-3 isomer initially separated by HPLC technique underwent dark back. 3.2. Photoisomerization experiments: the conversion percentage of isomers and fluorescence traces of ligands affected by the presence of DNA In order to evaluate the effect of complex formation between G-quadruplex and arylstilbazolium ligands 1–3 on their photoisomerization, the photoirradiation experiments were carried out in the presence and the absence of selected oligonucleotides. Progress of trans-cis photoisomerization was monitored using two alternative techniques: by recording fluorescence traces of ligand with time of light illumination and by HPLC separation of samples (isomeric mixtures) collected for different time intervals of irradiation. Fluorescence intensity should decrease upon trans-cis isomerization since fluorescence quantum yield of trans isomer is generally much higher than that for cis one [27]. In the HPLC-based method, the conversion percentage of starting trans isomer was calculated from peak areas of trans and cis isomers. Gradient elution enabled good separation of both trans and cis isomers of ligand as well as a peak representing oligonucleotide. Areas of peaks directly reflected concentrations of isomers since chromatograms were monitored at the isomer isosbestic point (391 nm for ligand 1). The following retention times were observed: 30.6 min and 30.2 min, for E,E-1 and E,Z-1 isomer, respectively. HPLC studies revealed that trans-cis photoisomerization of free E,E-1 proceeded very efficiently and led to almost 90% conversion toward E,Z-1 isomer (Fig. 3a). This considerable degree of trans → cis transformation was already achieved after 6 min of irradiation. In contrast, the presence of telomeric quadruplex (DNA #11) hampered the trans-cis photoisomerization

Fig. 3. Photoisomerization of E,E-1 in the absence and the presence of telomeric quadruplex (DNA #11) monitored with HPLC approach (a) and fluorescence measurements, em = 520 nm (b). Conditions: Cligand 1 = 10 ␮M, CDNA #11 = 20 ␮M, BPES buffer (pH 7), irr = 450 nm.

of compound 1 as can be inferred from the unchanged amount of E,E-1 during irradiation process (Fig. 3a). Fluorescence traces representing photoisomerization of free ligand 1 (Fig. 3b) possesses similar shape as that obtained from HPLC analysis (Fig. 3a) that proved our assumption on much higher quantum yield of the E,E-1 isomer. Interestingly, E,E-1 complexed by quadruplex #11 appeared to be only weakly fluorescent that is probably caused by fluorescence quenching phenomena, since in this case there is no transformation to E,Z-1 isomer as can be inferred from HPLC results shown in Fig. 3a. The question arises what is the origin of the restricted trans-cis photoisomerization of the quadruplex bound E,E-1 ligand. One possibility is the difference in binding affinities of both isomers toward quadruplex structure. However, two times higher binding constant with DNA #11 for E,E-1 over that for E,Z-1 may only partially explain observed effect. Alternative explanation involves the participation of electron transfer (ET) process in fluorescence quenching of ligand. Arylstilbazolium derivatives are known to undergo quenching due to ET mechanism from guanine moiety and for a ligand stacked on G-tetrad such a process is expected to be very efficient [37]. As ET quenching is regarded as redox reaction, the product adopts thermodynamically more favorable trans conformation (E,E-1 isomer). However, in order to restrict trans → cis transformation efficiently, both mechanisms should operate simultaneously: the binding selectivity shifts equilibrium toward E,E-1/quadruplex complex that cannot be transformed into E,Z-1 derivative due to ET mechanism. The E,Z-1/quadruplex complex that can be formed in a minor amount due to the free ligand isomerization is instantaneously converted into trans derivative (ET mechanism). To gain further evidence on the importance of both mechanisms that restrict trans to cis isomerization, we have studied photoisomerization process for ligand 2 and ligand 3. As reported previously, E,E,E-2 interacted with G-quadruplexes and triplexes with comparable affinity (Kapp ∼ 0.6 × 105 M−1 ) [34]. Furthermore, this compound formed also quite stable complex with dsDNA #7. Fig. 4 shows photoisomerization of ligand 2 in the absence and the presence of DNA samples #7, #8, and #11. Profiles of isomerization progress monitored using the HPLC approach and corresponding fluorescence traces recorded during photoisomerization experiments are shown in Fig. 4a and b, respectively. Free E,E,E-2 isomerizes slowly to achieve 60% conversion to the isomerization products after 50 min irradiation time that is roughly reproduced by the fluorescence trace. Three DNA samples were tested as mediators of the photoisomerization of ligand 2. These three DNA samples (quadruplex #11, triplex #8 and

I. Czerwinska, B. Juskowiak / International Journal of Biological Macromolecules 51 (2012) 576–582

Fig. 4. Photoisomerization of E,E,E-2 in the absence and the presence of DNA samples (#7 duplex, #8 triplex, and #11 quadruplex) monitored with HPLC approach (a) and fluorescence measurements, em = 452 nm (b). Conditions: Cligand 2 = 10 ␮M, CDNA = 20 ␮M, BPES buffer (pH 7), irr = 390 nm.

