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Unravelling the binding mechanism of a poly(cationic) anthracenyl fluorescent probe with high affinity towards ds-DNA Marco Deiana, Bastien Mettra, Katarzyna Matczyszyn, Delphine Pitrat, Joanna Olesiak-Banska, Cyrille Monnereau, Chantal Andraud, and Marek Samoc Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01113 • Publication Date (Web): 03 Oct 2016 Downloaded from http://pubs.acs.org on October 5, 2016

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Unravelling

the

binding

mechanism

of

a

poly(cationic) anthracenyl fluorescent probe with high affinity towards ds-DNA Marco Deianaa, Bastien Mettrab, Katarzyna Matczyszyn*a, Delphine Pitratb, Joanna OlesiakBanskaa, Cyrille Monnereaub, Chantal Andraudb, Marek Samoca a

Advanced Materials Engineering and Modelling Group, Faculty of Chemistry, Wroclaw

University of Science and Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw (Poland) b

Laboratoire de Chimie, CNRS UMR 5182, Ecole Normale Supérieure de Lyon, Université Lyon

1, Lyon, (France) KEYWORDS. anthracenyl derivative, DNA, fluorescence, polycationic, quenching, two-photon fluorophore.

ABSTRACT. We report the synthesis, spectroscopy, and the DNA binding properties of a biocompatible, water-soluble, polycationic two-photon absorbing anthracenyl derivative (AntPIm) specifically designed for bio-related applications. Detailed insights into the Ant-PIm-DNA binding interaction are provided by using several spectroscopic approaches, including UV-VIS absorption, circular dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), steady-

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state and time-resolved fluorescence techniques. Absorption and fluorescence quantitative data analysis show a strong Ant-PIm-duplex interaction with binding constants of: Kf = 4.7 ± 0.2 × 105 M-1, 7.1 ± 0.3 × 105 M-1 and 1.0 ± 0.1 × 106 M-1 at 298, 304 and 310 K, respectively. Spectral changes observed upon DNA binding provide evidence for a complex formation with off-on fluorescence pattern, which can be related to two consecutive binding equilibria. Results of DNA binders displacement and iodide quenching experimental assays unambiguously point to the groove binding mode of Ant-PIm to the DNA-helicate. Thermodynamic and chemical denaturation studies suggest that long-range interactions of hydrophobic nature regulate the association of Ant-PIm with the biopolymer. The ionic strength dependence of the binding constant shows that electrostatic component has an important contribution to the overall Gibbs free energy. FTIR and CD data provide evidence of partial modification of the B-DNA secondary structure, while the increase in the melting temperature clearly indicates the enhancement of the thermal stability of the duplex. Furthermore, the two-photon absorption cross section spectrum determined using the TPEF technique shows a strong 2PA maximum at 820 nm with a σ2 ˃ 800 GM which emphasizes the advantageous combination of biological and optical properties possessed by this positively charged bio-probe.

1. Introduction Multiphoton absorbing fluorescent molecules and materials are key components in the ongoing quest towards increasingly sophisticated, selective, sensitive and versatile nonlinear fluorescence microscopy procedures.1-3 Current research trends have been focused on increasing the brightness of two-photon fluorophores in the red and near-infrared (NIR) part of the electromagnetic spectrum, improving their selectivity towards target organs or biological


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compartments, either by specific (bioconjugation with antibodies) or nonspecific (EPR or charge effects) means, improving their biocompatibility and metabolization pathways, or making them responsive to various chemical, photochemical, or electromagnetic stimuli.4-8 Nevertheless, the conception of nonlinear fluorescent probes that combine high performance with a decent synthetic availability and production cost remains highly challenging. In a series of recent papers, we have described a polymer engineering strategy which afforded water soluble two-photon fluorophores with large two-photon brightness in the red/ far-red; we showed that the resulting probes were efficient non-specific markers of the cellular cytosol, and could even be used for intravital imaging of the blood vasculature.9,10 Even more recently, evidence was found that anthracene polymeric probes based on this approach presented a relatively high affinity for interaction with double-stranded DNA, through insertion of the probe within the groove of the duplex.11 The association of fluorescent molecular dyes with nucleic acids represents an astonishing tool to control DNA functions for gene-delivery, bio-imaging, therapeutic and diagnostic applications and offers the opportunity for a real-time monitoring of the structural reorganization of biomacromolecules in living cells.12-15 The noncovalent binding modes of bio-probes targeted at the DNA sites are mainly directed by a combination of both long and short-range forces including hydrophobic, van der Waals, electrostatic and hydrogen-bonding interactions.16,17 In general DNA binders may associate to the duplex template via two distinct modes: intercalation and groove binding (Scheme 1).18,19 Typically, well-known intercalators obeying the neighbourexclusion principle possess the binding site size-specificity of two/three base pairs that can discriminate only one out of 10/32 random sequences, respectively.20 Therefore, there is indeed a special interest in molecules that bind the DNA template in a sequence-specific manner.20


