Sensing of Different Human Telomeric G-Quadruplex DNA Topologies

Article pubs.acs.org/JPCB

Cite This: J. Phys. Chem. B 2018, 122, 10279−10290

Sensing of Different Human Telomeric G‑Quadruplex DNA Topologies by Natural Alkaloid Allocryptopine Using Spectroscopic Techniques Paulami Mandal,*,† Dibakar Sahoo,‡ Saumen Saha,† and Joydeep Chowdhury† †

Department of Physics, Jadavpur University, 188, Raja S. C. Mallick Road, Kolkata, West Bengal 700032, India School of Physics, Sambalpur University, Jyoti Vihar, Burla, Odisha 768 019, India

‡

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S Supporting Information *

ABSTRACT: This article describes how a natural alkaloid allocryptopine (ALL) is able to differentiate two forms of biologically relevant human telomeric (htel22) G-quadruplex DNAs (GQ-DNA) depending on the presence of K+ and Na+ ions by steady-state and time-resolved spectroscopic techniques. For both interactions, predominant involvements of static-type quenching mechanism with the negligible influence of dynamic collision are established by UV−vis absorption and fluorescence emission study, which is further supported by fluorescence lifetime measurements. ALL exhibits appreciable affinity toward both GQDNAs. Both the mixed-hybrid (3 + 1) quadruplex structures in K+ ions and the basket-type antiparallel quadruplex structure under Na+ condition are converted to parallel types in the presence of ALL. Fluorescence intercalator displacement assay experiment revealed modest selectivity of ALL to both quadruplexes over duplex DNA along with higher selectivity for antiparallel types among the two quadruplexes via groove and/or loop binding, which is distinct from the conventional π-stacking of the ligands on external G-quartets. ALL stabilized both GQ-DNA topologies moderately. The differences in the dynamics of ALL within both DNA environments have been demonstrated vividly by time-resolved anisotropy measurements using the wobbling-incone model. These results suggest groove binding with antiparallel G-quartet with high affinity and moderate loop binding with mixed-hybrid G-quartet accompanied by the partial end stacking additionally in both of the cases. Our conclusions are further supported by steady-state anisotropy measurements and molecular docking. The present investigation can be used in the development of a biocompatible antitumour/anticancer agent targeting particular GQ-DNA conformation. the presence of extrinsic monovalent cations like Na+ and K+.2,3 Different G-quadruplex topologies are shown in Figure 1a. In Na+ solution, the four-repeat human telomeric sequence d[AGGG(TTAGGG)3] adopts an antiparallel stranded baskettype G-quadruplex.5,6 However, in K+ solution, the same sequence forms multiple predominantly intramolecular (3 + 1) mixed-hybrid G-quadruplex configurations.3,7,8 Nevertheless, to date, not much is known about in vivo quadruplexes. However, among many activities, one of the important biological activities of the G-quadruplex structures is to inhibit the activity of telomerase enzyme. This enzyme is responsible for the eternal life period of cancer cells and is effective in approximately 85% of tumors. Thus, any small ligands that can facilitate and stabilize quadruplex generation can be treated as probable telomerase inhibitors and consequently an antitumour or anticancer drug.9 Apart from potential use in therapy, these quadruplex binders are also important in the

1. INTRODUCTION Since the past two decades, a wide range of new families of anticancer agents with novel action mechanisms have been introduced, but still the drugs that are able to function by interplaying with DNA are one of the significant research areas in our modern world. It is well known that DNA can exist in diverse structural forms like parallel-stranded DNA, singlestranded hairpins, triplexes, i-motif, and tetraplexes or quadruplexes apart from classical double helix, and that has evoked new molecular targets for novel families of anticancer agents.1 However, among these structures, only recently the Gquadruplex structure has been found to be biologically crucial. G-rich sequences are present in the telomeric DNA repeats and also at the promoters of numerous oncogenes/protooncogenes that assume non-canonical four-stranded forms recognized as G-quadruplexes.2,3 These structures are stacks of four guanine bases arranged in a square planar configuration and balanced by Hoogsteen hydrogen bonding.4 Also, Gquadruplexes persist in different topologies either parallel or antiparallel, or in both conformations (termed as mixedhybrid) depending on the DNA-strand orientation or loops in © 2018 American Chemical Society

Received: August 13, 2018 Revised: October 20, 2018 Published: October 22, 2018 10279

DOI: 10.1021/acs.jpcb.8b07856 J. Phys. Chem. B 2018, 122, 10279−10290

Article

The Journal of Physical Chemistry B

antitumor activity for having salient features like large variability in their chemical structures, huge availability from natural sources, cheap extraction processes, and most importantly, low toxicity. The present investigation involves naturally occurring isoquinoline alkaloid (IQA) allocryptopine (ALL) (Figure 1b) as a small molecule that is mainly isolated from the plant families Berberidaceae, Papaveraceae, Fumariaceae, and Rutaceae. ALL is the component of numerous phyto preparations. As other IQAs, it also helps plants in combating biotic stress.31 Moreover, IQAs possess a wide spectrum of biological activities, for example, anti-thrombotic, anti-spasmodic, anti-inflammatory, anti-fungal, neuro-protective, antibacterial, anti-parasitic, and anti-viral activities, apart from their multiple actions on the cardiovascular system.32 Importantly, ALL has antitumor activity, which can be exploited successfully in anticancer therapy.33 This article aims to describe the critical differences in effects of the alkaloid ALL upon the optical, structural, and dynamic aspects of the two different topologies of GQ-DNA in two K+ and Na+ ionic conditions using several steady-state and timeresolved spectroscopic tools and molecular docking. The present investigation can be further employed as a model to other ligand−GQ-DNA interactions. Herein, we have estimated whether the natural alkaloid ALL can distinguish antiparallel topology (under Na+ conditions) and mixed-hybrid topology (under K+ conditions) of htel22 quadruplex DNA and its selectivity toward quadruplexes over duplex DNAs. Altogether, this potential study helps us to differentiate the characteristics and actions of two important GQ-DNA topologies (antiparallel and mixed-hybrid) based on their configurational dissimilarities, and also supports us to understand activities of antitumor and antivirus agents, which would be very much useful in developing novel and highly effective gene-directed therapeutics.