duplex #7) exhibited comparable binding affinities to ligand 2 (Kapp = 0.5–0.7 × 105 M−1 ) [34]. Contrary to quadruplex #11, in which guanine tetrads were responsible for binding interactions, duplex #7 and triplex #8 DNAs possessed also AT-rich binding sites. As shown in Fig. 4a, complex formation with duplex and triplex inhibited partially conversion of E,E,E-2 to photoproducts, but the most effective inhibition is observed for quadruplex-bound ligand (DNA #11). Taking into account comparable values of binding constants for these three systems, one can conclude that the ET mechanism with the participation of guanine nucleobase is a dominating process that hampers trans to cis photoisomerization of arylstilbazolium ligands. Fluorescence traces shown in Fig. 4b confirm above conclusion since the number of guanines in DNA samples correlates well with partial quenching (duplex and triplex) and substantial quenching (quadruplex) effects. One should remember, however, that binding constant for the quadruplex/ligand system should be high enough to assure that sufficient amount of complex (bound ligand) is present to suppress a competitive isomerization of free ligand. Effect of binding affinity on photoisomerization was studied on the example of ligand 3 and its complexes with G-quadruplexes that exhibit low (#11) and high (#10) binding affinities, respectively. The extent of ET mechanism is expected to be the same for both quadruplexes since the guanine tetrad is a binding site in both cases. A nearly 90% conversion of free ligand was achieved within 10 min of irradiation time (Fig. 5a). As expected, the presence of particular tetraplexes differently influenced the efficiency of photoisomerization of ligand 3.

Fig. 5. Photoisomerization of E-3 in the absence and the presence of quadruplexes (DNA #10 and #11) monitored with HPLC approach (a) and fluorescence measurements, em = 515 nm (b). Conditions: Cligand 3 = 10 ␮M, CDNA = 20 ␮M, BPES buffer (pH 7), irr = 390 nm.

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System E-3/quadruplex #11 (low binding affinity) exhibited photoisomerization profile that resembled that for free ligand (Fig. 5a). Final conversion percentage of E-3 attained 80% that was slightly lower value than that for free ligand (90%). Fluorescence traces (Fig. 5b) also show similar runs for free E-3 and E-3/#11 system. These results can be easily explained in terms of binding equilibria since quadruplex #11 binds only ca. 30% of ligand 3 as one can calculate from binding constant of 0.3 × 105 M−1 and reagent concentrations used [34]. Thus, in the E-3/quadruplex #11 system the free ligand photoisomerization pathway dominates. In contrast, six-times higher binding constant for E-3/quadruplex #10 complex indicates that more than 75% of ligand exists in a quadruplex-bound form. As a result, the inhibitory effect of #10 on the photoisomerization of E-3 is more pronounced (Fig. 5a). The higher ligand binding affinity is also reflected by the efficient quenching effect of c-myc #10 on the E-3 fluorescence (Fig. 5b). Difference in fluorescence properties of E-3 in the presence of both investigated quadruplexes are so clear that ligand E-3 may be considered as a potential fluorescent probe for recognition of the parallel and antiparallel quadruplex structures. 4. Conclusions Binding preferences and selectivity of arylstilbazolium ligands to twelve samples of nucleic acids (ssDNA, dsDNA, triplex DNA, quadruplex DNA) were studied using the equilibrium dialysis method. The investigated ligands 1–3 are good candidates to be used as G-quadruplex stabilizing agents because of their high affinity to the quadruplex structures. Unlike other quadruplexbinding ligands, the arylstilbazolium derivatives possess ability to undergo photoisomerization that provides a new tool for tuning the DNA–ligand binding affinity. The trans and cis isomers of 1,4-bis(vinylquinolinium)benzene (E,E-1 and E,Z-1, respectively) interacted with parallel and antiparallel tetraplexes exhibiting different selectivity. The E,Z-1 revealed higher binding preference to c-myc DNA (a propeller-type quadruplex), whereas the E,E-1 favorably interacted with telomeric DNA (a basket-type quadruplex). The binding affinity that depends on the structure of isomer and on the topology of quadruplex strongly suggests that structural factors play the crucial role in the molecular recognition interactions in these systems. The E,E,E-2 that possesses three positive charges exhibited low quadruplex-binding selectivity that was accounted for the interactions dominated by electrostatic attraction forces and structural recognition phenomena. In contrast, a decrease in the number of positive charges and the presence of extended polyaromatic moiety (ligand E-3) resulted in a domination of steric hindrance factors and end-stacking interactions as E-3 showed high preference for the parallel quadruplex with exposed guanine tetrads. The photoisomerization process of ligands 1–3 was studied in the absence and the presence of selected DNA samples (mainly quadruplexes). The trans-cis photoisomerization of free E,E-1 proceeded very efficiently, whereas the presence of telomeric quadruplex DNA hampered photoisomerization of quadruplexbound ligand. Similar results were obtained for E,E,E-2 and E-3 compounds: free ligands isomerized easily and DNA–ligand complexes were more resistant for photoisomerization. Two factors: DNA binding affinity and the fluorescence quenching by electron transfer mechanism, were considered as crucial for explanation of obtained results. Ligand/DNA complexes with high binding constants (E,E-1/#11, E-3/#10) were efficiently quenched by the ET from guanine moiety to the ligand stacked on G-tetrad and the product of this redox reaction adopted thermodynamically more favorable trans conformation (E,E-1 or E-3). In contrast, systems with low (intermediate) stability constant or with different binding

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