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Groove binders usually are constituted by two aromatic rings and a crescent structural motif which allows the ligands to fit perfectly either to the deep major groove or to the shallow minor groove causing no or weak distortion of the DNA secondary structure.12,13,21-24 Their higher sequence selectivity than that of intercalators and their ability to not induce structural alterations in the target DNA scaffold make such probes promising candidates for molecular recognition strategy in the fields of chemistry and biology.25-27 Cationic and poly(cationic) molecules are known for their superior ability to provide strong DNA binding, and many examples of cationic binders, including the archetypical Hoechst 33258 and 33342 as well as the well-known aminoglycoside antibiotic Neomycin, have been used in this framework.28-32 We recently reported on a convenient two steps strategy which allowed converting the hydroxyl moieties of a poly(hydroxylethyl)acrylate polymer chain into a variety of cationic heterocyclic moieties.33 This prompted us to investigate whether this transformation could be taken advantage of the conception of biocompatible fluorescent probes with enhanced selectivity towards the DNA moiety, as the poly(cationic) character of the polymer side chains could participate in establishing additional interaction with the negatively charged phosphate of the DNA backbone. In this paper, we report on the synthesis and characterization of a poly(cationic) water-soluble two-photon fluorescent probe, Ant-PIm specifically designed for bio-related two-photon applications (Scheme 2). In particular, through a detailed series of biophysical experiments performed as functions of temperature as well as salt and DNA concentration, we provide an exhaustive description of Ant-PIm-DNA binding mode, and prove that, in striking contrast to the case of previously reported by us Ant-PHEA11, AntPIm:DNA binding occurs in a highly unusual successive binding mode through a dimerization process in the minor groove of the


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duplex. Moreover, stabilization of the B-form of DNA along with a compaction / condensation process was observed upon binding of the positively charged bio-probe. These findings differ significantly from those found for the neutral Ant-PHEA molecule leading to the conclusion that the interplay between the polycationic moiety and the anthracenyl template is the sole responsible feature for the different binding mechanism of the whole ligand.

Scheme 1. Chemical structures of well-known groove binders A) Distamycin, B) Netropsin, C) Neomycin and intercalators D) Acridine orange and E) Thiazole orange.

Scheme 2. Chemical structures of A) Ant-PIm, and the well-known duplex site-markers used in fluorescence displacement assays B) Hoechst 33258 and C) Ethidium bromide.


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2. Materials and Methods 2.1. General procedures. The absorption spectra were recorded on a Perkin Elmer Lambda 20 UV-Vis spectrometer. Emission spectra were recorded with a Hitachi F-4500 spectrofluorometer. Fluorescence lifetimes were determined with an Edinburgh

Instruments FLS 980

spectrophotometer via time correlated single-photon counting (TCSPC), with excitation from a 516 nm picosecond laser diode. CD spectra were measured with a Jasco J-815 spectropolarimeter (JascoInc, USA) equipped with the JascoPeltier-type temperature controller (CDF-426S/15). A 1.0 cm path length quartz cells were used throughout the whole measurements. IR spectra were recorded on the diamond crystal surface under vacuum (< 1 hPa), using a Bruker Vertex70v FTIR spectrometer. 2.2. Samples. Double-stranded DNA, Hoechst 33258 and Ethidium Bromide were from Sigma-Aldrich. Stock solutions of the commercially available dyes and Ant-PIm were prepared in double distilled water at concentrations on the order of 10-4 M. DNA stock solution was prepared in sodium cacodylate buffer (10 mM, pH 7.25) and stored in the refrigerator at 4 ºC. The DNA concentration and purity were determined according to the literature reports.34,35 The experimental results reported within the manuscript are derived from at least three different trials. 2.3. Steady-state and time-resolved fluorescence titrations. Ant-PIm concentration was held constant and concentrated DNA solution was added until the changes in the emission were no longer observed. Time-resolved fluorescence decay measurements were performed at different DNA/Ant-PIm molar ratios ranging from 0 to 4.


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2.4. Competition experiments. Site markers competitive experiments were performed according to the experimental conditions described previously.11 2.5. Iodide quenching experiments. Quenching experiments were conducted by adding different concentrations of sodium iodide (0.001 - 0.1 M) to Ant-PIm and Ant-PIm-DNA complex solutions, respectively. The changes in emission intensity were recorded and the quenching constants were evaluated from the resulted data. 2.6. UV-Vis measurements. The absorption titration was performed according to the experimental conditions described previously.11 2.7. FTIR spectroscopic measurements. IR spectra were recorded according to the experimental conditions described previously.11 Data analysis was carried out based on previous reports.11,36,37 2.8. Circular dichroism and melting studies measurements. CD measurements and quantitative data analysis were performed according to our previous report.11 2.9. Two-photon excited fluorescence (TPEF). TPEF measurements were performed at AntPIm concentration equal to 2.5 µM and using fluorescein as reference. The relative concentration was adjusted so that the linear absorbance at 2hν was kept below 0.1 in the whole wavelength range studied (780 – 920 nm). The excitation was achieved by using a Coherent Chameleon laser that delivered a train of ~100 femtosecond pulses with 80 MHz repetition rate. The TPEF spectra were recorded by an Ocean Optics 2000 fiber spectrometer.