Figure 1. (a) Different structures of GQ-DNA (b) ALL.

development of various DNA nanomachines within, for example, molecular diagnostics, biosensing, and imaging techniques. In all of the abovementioned applications, fluorescence is used as an effective sensing technique involving photostable and chemically stable fluorophores with high fluorescence quantum yields.10,11 In imaging, turn-on fluorescent probes are popular although fluorescence quenchers have also been proven to be significantly useful. Nonetheless, nucleic acids are fascinating construction pieces for guided assemblage of nanostructures.12 From a drug discovery perspective, designing new profoundly selective molecules that can target and stabilize or disrupt G-quadruplex configurations among the most abundant B-form duplex DNA is immensely desirable. Generally, small molecules bind with duplex DNA either by covalent binding (alkylation/interand intra-strand cross-linking) or by non-covalent binding (electrostatic/surface binding with the anionic phosphate backbone, groove binding via hydrogen bonding/by van der Waals interaction, and intercalation within the two bases). However, quadruplex and duplex structures of DNA differ completely from each other in terms of contrasting geometries and functional groups.13 Consequently, small molecules usually interact with the G-quartets by π−π stacking interaction or with the grooves/loops/negative phosphate backbones of Gquadruplex DNA (GQ-DNAs).14,15 However, in targeting Gquadruplexes, the challenge remains in designing ligands that can stabilize quadruplexes with high selectivity and specificity over diverse DNA forms.16−19 However, to date, a number of small molecules have been discovered that can bind and induce telomeric GQ-DNAs, for instance, anthraquinone derivatives,20 porphyrins,21 acridines,22 perylenes,23 triazines,24 aminoglycosides,25 and benzimidazole derivatives,26 and natural elements like ascididemin,28 telomestatin,27 alkaloids, and their derivatives29,30 are recognized as probable anticancer agents. Nowadays, among these, natural elements are coming up as a promising candidate in the development of novel drugs with

2. MATERIALS AND METHODS 2.1. Materials. Our study involves 22-mer human telomeric (htel22) DNA oligonucleotides (5′-AGGGTTAGGGTTAGGGTTAGGG-3′) of HPLC grade, which were bought from Eurofins Genomics India Pvt. Ltd without further purification. Alkaloid ALL (98% pure) was supplied from Aldrich and was used in our study after checking the purity of the alkaloid through impurity emission testing in the desired wavelength region. We took fresh TE buffer. Oligonucleotide solutions were prepared by dissolving it in 10 mM Tris-HCL, 1 mM ethylenediaminetetraacetic acid, and 100 mM KCl and NaCl at pH 7.4 separately.34 First, annealing of htel22 was done at 90 °C for 10 min and then stored at 4 °C for 48 h.35 The concentration of the GQ-DNA stock solution was 100 μM. The formation of (3 + 1) hybrid GQ-DNA topology under K+ ions and antiparallel stranded basket-type GQ-DNA topology under Na+ condition was confirmed through circular dichroism (CD) before each experiment. 2.2. Absorption and Fluorescence Emission Study. At 296 K (ambient temperature), we took the steady-state absorption spectra using an absorption spectrophotometer Shimadzu UV−vis 2401PC and also recorded fluorescence emission spectra of diluted sample solutions through F-7000 fluorescence spectrophotometer (Hitachi). We used rectangular quartz cells of 1 cm path length in both of the cases. 2.3. CD Study. We used CD spectrometer, model J-815150S, connected with a thermostable cell holder to record our CD spectra. We also used a quartz cuvette of 0.2 cm path 10280


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The Journal of Physical Chemistry B length at 20 °C, TE buffer, and 100 mM of both salts. We obtained our spectra as a function of wavelength (200−350 nm), which is the averages of three scans at a scan speed of 100 nm/min with baseline correction from the blank buffer. Equilibration was ensured by recording each spectrum after 10 min of the addition of both DNAs. 2.4. Fluorescence Intercalator Displacement Assay. Fluorescence intercalator displacement (FID) titrations were performed at room temperature in buffers. The DNA solutions were prepared by mixing 0.5 μM thiazole orange (TO) with 0.25 μM of both GQ-DNAs. An increasing amount of ALL was added successively in 5-min equilibration periods before the fluorescence spectrum (excitation at 501 nm and emission at 510−700 nm) was recorded. 2.5. CD Melting Study. DNA melting study in the absence and presence of the alkaloid was performed through JASCOJ815-150SCD spectrometer at the heating rate of 1 °C/min. All CD melting experiments were monitored at 295 nm and the temperature was increased from 20 to 90 °C. The CD melting experiments were conducted in TE buffer with 100 mM of both salts at pH 7.4. The Tm has been calculated by taking the first derivative of the melting profiles. Samples were tightly capped by Teflon stoppers.36 2.6. Studies of Steady-State and Time-Resolved Anisotropy. Steady-state anisotropy (r) was assessed using Varian Cary Eclipse (USA) spectro-fluorimeter. In our study, anisotropies were calculated by using the relation37 ÅÄÅ ÑÉ ÅÅI − IHV I ÑÑÑ ÅÅ VV ÑÑ VH IHH Å ÑÖ r = ÄÅÇ ÉÑ ÅÅÅI + 2 IHV I ÑÑÑ ÅÅ VV ÑÑÖ IHH VH Ñ ÅÇ

