This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.
Article Cite This: ACS Omega 2019, 4, 6347−6359
http://pubs.acs.org/journal/acsodf
Affinity of Anticancer Drug Daunomycin toward Tetrahymena Telomeric G‑Quadruplex DNA D‑[GGGG(TTGGGG)3] Zia Tariq and Ritu Barthwal* Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, India
ACS Omega 2019.4:6347-6359. Downloaded from pubs.acs.org by 37.44.252.202 on 04/04/19. For personal use only.
S Supporting Information *
ABSTRACT: The anthracycline drug daunomycin exhibits complex mechanisms of anticancer action, which are not well understood. It plays a role in telomere dysfunction and binds to G-quadruplex DNA besides duplex DNA. Using the surface plasmon resonance technique, we demonstrate that it binds to a 22-mer D-[GGGG(TTGGGG)3] telomeric DNA sequence from Tetrahymena thermophilia in K+-rich aqueous solution. Changes in absorption/circular dichroism spectra and efficient quenching of fluorescence accompanied by a minor change in wavelength establish external binding of daunomycin with no scope of classical intercalation as observed on its binding to duplex DNA. Daunomycin−DNA complexes with stoichiometries of 1:1 and 2:1 coexist in solution. The daunomycin dimers in free solution are disrupted on binding. Proton nuclear magnetic resonance (NMR) spectra show significant shifts in aromatic protons of ring B/D, daunosamine sugar protons, and 14 short intermolecular contacts, exhibiting specificity of interaction. Large downfield shifts in phosphorus-31 NMR spectra, expected on account of classical intercalation, are absent. Molecular docking confirms external binding by the formation of a daunomycin− DNA complex with negative binding energy. Differential scanning calorimetry experiments show binding profiles with melting temperature Tm increasing with the daunomycin to DNA ratio and total thermal stabilization, ΔTm = 10 °C, which is expected to interfere with telomerase access to its functional site at telomeres, causing telomere dysfunction. The findings have implication in the design of analogues with different chemical modifications that could produce de novo anthracycline that acts as a potent telomerase functional inhibitor with enhanced selectivity toward G-quadruplex and hence result in reduced cell toxicity.
■
INTRODUCTION G-quadruplex DNA is distributed in human genome particularly in telomeric DNA, oncogenes (e.g., c-myc, c-kit, K-ras, bcl-2), gene promoters, and transcriptome and correlates with the gene expression level by interacting with an array of proteins.1,2 Tumor cells become immortalized through activation of the telomerase enzyme that stabilizes the length of telomeres. Telomerase levels have been found to correlate with cancer progression and metastatic state, and the enzyme is not expressed in the normal human tissue but is present in at least 85% of tumor cells. The RNA template of telomerase and capping function require an extended single-stranded DNA primer for effective hybridization, and folding of telomeric repeats into higher-order DNA structures (e.g., G-quadruplex) would hinder these processes. DNA quadruplex, stabilized in vivo by the K+ ion, was shown1−4 to inactivate telomerase, and its regulatory potential toward cancer cell growth has been substantiated. The general consensus is that G-quadruplex binders, which stabilize G-quadruplex structures,1−4 interfere with DNA damage response activation, oncogene expression, and genomic stability and hence possess potential to act as regulatory elements of different processes. Consequently, Gquadruplex binding agents can serve as a viable therapeutic strategy2,5,6 because of their selectivity as they would not show © 2019 American Chemical Society