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The two-photon absorption (TPA) cross-sections were determined according to the following equation:

=

(1)

where σ2 is the TPA cross-section, c and n are the concentration and refractive index, respectively, and F is the integrated area obtained from the TPEF spectrum. The subscript r refers to the reference solution. 3. Results and Discussion 3.1. Synthesis and characterizations of Ant-PIm are detailed as Supporting Material (SI p. S2-4) 3.2. UV-Vis spectral studies of Ant-PIm-DNA adducts. It is well-established that the strength and mode of binding between exogenous chromophores and duplex DNA can be quantified and assessed through absorption spectroscopy.38 The UV-Vis spectrum of the uncomplexed Ant-PIm is characterized by a band at 515 nm and additional π-π* bands at shorter wavelengths (283 and 257 nm). Upon DNA addition the Ant-PIm band at 515 nm is perturbed and a significant hyperchromic effect is detected (Figure 1). It is known that the intercalative mode of binding usually gives rise to hypochromism and bathochromism due to the involvement of stacking interactions between the π electrons of the aromatic chromophore and the π electrons of DNA bases which causes a decrease of the π-π* energy level gap.39 On the other hand, groove and outside binders may give rise to hyperchromic and/or hypsochromic effects.40-42 This overall framework may hint at the possibility that the hyperchromism observed could be the direct outcome of the excitonic coupling of Ant-PIm with the DNA base pairs, located in the bottom


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groove region. Similar behavior is encountered for QCy-DT and Esculetin-DNA systems, for which a groove binding mode has been assigned.20,43 Moreover, a similar trend was observed for the neutral Ant-PHEA-DNA system, recently studied by our group.11 Thus, the absorption titration unambiguously points to the binding of the anthracenyl derivative in the groove of the duplex, ruling out the intercalative process.44 Based upon the variations in absorbance at 515 nm, the association constant Ka for the AntPIm-DNA system was derived by using the following equation45:

=

+

( )

× [] (2)

where A0 is the absorbance of Ant-PIm at 515 nm in the absence of the duplex, A∞ is the final absorbance of the complex when no more changes in the absorbance are observed and A is the recorded absorbance at different Ant-PIm / DNA ratios. The double reciprocal plot of 1/(A-A0) versus 1/CDNA allows to evaluate the association constant (inset in Figure 1). The Ka value was found to be 3.6 ± 0.3 × 105 M-1 (298 K), which matches well with those typical for minor groove binders and also with that found by us for the neutral Ant-PHEA chromophore.11 Since both the Ant chromophores studied by us showed a similar trend of changes in the absorption maximum we infer that the angle between the transition dipole moment of the main CT band of both the probes and the DNA helix axis may be similar, bringing evidence that the spatial orientation of the chromophore in respect to the long molecular axis of the biopolymer is strictly a function of the Ant core and does not depend on the different nature of the substituents. However, it is worth mentioning that this similitude represents one of the few common points found between the neutral and polycationic ligands.


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Figure 1. Absorption spectra of Ant-PIm (5 µM) treated with: 0.0, 1.5, 3.0, 4.5, 6.1, 7.6 and 9.1 µM (curves 1-7) of DNA at 298 K in sodium cacodylatetrihydrate 10 mM (pH 7.25). Inset: Plot of 1/(A-A0) against 1/[DNA] for Ant-PIm-DNA system at 298 K. A0 and A are the absorbance values of Ant-PIm at 515 nm in absence as well as in presence of DNA, respectively.

3.3. Steady-state fluorescence studies. Ant-PIm is a strong luminophore and shows an emission band centered at 570 nm when excited at 516 nm (Figure 2). Changes of the emission spectra of Ant-PIm upon incremental addition of ds-DNA were thus investigated. Upon incrementally increasing concentrations of DNA, a steady decrease of the fluorescence intensity (~70%) of Ant-PIm with a slight blue-shift of the maximum emission wavelength (from 570 nm to 568 nm) is initially observed, up to ca 27 µM of DNA. This brings evidence of the effective interaction between the fluorophore and the biopolymer; however this evolution is markedly different to that previously reported for the neutral Ant-PHEA under similar conditions, hence suggesting a consistently different binding, presumably due to the difference of electrostatic potential between the two anthracenyl derivatives. It is worth noting that the binding behaviour exhibited by our luminophore dramatically differs from that encountered for the well-known monointercalator dye Thiazole Orange (TO) hinting at a different coordination process between the two bio-probes.46 TO is an asymmetric cyanine fluorescent light-up probe with an intrinsic


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low quantum yield ( ~ 2 × 10-4) in its uncomplexed state. However, upon binding to DNA its quantum efficiency is reported to increase ~ 19,000 times.47 The reasons of this phenomenon are ascribed to the intercalative behaviour of the dye and to its relatively high binding affinity for duplex DNA (KTO-CT-DNA = 3.2 × 106 M-1 in 10 mM NaCl).48-51

Figure 2. Fluorescence emission spectra of Ant-PIm (5.0 µM) at 298 K treated with: 0.0, 3.9, 7.6, 11.0, 15.0, 19.0, 23.0 and 27.0 µM (curves 1-8) of DNA.