the DFT calculations, B3LYP functional inclusive of 6-31+ +G(d,p) basis set was adopted. The 6-31++G(d,p) is a split valence basis set with polarization functions “d” and “p”. The polarization functions allow the electron distribution to remain polarized in the computational run toward the estimation of the optimized molecular structure of ALL. ALL contains nitrogen and oxygen atoms, both having lone pair electrons. Lone pair electrons on these atoms are loosely bound, and they settle at larger distances from the corresponding nuclei in comparison to bonding or core electrons. DFT calculations take into account the effect of lone pairs to estimate the optimized geometry of the molecule. Gradient-correlated Becke’s three-parameter hybrid exchange (B3) and Lee− Yang−Parr correlation (B3LYP) functional were used in the DFT calculation.38,39 However, the vibrational frequency estimations do not exhibit any imaginary value, signifying the attainment of local minima on the potential energy surface of the ligand molecule. The optimized molecular structure of ALL is shown in Figure S1. DFT calculations were accomplished by Gaussian 03 software suit in the Windows operating system. The optimized structure of the ligand ALL was docked one by one with antiparallel basket type and (3 + 1) hybrid-type GQDNA with the aid of AutoDock 4.2 software. The crystal structures of antiparallel basket type and mixed-hybrid GQDNA were obtained from the Protein Data Bank (PDB)40,41 with the PDB identifier 143D (antiparallel GQ-DNA) and 2HY9 (mixed-hybrid GQ-DNA), respectively. Docking calculations were accomplished using AutoDock 4.2 software. It deploys an empirical scoring function on the basis of free energy binding.42 AutoDock Lamarckian Genetic Algorithm (LGA) was used to estimate the binding stance of the ligand molecule.

where IVV and IVH are the intensities of parallel and perpendicular polarized emission with vertically polarized excitation, and IHH and IHV are the intensities of horizontally and vertically polarized emission while excited with horizontally polarized light. IHV/IVV describes instrumental correction factor G. This correction is contrived because of any alteration in the emission channel sensitivity for the vertically and horizontally polarized components. The time-resolved fluorescence decays and polarized emission decays are measured by the single-photon-counting technique. The time-dependent fluorescence anisotropy function r(t) is demonstrated below37

3. RESULTS AND DISCUSSION 3.1. Steady-State UV−Vis Absorption Spectra. In order to reveal ground-state interaction between the natural alkaloid ALL and two forms of GQ-DNAs in the presence of K+ and Na+ ions, respectively, UV−vis absorption technique was used. It was employed to monitor the modifications in absorption spectra of ALL as we enhanced the concentrations of two GQDNAs gradually (up to 8 μM) (Figure 2). The concentration of ALL was kept fixed at ∼100 μM all over the UV−vis experiment. ALL absorption spectra are found to consist of a wide band with a maximal at 283 nm. The stepwise addition of the two GQ-DNAs to the alkaloid solution contributed a

( ) ( )

r (t ) =

IVV(t ) − IVH(t ) IVV(t ) + 2IVH(t )

Here, IVV(t) and IVH(t) describe the polarized fluorescence decays. Here we have excited the samples at 340 nm by a picosecond diode (IBH NanoLED-07). Corresponding emission was recorded at a magic angle polarization using a Hamamatsu MCP photomultiplier (2809U). The timecorrelated single-photon counting set-up has an ORTEC 9327 CFD and a Tennelec TC 863 TAC. Data are collected by a PCA3 card (Oxford) as a multichannel analyzer. Twenty-five picosecond is the full width at half-maximum of the system response. The width of the channel is 12 ps/ channel. Deconvolution of fluorescence decays has been made by IBH DAS6 software. 2.7. Docking Studies. The ligand ALL molecule was optimized using density functional level of theory (DFT). In

Figure 2. Absorbance spectra of ALL (conc. ≈ 100 μM) in the presence of increasing GQ-DNA concentrations in K+ condition from 0 to 8 μM (inset: GQ-DNA concentrations in Na+ condition from 0 to 8 μM). 10281


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The Journal of Physical Chemistry B hyperchromic effect to the 283 nm absorption band of ALL. In addition, there is a blue shift of 5 nm in K+ and 3 nm in Na+ at 283 nm peak position. These significant modifications in the ALL absorption spectra indicate the occurrence of a noticeable change in the electronic environment of ALL as it binds with both GQ-DNAs.43 Moreover, the blue shifts in both spectra suggest that the polarity around ALL molecules alters from lower to higher because of its interaction with two DNAs. Absence of the isosbestic point in the absorption spectra implies that ALL binds with both GQ-DNAs probably via more than a single mode.44 Thus outcome of our absorption study surely indicates an influence of ALL on the two types of GQ-DNAs in their ground states. 3.2. Fluorescence Emission Studies. To add valuable information regarding the structure and dynamics of both GQDNA topologies, we measured the intrinsic fluorescence changes of the alkaloid ALL in the presence of both htel22 GQ-DNA structures in the K+ and Na+ ions. In our study, ALL was excited at 285 nm in the presence of GQ-DNAs (Figure 3). It should be noted that the absorbance of both GQ-DNAs at 285 nm was very negligible. From Figure 3a,b it is apparent that with the increasing concentrations of GQ-DNA from 0 to 8 μm in both K+ and Na+ ionic conditions, the fluorescence emission spectra of the alkaloid was quenched gradually. Throughout the experiment, the concentration of ALL was kept constant at 50 μM. However, it is logical to presume that at high concentrations of DNAs, inner-filter effect may occur during the quenching of ALL fluorescence. Therefore, corrections of the steady-state fluorescence intensity for the inner-filter effect were accomplished by the following equation37 i ODqex ODqem zy zz fcorrected = fobs antilogjjjj + 2 z{ k 2

where ODqex and ODqem are the absorbance and emission intensity of DNAs at the excitation wavelength 285 nm. However, corrections were found to be negligible. Gradual mixing of the two DNAs (Figure 3a,b) caused a quenching in the emission profile of ALL (∼49% in K+ and 71% in Na+ at 8 μM of each GQ-DNA). However, the fluorescence quenching of ALL in the presence of two GQ-DNAs indicates an appreciable binding interaction, but in the presence of Na+ ions, the binding interaction is more pronounced than in the presence of the other ion. In both of the cases, to find the binding modes of ALL with GQ-DNAs, we employed Stern−Volmer equation37