cytotoxic effect outside tumor. This paved the way for the discovery of novel anticancer agents1−3 by focusing much research activity on design approaches based on molecular interactions of ligands specific with G-quadruplex DNA sequences. Subsequently, G-quadruplex-selective probes interacting with G-quadruplex DNA for detection of small molecules have also been developed for potential applications in clinical diagnosis of metabolic and genetic disorders.7−11 Gquadruplex-based assays have also been developed for detection of hazardous chemicals such as arsenic in natural water sources.12 It has also been proposed to monitor the realtime formation of G-quadruplex and switch in its conformational states using nanocube-based biosensors.13 Daunomycin (Figure 1), the first anticancer drug isolated from Streptomyces peucetius, is active against acute lymphoblastic or myeloblastic leukemias.14,15 Several mechanisms of action for daunomycin have been proposed such as duplex DNA intercalation, topoisomerase II poison, free-radical generation, DNA damage,14,15 cell viability, and dissociation of H1.1 linker histones from DNA, resulting in loss of higherReceived: February 5, 2019 Accepted: March 25, 2019 Published: April 4, 2019 6347
DOI: 10.1021/acsomega.9b00331 ACS Omega 2019, 4, 6347−6359
ACS Omega
Article
It has been shown that G-quadruplexes fold in different conformations depending upon cation, concentration, and flanking bases.1 The 22-mer human telomeric sequence (HTel22) adopts a mixture of two forms34 of 3 + 1 hybrid conformation in K+ solution with two lateral loops and one double chain reversal loop formed by the first linker (form 1)35 and third linker (form 2).36 On the other hand, HTel22 in Na+-rich solution adopts a well-defined antiparallel basket conformation having one diagonal and two lateral loops.37 Apparently, the binding mechanism for the two conformations is quite different.28,29 This is understandable as ligands recognize the loops, flanking bases, and intricate conformational aspects of the G-quadruplex. This has been further demonstrated by a recent study38 showing that mitoxantrone, an anticancer drug, shows different affinities for G-quadruplex structures formed by hteloG and hteloC; corresponding thermal stabilization of DNA due to binding is significantly different, being 23 and 45 °C. Because by increasing the melting temperature, these drugs hamper telomerase access to its functional site at telomeres,2 the extent of thermal stabilization of G-quadruplex structures is a crucial factor for the efficiency of the anticancer action. In addition, differences in the binding affinity of a drug toward different DNA quadruplex sequences may bring about selectivity toward a particular tumor/promoter of oncogenes. Recent modeling and spectroscopy studies on binding of daunomycin to parallel intermolecular G-quadruplex DNA, containing the sequence TTGGGG from the telomeric DNA of T. thermophilia, have suggested end stacking and groove binding as modes of interaction.39−41 There are no studies on intramolecular Gquadruplex DNA having four guanines and loops, for example, D-TTGGGG, the telomeric DNA sequence from T. thermophilia, which forms more stable structures. Henceforth, we have carried out a series of experiments using surface plasmon resonance (SPR), absorbance, fluorescence (steady state and lifetime), CD, and NMR spectroscopy to ascertain real-time binding and mode of interaction of daunomycin with Gquadruplex 22-mer D-[GGGG(TTGGGG)3] from T. thermophilia telomeric DNA in the presence of 100 mM K+. Thermal melting profiles have been obtained using differential scanning calorimetry (DSC) to determine the extent of stabilization of DNA.
Figure 1. Chemical structure of daunomycin.
order chromatin structures,16 and so forth. Biophysical studies using absorption, fluorescence, and circular dichroism (CD) have yielded affinity constant ∼106 to 107 M−1 on binding of daunomycin to salmon sperm, calf thymus DNA, and transfer RNA.17−21 Apart from binding to duplex DNA, daunomycin molecules also interact with G-quadruplex DNA.22 It was shown that daunomycin increases endogenous ceramide levels in lung adenocarcinoma cell line, which results in a decrease of telomerase and c-Myc transcription factor.23 Also, anthracyclines, doxorubicin and epirubicin, disrupt telomere maintenance through degradation of the PINX1 protein responsible for telomerase binding onto telomeres.24 Adriamycin-induced senescence in breast cancer cells is caused by an increase in activity of tumor suppressor protein p53 and a decrease in telomerase enzyme activity.25 It was also found that antitelomerase therapy may be useful in acute leukemia in combination with daunomycin.26 