Further quantitative insights in the quenching of fluorescence in the DNA-Ant-PIm complex were provided by using the Stern-Volmer equation at three different temperatures52,53: F /F = 1 + K τ [Q] = 1 + K !" [Q]

(3)

where F0 and F denote the steady-state fluorescence intensities in the absence and presence of DNA, respectively. Ksv is the Stern-Volmer quenching rate constant, [Q] is the DNA concentration, Kq is the apparent quenching rate constant of the biomolecules and τ0 is the average excited-state lifetime of biomolecules without a quencher assumed equal to 10-8 s.54 It is well known that the fluorescence quenching can be ascribed either to ground-state complex formation (static mechanism) or diffusive collisions (dynamic mechanism).54 One can see in Figure 3 that the Stern-Volmer plot exhibits an upward curvature for DNA/Ant-PIm mole ratio greater than 4, while a good linearity is observed when CDNA/CAnt-PIm< 4 suggesting a


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single component donor quenching system that would be expected for one major binding mode.54 The upward deviation from the linear Stern-Volmer plot is usually ascribed either to the presence of combined dynamic and static quenching or just pure static quenching.54 It is known that the intrinsic curvature of the plots can be ignored if [FQ] < [Q], which allows the extrapolation of the Ksv and consequently the Kq values, reported in Table 1, from the linear portion of the SternVolmer plot.54,55

Figure 3. Plots for the fluorescence quenching of Ant-PIm by DNA at 298, 304 and 310 K.

Table 1. Quenching parameters for the interaction between temperatures. T (K) KsvM-1 Kq M-1 s-1 298 3.1 ± 0.1 × 104 3.1 ± 0.1 × 1012 4 304 2.4 ± 0.2 × 10 2.4 ± 0.2 × 1012 310 2.2 ± 0.2 × 104 2.2 ± 0.2 × 1012

Ant-PIm and DNA at various n 1.2 1.3 1.4

Kf M-1 4.7 ± 0.2 × 105 7.1 ± 0.3 × 105 1.0 ± 0.1 × 106

Static and dynamic quenching exhibit different dependence on temperature.54 Higher temperatures lead to a faster diffusion and thus stronger collisional quenching, while in the case of static quenching the temperature increase typically results in the dissociation of weakly bound complexes. Table 1 shows that less quenching occurs at higher temperatures, pointing out to the formation of a ground-state complex between the bio-probe and the duplex. Further evidence


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was also provided by the analysis of the Kq values, which were found to be much higher than the maximum diffusion collisional quenching rate of various quenchers with biopolymers ≈ 2.0 × 1010 M-1 s-1, confirming a static quenching mechanism. Static complex formation can be further confirmed by the modification of the absorption and circular dichroism bands (vide infra) definitely ruling out any fluorescence quenching process through dynamic collisions, which do not cause changes in the absorption bands.54 3.4. Time-resolved fluorescence. Fluorescence lifetime studies are the most definitive method to distinguish between static and dynamic quenching: static quenching results in a nonfluorescent complex so that the observed fluorescence comes only from the uncomplexed ligand. Since the uncomplexed fraction is unperturbed, the lifetime of the fluorophore in absence (τ0) and in presence (τ) of the biomacromolecule should be constant in the case of static quenching.54 Fluorescence lifetime measurements were thus performed at various DNA/Ant-PIm mole ratios ranging from 0 to 4. The free Ant-PIm was found to exhibit a mono-exponential decay pattern with a decay constant τ0 = 3.30 ± 0.03 nsec. As shown in Figure 4, the fluorescence decay function is almost unaffected by the presence of DNA proving that the Ant-PIm-DNA association is driven by static quenching complex formation.54 Further insights were also provided by calculating the bimolecular quenching rate constant Kq54: #$ =

%& '

(4)

where τ0 is the lifetime of the fluorophore in the absence of DNA.


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The bimolecular quenching constant reflects the accessibility of the fluorophore to the quencher and this probability is proportional to the rate of diffusion, size and concentration as depicted by the following formula54: #$ = 4)*+, × 10. (5) where D is the diffusion coefficient, σ is the collisional radius and Na is the Avogadro’s number. Since the Kq value calculated for the Ant-PIm-DNA system was equal to 9.4 × 1012 M-1 s-1, we can speculate that a combination of specific long and short-range interactions between the fluorophore and the DNA binding sites regulate the binding pathway as demonstrated further on in the energetic analysis.

Figure 4. Representative time-resolved fluorescence decay profiles (λex = 515 nm) of AntPIm treated with: 0.0, 5.0, 10.0, 15.0 and 20.0 µM of DNA. Inset: plot of τ0/τ against [DNA] for Ant-PIm-DNA system.

3.5. Fluorescence binding data analysis. By exploiting the fluorescence titration data and assuming static quenching, we evaluated the association constant (Kf) and the number of binding sites (n) by using the following equation53: log

[(2 2)] 2

= log K 3 + n log[Q] (6)


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Figure 5. Plot of log (F0-F)/F versus log [DNA]. The obtained data, reported in Table 1 and shown in Figure 5, clearly evidence a strong and specific binding affinity of the ligand for double-stranded DNA. The Kf value is higher than those reported for classical intercalators56 but matches well with those found for minor groove binders such as Hoechst 33258-DNA30, Distamycin-DNA57 and the new NIR fluorophore QCyDT-DNA20. 3.6. Off-on state of the Ant-PIm-DNA fluorescence pathway. Surprisingly, increase of DNA concentration in the fluorophore solution (5.0 µM) above 30 µM resulted in an enhancement of the fluorescence intensity alongside with an overall hypsochromic shift of 12 nm (Figure 6).