Figure 3. Emission spectra of ALL (50 μM) in the presence of increasing concentrations of (a) GQ-DNA under K+ condition from 0, 1, 2, 3, 4, 5, 6, 7, 8 μM (inset: SV plot of ALL−GQ-DNA interaction under K+ condition) and (b) GQ-DNA under Na+ condition from 0, 1, 2, 3, 4, 5, 6, 7, 8 μM (inset: SV plot of ALL− GQ-DNA interaction under Na+ condition). (c) Plot of log[(F0 − F)/ F] vs log[GQ-DNA] for the interaction of ALL and GQ-DNA in the presence of Na+ ions. (Inset: Plot of log[(F0 − F)/F] vs log[ct-DNA] for the interaction of ALL and GQ-DNA in the presence of K+ ions.)

are almost linear in nature, which indicates predominant involvement of either static or dynamic quenching processes in both cases. The values of KSV and kq are listed in Table 1 and

F0 τ = 1 + KSV[Q] = 0 F τ

Table 1. Stern−Volmer Quenching Constants for ALL−GQDNA Interactions in the Presence of K+ and Na+

Here F0 and F define the steady-state fluorescence emission intensity in the absence and presence of quencher, respectively. KSV (=kqτ0) is the SV quenching constant and [Q] is the concentration of DNA. kq is the bimolecular quenching constant and τ0 is the unquenched lifetime of the molecule, which is 2.445 ns for ALL. KSV was estimated in both of the cases to assess the efficiency of fluorescence quenching of ALL by DNAs. The linearity of SV plots hints the involvement of only one type of quenching process, that is, either dynamic or static kind. In contrast, upwardly curved SV plots reveal the existence of both static and dynamic quenching processes.45 The present results show that for the two different ion conditions, the corresponding SV plots (insets of Figure 3a,b)

system ALL + GQ-DNA +

K Na+

KSV (L·mol−1)

kq (L·mol−1·s−1)

R2

1.003 × 10 3.44 × 105

4.23 × 10 1.45 × 1014

0.99188 0.97742

5

13

the kq for both of the cases are found to be much higher compared to the maximum scatter collision quenching constant (2 × 1010 L·mol−1·s−1) for various quenchers with bimolecules.45 This confirms that in the two interactions, there prevails mostly a static kind of quenching. Thus, SV results estimate that ground-state strong complex formation is the key cause behind the appreciable fluorescence quenching, whereas 10282


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The Journal of Physical Chemistry B dynamic collision might have some negligible influence.46,47 However, these values reveal that apart from dynamic contributions, ground-state complex formation in between the alkaloid ALL and GQ-DNA topology in the Na+ ion conditions is much stronger and more stable than that in the K+ ion condition. Moreover, the KSV and kq values for both GQ-DNAs lead us to speculate a few probable reasons behind this fluorescence emission quenching of ALL, such as: (i) intercalation of ALL in between the GQ-DNA quartet or (ii) end stacking at the top or bottom end and/or (iii) binding in the external loops and/or grooves of GQ-DNA.25,35 However, the unfolding of an intercalation site, buried in the stack of Gquartets, is very low energetically. Nonetheless, there is no superposition of the emission of ALL and absorption spectra of any nucleobases, which makes us cancel out the probability of the resonance energy transfer (FRET). Therefore, primarily our steady-state measurements attribute to the prospect of exterior loop and/or groove binding accompanied by the partial end stacking of ALL to both G-quadruplexes in the presence of the two ions.48 3.3. Time-Resolved Measurements. To have a deep insight into the binding mechanism, we also performed different time-resolved measurements upon the ligand− quadruplex interactions. From the time-resolved lifetime measurements, it is found that the lifetime decay profile of alkaloid follows a double exponential fitting (Figure S2). In our study, we used the average lifetime of ALL of these two lifetime components, which is found to decrease gradually with the increasing concentrations of DNAs in the presence of both K+ and Na+ ions (Table S1), which is indicative of some sort of involvement of dynamic quenching effect in both of the cases. In order to gain more information about the lifetime data, we calculated the dynamic quenching constant of these interactions by using the Stern−Volmer equation37

(F0 − F ) = log Kb + n log[DNA] F where F0 and F are the fluorescence intensities of ALL without and with the two GQ-DNAs, respectively. Concentrations of the two G-quadruplexes are denoted by [DNA]. Kb and n have their usual meaning. It can be observed from Figure 3c that the log(F0 − F)/F versus log[DNA] plot is linear in nature. Corresponding Kb and n values are presented in Table 2. The value of n for the log

Table 2. Binding Parameters for ALL−GQ-DNA Interactions in the Presence of K+ and Na+ system ALL + GQ-DNA

Kb (L·mol−1)

n

R2

in K+ in Na+

7.74 × 104 2.44 × 108

0.97912 1.55

0.9867 0.9949

alkaloid−DNA interaction under the K+ condition reflects the presence of a single binding site and less stable complex formation, whereas that in the presence of Na+ signifies involvement of more than one binding sites and stronger as well as more stable complexation in comparison to the preceding one. The values of Kb for the ALL−GQ-DNA interactions suggest that in the K+ ionic condition, there was a modest ligand−quadruplex interaction, but in the presence of Na+, the interaction was much stronger than the previous one.50 Thus, much more binding affinity of ALL toward GQDNA species in Na+ than that toward the GQ-DNA structures in the K+ ion conditions is revealed from the Kb values. The reasons behind the affinity of our alkaloid toward both GQDNAs might be attributed to the 10-member heterocyclic ring of ALL, consisting of one tertiary nitrogen and carbonyl group, stabilized by strong electrostatic interaction, which helps the alkaloid to bind with the bases of both DNAs via hydrogen bonding, van der Waals interaction, and π−π stacking and probably by electrostatic interaction with the phosphate backbone as well. Differences in binding constants and binding sites demonstrate the specificity and selectivity of the ligand toward the structural differences of both GQ-DNAs (antiparallel and mixed hybrid) under the two different ion conditions. 3.5. CD Spectroscopic Studies. In order to further understand the differences between the ALL and DNA interactions in the presence of two different ions, we employed CD spectroscopy. In our study, CD spectroscopy discloses the changes that occurred in the different GQ-DNA topologies, the effects of metal ions, and the stability of GQ-DNAs because of the interaction with ALL in the far-UV region (190−350 nm). Usually an antiparallel quadruplex structure is represented by the positive peaks at nearly 295 and 240 nm, accompanied by a negative band at 260 nm, whereas a negative peak close to 240 nm and a positive peak around 260 nm signify a parallel topology of quadruplex DNA. Mixed-hybrid structure is characterized by two positive maxima at 290 and 270 nm. Figure 4a demonstrates the CD spectrum of the oligo htel22 under the K+ condition, where the DNA concentration was fixed at 10 μM. Here we obtain a broad positive band at around 290 nm, with a tiny positive peak at 273 nm along a negative band around the 234−240 nm region, which possibly suggests the formation of mixed-hybrid (3 + 1) quadruplex configuration.35,41 There is also a hump at around 250 nm, which seemingly indicates the presence of a meager portion of

τ0 = 1 + KD[Q] τ

where τ0 and τ are the lifetime of ALL in the absence and presence of two types of GQ-DNA, respectively, and KD and [Q] are the dynamic quenching constant and the quencher’s concentrations, respectively. By employing this SV equation, we plotted the lifetime data of ALL at different concentrations of GQ-DNA (inset of Figure S2) for both ionic conditions. The plots are linear in nature, and the corresponding dynamic quenching constants are calculated as 3.25 × 104 in the presence of K+ and 1.03 × 105 in Na+ condition. The corresponding bimolecular quenching constants are found to be much higher than the theoretically estimated value generally arising from the diffusion properties.49 These results surely reveal the assured presence of some other additional quenching mechanism other than static-type quenching in our study. Hence, lifetime results further corroborate our previous predictions about the dominating presence of the static quenching process along with insignificant contribution of dynamic quenching processes in the two interactions. 3.4. Estimation of Binding Parameters. Next, to further assess the binding constant (Kb) and the corresponding stoichiometry (i.e., n: number of binding sites) for the interactions of ALL with two GQ-DNAs in the presence of two ions, K+ and Na+, fluorescence titration data was employed in the following equation43 10283


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(Tm: midpoint of the melting transition is the melting temperature) of DNAs at 295 nm wavelength. In our case, melting investigations were carried out for htel22 quadruplexes in K+ (mixed-hybrid topology) and Na+ (antiparallel topology) ions, respectively, by following the changes in ellipticity at 295 nm (Figure 5). In the absence of the ligand ALL, the Tm of the

Figure 5. CD melting profiles of G-quadruplex (10 μM) at 295 nm: in the presence of K+ (1) no ligand, (2) 10 μM ALL (inset: in the presence of Na+ (1) no ligand, (2) 10 μM ALL).

GQ-DNA in the K+ and Na+ conditions are 59.8 and 54 °C, respectively. In the presence of ALL (concn ≈ 10 μM), the melting temperature of GQ-DNA in the presence of K+ increased by 3.6°, whereas under Na+ condition, it was enhanced by 3 °C, indicating a moderate stabilization of both GQ-DNAs upon binding with ALL.5 Therefore, thermal melting results refer to the almost similar propensity of ALL to form stable interactions with both GQ-DNA topologies by enhancing the stability of both GQ-DNAs with respect to the native GQ-DNAs. 3.7. FID Assay Experiment. Next, to get a deep insight into the nature of binding of ALL to both GQ-DNA species in the presence of K+ and Na+ ions and at the same time to evaluate the affinity and selectivity of ALL to the GQ-DNAs above the duplex DNA, an elementary and rapid technique, FID assay,53 was exploited in our study. It is the fall of fluorescence of TO because of competitive displacement of DNA by the putative ligand. DC50 estimates the affinity of a molecule for a particular DNA topology. This is actually the amount of ligand needed to produce a 50% fluorescence reduction, which is reflected by a 50% displacement of the fluorescence probe. Here, we have analyzed the binding capacities of ALL with two different topologies of quadruplex DNA. Figure 6a displays the fluorescence emission spectra of TO−GQ-DNA mixtures without and with ALL showing quenching of emission intensity of the mixture because of gradual titration of ALL into TO−GQ-DNA solutions. Our investigation revealed that 2.5 μM ALL failed to dislocate TO entirely from TO−GQ-DNA complexes. This ligand concentration is conventionally employed in these types of assay studies. In our case, only 37% TO was dislocated from TObound GQ-DNA under K+ condition by 16 μM of ALL, whereas only 26.5% of TO from TO−GQ-DNA complex under Na+ condition was displaced at 11.8 μM of ALL. Thus, here the determination of GQDC50 was not possible for both of the cases. Moreover, 15 μM ALL displaced only 46% TO from TO−duplex DNA complex (Figure 6b). Hence, the selectivity could not be evaluated conventionally by the dsDC50/GQDC50

Figure 4. CD spectra for (a) (3 + 1) hybrid GQ-DNA (10 μM), (b) antiparallel GQ-DNA (10 μM) at 2, 5, 7, 9, 11, 13, 15 μM concentration of ALL.

unvaried single-strand GQ-DNA.51 As we add ALL regularly to the DNA solution, the 234 nm trough along with a hump at 270 nm becomes more salient accompanied by a decline at 290 nm peak. Moreover, the 250 nm band gets diminished gradually. Nonetheless, as a peak at 270 nm and a dip at 240 nm commonly attribute to parallel GQ-DNA,52 here we assume that a larger portion of parallel quadruplexes were created because of the interaction of ALL with hybrid GQDNA. Hence in conclusion, we presume that under K+ ions when ALL is absent, mixed (3 + 1) quadruplex topologies were formed along with a tiny portion of unvaried single-strand GQDNA. These single-strand GQ-DNAs were further converted to parallel ones upon the addition of ALL (Figure 4a). Most likely, under K+ condition, GQ-DNA was transformed from mixed to parallel topology by ALL, and at the same time, further parallel GQ-DNA generation was facilitated. In contrast, the CD spectrum of the htel22 G-quadruplex under the Na+ ions condition (Figure 4b) consists of a 263 nm negative band along with 294 and 240 nm positive bands, which confirm the formation of the basket-type antiparallel structure of the free GQ-DNA in this ion condition.43,53 The addition of ALL lead to the gradual decrement of 240, 263, and 294 nm bands with a slight red shift of 1 nm in the 294 peak. Also, the intensity of the band around 280 nm increased slightly. These modifications in CD spectra suggest complexation of ALL with GQ-DNA in Na+ condition, which further induced a change in GQ-DNA from the antiparallel configuration to a parallel one keeping its global secondary structure unaltered.53 3.6. CD Melting Studies. Thermal stability of the quadruplexes in the presence of ligands was assessed from the CD spectroscopy by comparing the melting temperatures 10284