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Figure 6. Fluorescence emission spectra of Ant-PIm (5.0 µM) at 298 K treated with: 30.3, 34.1, 41.6, 56.8, 71.9, 87.1, 102.2, 117.4, 132.5, 151.5, 189.3 and 227.2 µM (curves 1-12) of DNA. Inset: plot of 1/(I-I0) against 1/[DNA] for the Ant-PIm-DNA system at 298 K by using the equilibrium DNA concentration that corresponds to the free DNA present in solution once that the first binding equilibrium has reached the saturation.

This overall framework of fluorescence changes hinted at the possibility of two consecutive equilibria, the former being driven by dimeric aggregation of the dye in the DNA binding site and the latter the outcome of a monomeric binding fashion. As the monomeric structure of the studied chromophore consists of four heteroaromatic rings linked through methylene spacers to an anthracene moiety, the strong hydrophobic character of the molecule may favor formation in aqueous solution of either oligomers or aggregates with specific structures (J or H). H- and Jaggregates typically give rise to hypsochromic or bathochromic shifts of the absorption maxima, respectively.58 In our case, the blue shift observed for the Ant-PIm-DNA complex suggests a dimerization of the dye in the minor groove of the DNA in a parallel eclipsed stacking arrangement in which only one side of the chromophore can participate in surface binding driven by a combination of both specific (Ant-PIm-nucleobases) and non-specific (Ant-PIm-phosphonates) interactions with the biopolymer while the opposite side its oriented towards the external hydrophilic environment. The resulting complexation, at high chromophore/DNA ratio, leads to formation of a supramolecular chromophore:DNA complex. Then, upon subsequent DNA addition the equilibrium is shifted towards the formation of the classical monomeric adduct, and the fluorescence is restored. In order to test this hypothesis, fluorescence measurements were undertaken on aqueous solution with increasing concentrations of Ant-PIm ranging from 2.5 to 250 µM. A decrease of fluorescence intensity alongside with a progressive red-shift of the emission band was clearly visible upon increasing Ant-PIm concentration, which could be


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related to aggregation induced luminescence quenching of its luminescence, in good agreement with our hypothesis (SI p.S6). A proposed mechanism of the way in which the anthracene derivative binds the duplex is shown in Scheme 3.

Scheme 3. Cartoon showing schematically the successive complexation process occurring between Ant-PIm and the DNA template in the groove of the duplex.

At low DNA/Ant-PIm molar ratio, a supramolecular binding mode leads to a progressive decrease of the fluorescence intensity of the free dye until maximal quenching occurs. At higher DNA concentrations, the system is shifted towards a second independent binding equilibrium, in which the monomeric state of the chromophore is restored. In order to provide quantitative information on the strength of the second binding equilibrium, by assuming independent binding sites, the modified Benesi-Hildebrand equation was used59:

(55 )

= (5

5 )67 [89:]

+ (5

(7)

5 )

where I0 and I are the emission intensities of the Ant-PIm-DNA complex when the first binding equilibrium has reached stoichiometry and when further additions of DNA provide new accessible sites for the fluorophore, respectively, I∞ is the saturated fluorescence intensity. The plot of 1/(I-I0) versus 1/CDNA is linear in the whole concentration range studied, in good


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agreement with the model stated above (Inset Figure 6). The Kf value for the second binding equilibrium is found to be 2.8 ± 0.3 × 104 M-1 and a binding size of ~ 4.7 bp (vide infra), consistent with the proposed association mechanism discussed above. 3.7. Insights on the binding site size. The mole ratio method was used to evaluate the size of the binding site between the bio-probe and the DNA-helicate and the respective plot is shown in Figure 7. From the inflection point, the molar ratio DNA/Ant-PIm is found to be 5 and 4.7 for the first and second binding equilibrium, respectively, which indicates, by assuming identical independent binding sites, the number of DNA base pairs excluded to another free dye by each complexed dye molecule to have a size of ≈ 17 Å. The number of excluded base pairs found for Ant-PIm corresponds well to that expected for a minor groove binder such as Hoechst 33258.31 This observation also agrees very well with the number of Hoechst 33258 molecules (n) displaced upon Ant-PIm binding (SI p.S7) and matches well with that found for Ant-PHEA.11

Figure 7. A) Plot of DNA-Ant-PIm emission intensity vs. the mole ratio. B) Plot of DNAAnt-PIm emission intensity vs. the mole ratio by using the equilibrium DNA concentration that corresponds to the free DNA present in solution once that the first binding equilibrium has reached the saturation.