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that ALL shows higher selectivity toward the telomeric quadruplex form in Na+ than that in the presence of K+. Scheme 1. Probable Binding Position of Allcryptopine with the Antiparallel and Mixed-Hybrid GQ-DNA

3.8. Steady-State Anisotropy. Anisotropy measurement allows us to estimate the extent of rigidity offered by the surrounding environment.37 The change in the steady-state anisotropy of ALL with the change in concentrations of the two GQ-DNAs is presented in Figure 7a. We obtained a sharp rise in the steady-state anisotropies with the subsequent mixing of two types of quadruplexes, which indicates that ALL were rotationally far more restricted inside of both DNA environments compared to its free state. In the presence of 7 μM of GQ-DNA, under K+ condition the increment in the rotational restriction of ALL is almost two times, whereas in the Na+ condition, it is six times its free state. 3.9. Time-Resolved Anisotropy. In order to be more aware of the rotational dynamics of ALL inside of both Gquadruplexes, we carried out time-resolved anisotropy decay analysis of ALL in the buffer solution. Figure 7b presents timeresolved anisotropy decay curves, and the corresponding data are compiled in Tables 3 and 4. Without G-quadruplexes, we obtained single exponential anisotropy decay of ALL with rotational relaxation time of 608 ps because the buffer provides a uniform surrounding for ALL. In contrast, mixing of two GQDNAs to ALL delivered bi-exponential decay profiles of ALL with the two distinct rotational relaxation times arising out of free and constrained ALL, respectively. For both of the quadruplexes, when ALL is constrained to DNA, a slow component emerges, and this is the rotational time appearing from the DNA-constrained ALL. Moreover, there is a fast component signifying the rotational time of the unbound alkaloid. Corresponding slow and fast components of rotational relaxation times for both of the cases are enlisted in Table 3. The hydrodynamic volumes of unbound ALL and DNA-fastened ALL could be derived from the rotational relaxation times (τr) through the Stokes−Einstein’s relation given below35

Figure 6. Fluorescence displacement of TO-bound (a) GQ-DNA in the presence of Na+ by 1, 2, 3, 5, 6, 7, 8, 9, 10 μM of ALL (inset: TObound GQ-DNA in the presence of K+ by 1, 2, 3, 5, 6, 7, 8, 9, 10 μM of ALL), (b) duplex DNA by 1, 2, 3, 5, 6, 7, 9, 11, 14, 16 μM of ALL.

as it would implicate unreliable extrapolation of the plot. Alternatively, we introduced estimated selectivity, which is defined as follows:54 First, dsDC2.5μM value for the duplex DNA−TO complex was calculated and was found to be 10% (Figure 6b); then the necessary ALL concentration (GQC) to attain equal amount (10%) of dislocation of TO from TO− GQ-DNA under K+ and Na+ conditions was measured, and it was found that GQCK = 1.9 μM and GQCNa = 0.8 μM; in the end, we defined the estimated selectivity as 2.5/GQC, and in this study, this was computed to be 3 in the Na+ ions and 1.3 in K+ conditions (Table S2). These values hint the modest selectivity of ALL toward both quadruplex structures over duplex DNA. However, ALL specifically shows more selectivity to the antiparallel topology of GQ-DNA in the presence of Na+ than the mixed-hybrid structures in the presence of K+ over dsDNA. Thus, from our FID experiment, it could be demonstrated that ALL fastens to both GQ-DNAs with reduced affinity than TO, and/or ALL probably ties to GQ-DNAs in a mode different from that of TO. Our fluorescence quenching results have already indicated moderate affinity of ALL toward mixedhybrid GQ-DNA in K+ and strong affinity to the antiparallel basket-type GQ-DNA in Na+. Henceforth, this fractional dislocation of TO from the GQ-DNA−TO composite firmly implies the presence of an indirect fight in the binding of ALL to a site like quadruplex grooves and/or loops. This binding mode of ALL is clearly distinct from that of TO, which typically binds to GQ-DNA via π-stacking on its external quartets.54 Although both mixed-hybrid (3 + 1) and antiparallel basket-type quadruplexes of 22 mer human telomeric DNA have accessible quartets at the exterior, which can easily welcome planar binders, ALL might not adopt a stretched configuration favoring a nontypical binding mode (Scheme 1) in our case. Thus, our FID results suggest

τr =

ηV 1 = 6Dr kT

where Dr, η, and V are the rotational diffusion coefficient, viscosity of the medium, and hydrodynamic volume of the complex at absolute temperature T, respectively. The hydro10285


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time, and r0 = limiting anisotropy. S2 satisfies the inequality 0 ≤ S2 ≤ 1, where S2 = 0 denotes unrestricted reorientation and S2 = 1 when no wobbling-in-cone orientational motion occurs. The present model demonstrates that the faster rotational relaxation (τfast) is the motion of a constrained rotor or probe that has its transition dipole moment (μ) enduring an orientational diffusion in a semicone of angle θω about a hypothetical axis. Hence, faster and slower rotational lifetime constants can be described as follows 1 1 1 = − τω τfast τslow

where τω is the decay time constant of the wobbling motion of the probe. The semicone angle θω is obtained from the order parameter in the following way ÄÅ ÉÑ2 Å1 Ñ S2 = ÅÅÅÅ (cos θω)(1 + cos θω)ÑÑÑÑ ÅÇ 2 ÑÖ The wobbling-in-cone diffusion constant Dω when θω ≤ 30° can be derived from the relations given below Dω ≈ Figure 7. (a) Variation of steady-state fluorescence anisotropy of ALL (50 μM) as a function of increasing concentration up to 7.5 μM of GQ-DNA in the presence of Na+ (inset: variation of steady-state fluorescence anisotropy of ALL (50 μM) as a function of increasing concentration up to 7.5 μM of GQ-DNA in the presence of K+). (b) Time-resolved anisotropy decay of ALL (50 μM) in the (■) absence of DNA, (●) presence of GQ-DNA (7.5 μM) under K+ (inset: timeresolved anisotropy decay of ALL (50 μM) in the (■) absence of DNA, (●) presence of GQ-DNA (7.5 μM) under Na+) (λex = 285 nm, λmon = 323 nm).

7θω 2 24τω

where θω is in radians. Nonetheless, when θω ≥ 30° ÅÄÅ 2 Å x (1 + x 2) l o ij 1 + x yz zz Dω = {τω(1 − S2)}−1 ÅÅÅÅ mlnjj ÅÅÇ 2(x − 1) o n k 2 { i 1 − x yz| o 1−x zz} + + jjj (6 + 8x − x 2 − 12x 3 24 k 2 {o ~ ÉÑ Ñ Ñ − 7x 4)ÑÑÑÑ ÑÑÖ

dynamic radius of ALL is found to be 9.1 Å, whereas after binding with GQ-DNA in K+ and Na+, it is estimated to be 17.94 and 13.22 Å, respectively (Table 4). These increments in the hydrodynamic radius of our alkaloid ALL in the presence of both DNAs further corroborate our presumption of the formation of association complex between ALL and the two GQ-DNA topologies. Additionally, the wobbling motion of ALL inside of the two quadruplexes has also been investigated employing the wobbling-in-cone model. Conferring to the present model, the rotational anisotropy decay function is given below44,55 ÅÄÅ ÑÉÑ ÅÅ jij ( −t ) zyz jij ( −t ) zyzÑÑÑ Å r(t ) = r0ÅÅβ expjj z + (1 − β)expjj z j τslow zz j τfast zzÑÑÑÑ ÅÅ k { k {ÑÖ ÅÇ 2 where S = β is the order parameter defining the degree of restriction on the wobbling-in-cone orientational motion, β = pre-exponent factor of the slow part of rotational relaxation

where x = cos θω In Table 4, the calculated values for Dr, τr, τω, Dω, and S are provided. Upon binding with quadruplexes, ALL experiences a reorientation because of constrained internal rotational motion above the overall rotation. As discussed earlier, S refers to the spatial restriction of the dynamics of the fluorescence probe in the restricted space. However, free ALL in buffer possess fast unrestricted motion and the order parameter (S) becomes zero in this case. However, DNA-bound ALL faces an exceedingly aligned distribution within both GQ-DNA environments under K+ and Na+ ionic conditions, and therefore, here the order parameters become 0.256 and 0.433, respectively. We could thus anticipate the angular range of the constrained internal motion of the quadruplex-bound ALL from the calculated values of semicone angle θω (Scheme 2). However, we obtained two different sets of parameters of ALL internal

Table 3. Fitted Parameters of Anisotropy Decays of ALL and ALL−GQ-DNA Complexes in the Presence of K+ and Na+ Ions sample ALL (50 μM)

rotational relaxation time (τfast) (ps)

pre-exponential factor (β)

rotational relaxation time (τslow) (ns)

pre-exponential factor (β)

χ2

in Buffer K+ + 8 μM GQ-DNA Na+ + 8 μM GQ-DNA

608 603 623

1 0.93 0.81

4.63 1.85

0.07 0.19

1.0723 0.9973 0.9196

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The Journal of Physical Chemistry B Table 4. Calculated Parameters of the Wobbling-in-Cone Model sample ALL ALL + 8 μM GQ DNA in K+ ALL + 8 μM GQ-DNA in Na+

τr/ns 0.608 4.635 1.85

hydrodynamic radius (Å)

Dr/s−1

9.121 17.94 13.22

2.74 × 10 3.59 × 107 9.01 × 107

anisotropy 8

0.263 0.273 0.338

τω/ns 0.693 0.94

Dω/s−1 2.62 × 108 0.85 × 108

θω (deg)

S

59.22 29.95

0 0.256 0.433

(Dω = 2.62 × 108), and angular range (θ = 59.22°) (Table 4).58 In contrast, ALL was possibly accommodated within antiparallel basket-type GQ-DNA in one of its grooves (wide, narrow, and medium) and possibly fenced by the sugar backbone, leading to the hugely restricted diffusion of ALL inside G-quartet groove. This in turn get reflected by the higher order parameter (S = 0.433), the lower diffusion constant (Dω = 0.85 × 108), and the narrower angular range (θ = 29.95°) compared to the other GQ-DNA topology in K+ ions (Table 4).58 Hence, our investigation shows that the binding affinity becomes higher when the binding mode is more restrictive. We could therefore consider the order parameter (S) as a substitute in evaluating the selectivity of the ligand for GQDNA. Altogether, our time-resolved fluorescence anisotropy results have been interpreted successfully using wobbling-incone model. 3.10. Molecular Docking. Having information about the probable binding site inside of the DNA is truly crucial in predicting the efficiency of a biologically active drug molecule in the medicinal application. The molecular docking calculations allow us to estimate the binding location of the ligand in the DNA environment. Figure 8 shows the most probable docking pose depicting the binding of ALL with mixed-hybrid GQ-DNA in K+ condition and antiparallel GQDNA under Na+ ionic condition. The binding stance indicates that the ligand ALL undergoes loop binding through TTA bases of the mixed-hybrid GQ-DNA via hydrogen bonding and van der Waals interaction. The specific region is highlighted in Figure 8a. In the case of antiparallel quadruplex DNA, ALL is attached through medium groove binding and fenced by the sugar backbone. The stance is shown in Figure 8b. Here this is a conceivable binding mode because ALL is unlikely to have end stacking because of its nonplanar structure. Thus, docking results are consistent with the above experimental findings.