3.8. Competitive binding studies with Hoechst 33258. Hoechst 33258 binds with specificity to A-T sequences in the minor grooves of DNA, exhibiting association-induced fluorescence


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enhancement.60 If a competition for the same binding site occurs between the anthracenyl derivative and the commercially available probe, a decrease in the fluorescence intensity must be observed. As depicted in Figure 8, the fluorescence of the complexed Hoechst is efficiently reduced and 4 nm blue shifted by the addition of the Ant-PIm molecules which highlights the tendency of the anthracenyl probe to bind the duplex in the A-T rich regions where the Hoechst molecules are located.

Figure 8. Emission spectra of the competition between the bound site-marker Hoechst (λexc: 350 nm) and Ant-PIm treated with: 0.0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1 and 2.4µM (curves 1-9) of Ant-PIm. [Hoechst] = 5µM and [DNA] = 45 µM.

These results match well with those found for the Ant-PHEA-DNA system in the presence of the site-specific probes showing the outstanding capacity of the fluorophores to bind the DNA groove independently in the presence or absence of positive charges distributed along the polymeric hydrophilic arms, although the equilibria involved in the binding process appear to differ strongly.11 3.9.

Thermodynamic

analysis.

Non-covalent

forces

that

dominate

ligands

and

biomacromolecules binding in aqueous solution include electrostatic and hydrophobic interactions, hydrogen bonds and van der Waals forces. The sign and magnitude of the changes in the thermodynamic parameters are good tools to assess the nature of the interactions that


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regulate the association process between Ant-PIm and DNA. Positive values of enthalpy and entropy can be ascribed to short-range interactions of hydrophobic nature, whereas negative values of both enthalpy and entropy indicate long-range interactions of ionic nature such as van der Waals forces or hydrogen bond formation.61 The thermodynamic parameters were calculated by using the van’t Hoff equation (Figure S8): log #; = −

∆> ...@A

+

∆B ...@

(8)

∆C = −2.303 FG HIJ #; = ∆K − G∆L (9)

The obtained negative values of ∆G0 ( -32.3 ± 0.1, -33.4 ± 0.1 and -34.2 ± 0.2 kJ mol-1 at 298, 304 and 310 K, respectively) clearly indicate the spontaneity of the Ant-PIm-DNA complexation. Both ∆H0 ( 47.7 ± 6.2 kJ mol-1) and ∆S0 ( 268.7 ± 20.5 J mol-1 K-1) being positive indicates that in spite of the poly(cationic) nature of the probe the Ant-PIm-DNA complex is quite surprisingly stabilized mainly by non-ionic interactions even though long-range forces cannot be excluded. Since the thermodynamic studies showed positive values of entropy for the Ant-PIm-DNA adduct, the binding may entail the release of bound ions. Therefore, additional ionic strength investigations to distinguish between the electrostatic and non-electrostatic contributions to the overall Gibbs free energy were carried out and are presented further on. 3.10. Effect of chemical denaturation and hydrophobic contacts. Chaotropic agents, such as urea and guanidine hydrochloride (GuHCl), can denaturate biomacromolecules such as protein and nucleic acids by interfering with intramolecular interactions mediated by non-covalent forces and disrupting the hydrophobic networks.38 Therefore, we investigated the effective role played by hydrophobic forces in regulation of the Ant-PIm-DNA adduct by calculating the binding


20

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parameters in the presence of denaturants according to Equations 3 and 6 (Figure 9). The corresponding results are reported in Table 2. Table 2. Quenching parameters of the interaction between Ant-PIm and DNA in the presence of denaturant agents. Denaturant KsvM-1 Kq M-1 s-1 n Kf M-1 4 12 Free 3.1 ± 0.1 × 10 3.1 ± 0.1 × 10 1.2 4.7 ± 0.2 × 105 GuHCl 2.5 ± 0.3 × 104 2.5 ± 0.3 × 1012 1.1 8.6 ± 0.3 × 104 4 12 Urea 2.3 ± 0.4 × 10 2.3 ± 0.4 × 10 1.0 2.4 ± 0.3 × 104

As expected, the binding parameters decreased in the presence of the chaotropic agents suggesting that both GuHCl and urea were able to denaturate the DNA helix weakening the ability of the fluorophore to establish hydrophobic contacts with the bottom of the groove’s region. Furthermore, to determine the presence of hydrophobic contact in the Ant-PIm-DNA adduct, the spectral changes of the polymer symmetric and antisymmetric CH2 stretching vibrations, in the range 3000-2800 cm-1 were carried out by infrared spectroscopy (data not shown). The CH2 bands of the free Ant-PIm located at 2863 and 2956 cm-1 were shifted to 2869 and 2962 cm-1, respectively, in the complex with the DNA/Ant-PIm molar ratio equal to 5. Such shifts highlight the presence of hydrophobic contacts through the anthracenyl polymer aliphatic chain and the hydrophobic cavities of the duplex, in good agreement with the thermodynamic studies.


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Figure 9. A) Stern-Volmer and B) log-log plots for the Ant-PIm-DNA system in the presence of denaturants. [Urea] = 1.2 × 10-3 M and [GuHCl] = 1.2 × 10-3 M.

3.11. Effect of varying concentration of monovalent salt on affinity of Ant-PIm to dsDNA. Quantitative data analysis of the effect of changes in salt concentration on the equilibrium binding constant, Ka, for the Ant-PIm-DNA complexes was carried out according to the theory of Record et al and the data are shown in Table 3.62,63 The theory claims that when a ligand with Z positive charges binds to a nucleic acid, some number of phosphates (equal to Z) are neutralized. As a result, the condensed counterions which were associated with the Z phosphates are released into solution as well as the ions involved in long-range screening interactions. Thus, in the presence of a monovalent salt MX, the number of ionic interactions and counterions release involved in a ligand-nucleic acid adduct can be estimated by measuring the derivative δlog (Ka) / δlog [M+]. In a sufficiently diluted solution of the monovalent salt, the predicted quantitative dependence of the association constant on the ionic strength is63,64: M NOP MQRS [TU ]

= −VW

(10)

where Ψ is a constant (0.88 for B-DNA form) and the linear plot of log Ka vs. log [M+] allows to determine Z. Table 3. Apparent dissociation constant (Kd) of binding of Ant-PIm to DNA in the absence and in the presence of 50 mM NaCl. ZΨ is the slope of the dependence log Ka vs. log [NaCl]. Ant-PIm-DNA at 298 K Ant-PIm-DNA at 298 K in 0.05 M [Na+] Kd, µM ∆G kJ/mol Kd, µM ∆G kJ/mol ∆(∆G) kJ/mol ZΨ 2.8 ± 0.3

-31.7 ± 0.3

166.6 ± 0.2

-21.5 ± 0.1

-10.2 ± 0.4

1.9

The least squares slope in Figure S9 was 1.9 implying that ~ 2 ions are released in the interaction of each monomer of the fluorophore with the biopolymer within the salt


22

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concentration range studied. Consequently, the number of phosphate groups on the DNA involved in the ionic interactions with the positive charged ligand was ~ 2. Although these data confirm participation of the phosphate-imidazolium interaction in the binding process of AntPim and DNA, this value is lower than that expected: at a neutral pH, each Ant-PIm probe has four net positive charges (imidazolium moieties) distributed along each of its star shape hydrophilic polymeric arms. We infer that not all of the four arms participate in charge-charge interactions but just those which directly face the external edges of the duplex lie buried along the groove and thereby are responsible for electrostatic interaction and subsequent counterion release. The Gibbs energy of Ant-PIm-DNA association in the presence of the monovalent salt at a concentration equal to 50 mM can be written in terms of its non-electrostatic and electrostatic components as: ∆G = ∆Gnel+ ∆Gel = -31.7 ± 0.3 kJ/mol, where ∆Gnel = 21.5 ± 0.1 kJ/mol and therefore ∆Gel = -10.2 ± 0.4 kJ/mol. This suggests the fundamental role of electrostatic component (long-range interactions between the imidazolium group of the polymer arms and the phosphates on the DNA backbone) in stabilization of the Ant-PIm-DNA adduct even though a larger contribution arises mainly by hydrophobic contacts (short-range interactions) pointing out the specific aspect of their binding. Further insights on the effect of the ionic strength on the Ant-PIm-DNA complex were provided by analyzing the quenching constant, again using the Stern-Volmer equation (Equation 3). The corresponding results shown in Figure S10 and reported in Table 4, clearly pointed out the effective competition for the phosphonates of the duplex between the electrolyte and the positive charges of the ligand confirming the role of the electrostatic components for maintaining conformational stabilization of the Ant-PIm-DNA complex.


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Table 4. Stern-Volmer (Ksv) and quenching rate constant (Kq) of the interaction between AntPIm and DNA in the presence of varying NaCl concentrations. [Na+] mM 0.0 5 10 20 50

KsvM-1 3.1 ± 0.1 × 104 2.9 ± 0.3 × 104 2.2 ± 0.3 × 104 2.1 ± 0.4 × 104 1.9 ± 0.1 × 104

Kq M-1 s-1 3.1 ± 0.1 × 1012 2.9 ± 0.3 × 1012 2.2 ± 0.3 × 1012 2.1 ± 0.4 × 1012 1.9 ± 0.1 × 1012

3.12. Circular dichroism spectral study. The changes in the secondary structure of salmon testes DNA upon ligand interaction were studied by exploiting the circular dichroism technique. The DNA spectrum in the canonical B-form displays a positive peak at ~ 275 nm due to base stacking and a negative peak at ~245 nm arising from the helical structure that provides asymmetric environment for the bases.65,66Ant-PIm does not possess a chiral centre and is thus CD inactive. However, the addition of the duplex to the bio-probe results in a positive ICD band, in the region between 320 and 370 nm, because of the coupling of the electric transition moment of the dye and the nucleobases (Figure 10). It is known that groove binders give rise to positive ICD signal, whereas intercalators exhibit either negative or bisignate ICD band depending on the chemical structure of the ligand.67,68 It turns out that the presence of the positive ICD band is an unambiguous proof that the anthracenyl derivative binds the duplex in the groove region with a transition dipole moment oriented at ≈ 45º with respect to the DNA long molecular axis. Further insights were provided by analysing the intrinsic DNA bands which can provide useful information on the morphological changes occurring on the duplex upon ligand complexation. An enhancement of the positive DNA band, without any shift of the maximum, and a noteworthy decrease in molar ellipticity of the negative band followed by a bathochromic shift of 3 nm was detected upon ligand addition. Further, a clear isoelliptic point at 268 nm was observed revealing the formation of a complex between the guest and host molecules. The CD data categorically