motions, like order parameter, diffusion coefficients, and so forth, for the two distinct GQ-DNAs. Scheme 2. Schematic Representation of Time-Resolved Fluorescence Anisotropy of GQ-DNA and ALL Complexes Using Wobbling-in-the-Cone Model

In Na+, our htel22 sequence forms an antiparallel stranded basket-type G-quadruplex, which consists of one narrow, one wide, and two medium grooves. Here, the loops are edgewise− diagonal−edgewise.5 Most probably, here ALL resides within one of the grooves (wide, narrow, and medium) and possibly fenced by sugar backbone, which resulted in electrostatic interaction, further restricting its rotational motion within the groove. Otherwise ALL could also be accommodated either in diagonal loop or in edgewise loop along with end stacking on external quartets, which originates a restrictive force on the alkaloid. However, in K+ solution htel22 sequence gets stabilized to mixed-hybrid quadruplex structure containing one double-chain reversal and two edgewise loops along with one wide groove, two medium-sized grooves, and one narrow groove. Here the grooves are generally guarded by the doublechain reversal loops, which make either the edge groove or the double-chain reversal loop and/or lateral loops the favorable binding sites for ALL. Nevertheless, groove size is another determinant factor for the ligands to fit inside it.56 Therefore, in this case, ALL might be collaborating with the loops of the hybrid GQ-DNA via either stacking or creating H-bond on one/two-loop bases accompanied by the poor fractional end stacking at the closing part of the G-quartet. However, the corresponding affinity of this binding mode is much lower in comparison to the end stacking or sandwich stacking or groove binding. The flexibility of the loops and the limited π−π stacking interface are the reasons behind this low affinity.57 For the mixed-hybrid (3 + 1) quadruplex structure, as discussed above, the alkaloid was possibly attached in the loop and/or grooves along with partial end stacking, which is revealed by the order parameter (S = 0.256), diffusion constant

4. CONCLUSIONS Here, by using various spectroscopic techniques, the interactions of the natural alkaloid ALL with the two different GQ-DNA topologies, mixed-hybrid under K+ ions and baskettype antiparallel under Na+ condition, were extensively studied. Our steady-state and time-resolved study revealed predominant involvements of static-type quenching mechanism in both of the interactions because of ground-state complex formation with negligible dynamic contribution. Binding constants and the number of binding sites hint that ALL has a stronger affinity to the antiparallel G-quadruplexes compared to the mixed-hybrid GQ-DNA. Our FID assay results also exhibit higher selectivity for basket-type antiparallel GQ-DNA compared to the mixed-hybrid quadruplex structure. Moreover, ALL shows modest selectivity for both of the quadruplexes over duplex DNA. Possibly, the tertiary nitrogen and carbonyl group of ALL is facilitating hydrogen bonding, van der Waals interaction, and π−π stacking with the bases of both GQ-DNAs, and also favoring the electrostatic interaction 10287


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expected to contribute to the emergence of novel anticancer therapies with diminished toxicity.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.8b07856. Figure of the optimized geometry of the molecule ALL (Figure S1), fluorescence lifetime decay curve of ALL in the presence of increasing concentrations of (a) GQDNA under K+ condition and (b) GQ-DNA under Na+ condition (Figure S2a,b), table of the fluorescence lifetimes of ALL in the free form and ALL−GQ-DNA complexes (Table S1), table of comparative FID results for ALL (Table S2), and fitted parameters of anisotropy decays of ALL and ALL−GQ-DNA complexes in the presence of K+ and Na+ ions (Table S3) (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Paulami Mandal: 0000-0001-9773-1296 Dibakar Sahoo: 0000-0002-0201-1411 Joydeep Chowdhury: 0000-0001-9952-9956 Notes

The authors declare no competing financial interest.

■

Figure 8. Docked structure of (a) ALL−antiparallel GQ-DNA complex (representative snapshot of groove binding), and (b) ALL−mixed hybrid GQ-DNA complex (representative snapshot of loop binding). ALL is colored green-red-blue-gray in the ball and stick mode.

ACKNOWLEDGMENTS We are heartily thankful to Prof. Nikhil Guchhait of the Chemistry Department, University of Calcutta, Kolkata, for providing the opportunity to handle the time-correlated singlephoton counting machine. P.M. is grateful to Science and Engineering Research Board (SERB), New Delhi, India, for granting the financial assistance in the form of grants and fellowships (project file no. SB/YS/LS-199/2013).

with the phosphate backbone of DNAs. FID results also suggest that during the binding of ALL with both GQ-DNAs, ALL undergoes an indirect fight to bind in grooves and/or loops, clearly distinct from usual π-stacking on GQ-DNAs’ exterior quartets. We have found modest stabilization of both GQ-DNA topologies upon binding with ALL. The differences in the dynamics of ALL within both GQ-DNA environments were demonstrated thoroughly via wobbling-in-cone model in the time-resolved anisotropy analysis. This analysis provides us with a fair account of the dynamics of the constrained inner rotation of the alkaloid inside of the two GQ-DNA environments. The alkaloid was binding with the antiparallel G-quartet possibly by partial end stacking and groove binding with high affinity. On the other hand, ALL binds to the mixedhybrid structure moderately in the loops of DNA along with fractional end stacking. Our docking results further confirmed the above findings. Hence, our present investigation has shown that the small planar natural alkaloid ALL binds in two completely different modes with the two different GQ-DNA structures, antiparallel and mixed hybrid, with different affinity. Thus, ALL could discriminate two different GQ-DNA structures. This could be further exploited in the development of the therapeutic agents in targeting particular GQ-DNA conformation. Additionally, the present investigation can be extended to the development of other derivatives of ALL with improved quality of selective targeting of GQ-DNA in cancer cells and with greater stabilization properties, which are

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REFERENCES

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