24

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establish that the presence of the ligand produces stabilization of the right-handed B-form of DNA and the insertion of the Ant-PIm molecules preferentially occurs in the bottom of minor groove richest in AT-base pairs, which possesses a very high negative electrostatic potential and therefore can direct and orientate the positive charges of the ligand along the wall and/or the bottom of the groove. It is worth noting that the trends of changes in the DNA ellipticity at 245 and 275 nm were opposite to that found for the B to A-like DNA transition, in which the negative CD band increases towards zero, and to that encountered for the B to Z transition, in which the magnitude of the positive CD signal decreases towards negative ellipticity values.69,70 These results differ from those found for the neutral Ant-PHEA molecule and could be expected taking into account the cationic nature of the Ant-PIm polymer chain, which enhances the interaction with the anionic phosphate substituents located in the wall of the groove inducing the broadening of the DNA secondary structure.

Figure 10. CD spectra of DNA (50.0 µM) treated with: 0.0 (black line), 10.0 (red line) and 20.0 (blue line) µM (curves 1-3) of Ant-PIm at 298 K. The optical inactivity of the Ant-PIm is shown by the straight green line.

3.13. Circular dichroism melting study. Melting of the duplex was monitored by CD spectroscopy, following the changes in the molar ellipticity at 245 nm. The melting profiles of


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the free and bound DNA are reported in Figure 11. The melting temperature (Tm) of DNA was found to be 66 ± 0.5 °C while a Tm value of 72 ± 1 °C was detected for the Ant-PIm-DNA system, with the DNA/Ant-PIm molar ratio equal to 5. A similar result was reported recently in the literature for an NIR-fluorescence probe, designed for sequence-specific recognition of the DNA minor groove.20 These results can be expected in view of the partially bend shape of the polymeric arms of the bio-probe that, by fitting the external electronegative edge of the duplex, can partially unwind the DNA strand stabilizing the B-type morphology and thus increasing its thermal stability. It is worth mentioning that the observed difference is much more pronounced than what had been observed with the parent Ant-PHEA molecule, further indicating that the presence of the cationic moieties in Ant-PIm strongly contributes to stabilizing the secondary DNA structure.

Figure 11. Melting profiles of the free (black line) and complexed DNA (red line) in 5 mM sodium cacodylate trihydrate (pH 7.25). [DNA] = 50 µM ; [Ant-PIm] = 10 µM.

4. Conclusions In summary, the DNA-binding properties of a newly synthesized two-photon fluorescent probe have been comprehensively investigated as functions of temperature, salt and DNA concentration. By comparison with our previously report on Ant-PHEA, this study clearly


26

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establishes that the substitution of the pendant neutral groups on the polymer side chain by cationic moieties strongly affects not only its binding properties towards ds-DNA, but also the structural conformation of the duplex upon complexation. These effects can be unequivocally related to the intrinsic difference in electrostatic charges between the neutral and polycationic probes. More specifically, it turns out that the introduction of positive charges along the ligand template strongly enhances its ability of targeting selectively specific A-T rich areas which, possessing a high negative electrostatic potential, can constrain the guest molecules to be accommodated, in a unidirectional fashion, in specific restricted zone of the duplex giving rise to the formation of a supramolecular DNA-ligand structure. A detailed thermodynamic and FTIR analysis provides information on the nature of the secondary forces and specific binding sites involved in the complexation process and emphasizes the crucial role of the positive charges in establishing interactions with the DNA functional groups located in the wall and/or bottom groove region. The circular dichroism and melting data clearly demonstrate the ability of the probe to induce a major stabilization of the B-DNA secondary structure. Moreover, the effect of changes in salt concentration on the equilibrium binding constant allowed us to separate from the overall Gibbs free energy the electrostatic and non-electrostatic contributions, bringing evidence that the combination of both short and long-range interactions plays a pivotal role in stabilization of the Ant-PIm-DNA adduct. We are currently working on investigating the influence of the probes efficiency and selectivity towards imaging of the cells nuclei since we believe that the newly synthesized compounds constitute promising candidates for future biological applications.

ASSOCIATED CONTENT


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Supporting

Information.

Ant-PIm

synthesis;

characterization;

Page 28 of 34

aggregation

induced

luminescence quenching; TPEF measurements; quantitative data analysis for the Hoechst-DNAAnt-PIm system; Et-Br displacement assay; fluorescence quenching studies by using NaI; van’t Hoff plot; log-log plot of Ka vs. [NaCl]; Stern-Volmer [NaCl] dependence and Fourier transform infrared (FTIR) spectroscopy. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The financial support from NCN OPUS project DEC-2013/09/B/ST5/03417 and a statutory activity subsidy from the Polish Ministry of Science and Higher Education for the Faculty of Chemistry of WUT are acknowledged. REFERENCES (1) Denk, W.; Strickler, J.; Webb, W.W. Science 1990, 248, 73-76.


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Unravelling the Binding Mechanism of a Poly ... - ACS Publications

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