Adriamycin is also found to influence the functioning of immuno-modulatory genes.27 Recently, few studies have been conducted to deduce the mechanistic and biophysical insights of interaction of daunomycin with human G-quadruplex 21-/22-mer DNA. The interaction of doxorubicin and sabarubicin with 21-mer human telomeric sequence in K+- and Na+-rich solutions showed28,29 coexistence of 1:1 and 2:1 ligand/DNA complexes. In K+-rich solution, the decrease in absorbance accompanied by a red shift (∼25 nm) and dramatic quenching of fluorescence were observed. The CD spectra of the selfassociated dimeric form of 50 μM daunomycin changed to a positive band at 500 nm on binding, which led to an increase in melting temperature of DNA by 5 °C.28 The interaction in Na+-rich solution,17 on the other hand, did not show any stabilization of DNA, although fluorescence emission and CD bands were affected significantly.29 Using multiexponential decay for data fitting in femtosecond fluorescence spectroscopy experiments,30 it was shown that fast and slow transfer of electron is dependent on the orientation and distance of doxorubicin molecules from DNA. In another study by spectroscopy and molecular docking,31 it was shown that one ligand molecule stacks on to the top of G tetrad of 22-mer human telomeric DNA sequence by removal of water from Gquadruplex DNA through favorable enthalpy as well as entropy process. Yet, in another study by one-dimensional (1D) proton nuclear magnetic resonance (1H NMR), it was shown that daunomycin stacks on top of G-quadruplex fold of the purinerich sequence Pu24I from the MYC promoter region.32 Recent studies on interaction of analogues of daunomycin, for example, doxorubicin and nemorubicin, with several Gquadruplex sequences and c-MYC promoter element Pu22 through NMR spectroscopy indicate formation of a drug− DNA complex with stoichiometries of 1:1 and 2:1.33
■
MATERIALS AND METHODS All chemicals, that is, desalted oligonucleotide sequence D[GGGG(TTGGGG) 3 ], D -[AGGG(TTAGGG) 3 ], D [TTAGGGT], 5′ biotin-labeled desalted oligonucleotide sequence D -[GGGG(TTGGGG) 3 ], daunomycin, KCl, K2HPO4, and ethylenediaminetetraacetic acid (EDTA), were purchased from Sigma Aldrich Co., U.S.A., Ltd. DNA/ daunomycin solutions were prepared in buffer containing 10 mM K2HPO4, 1 mM EDTA, and 100 mM KCl (pH 7.0) using their respective molar absorption coefficient values by following standard procedures.40,41 For CD measurements, samples were also prepared in buffer containing 0, 25, 150, and 200 mM KCl. SPR experiments were carried out on a Biacore T200 instrument optical biosensor system (make GE Healthcare, Chicago, USA) with quadruplex DNA D -[GGGG(TTGGGG)3] immobilized on a streptavidin-derivatized sensor chip (BIACORE SA, make GE Healthcare Life Sciences, UK) as described earlier.41 Absorption studies were carried out using a UV−visible spectrophotometer (model 6348
DOI: 10.1021/acsomega.9b00331 ACS Omega 2019, 4, 6347−6359
ACS Omega
Article
Figure 2. SPR results for interaction of daunomycin with D-[GGGG(TTGGGG)3] using HEPES buffer (pH 7.4) with 100 mM KCl at 25 °C; (A) sensograms obtained for the increasing concentration of daunomycin from 3.75 μM (bottom) to 960 μM (top) and (B) steady-state binding plot showing response unit (R.U.) with respect to the concentration of daunomycin (μM).
propionic acid /85% phosphoric acid as the reference using a 500 MHz Bruker Avance NMR spectrometer equipped with TXI (triple inverse) probe), BBO (broad band observed) probe, and BVT (variable temperature unit) at NMR Facility, IIT Roorkee, India. The pulse scheme and parameters in NMR measurements were set as described earlier.41 Thermal transitions of free 50 μM D-[GGGG(TTGGGG)3] and its complexes with daunomycin were obtained using a differential scanning calorimeter (model VP-DSC, make MicroCal, Northampton, MA). The thermograms were then deconvoluted and fitted in “two-state” models using Origin 7.1 software.41 Molecular docking experiments for daunomycin (PubChem CID: 30323) binding with 24-mer G-quadruplex DNA D-[(TTGGGG)4], PDB ID: 186D, were performed using Autodock 4.2 software package and the docked poses were analyzed in PyMOL software.40
CARY-100 Bio, make Varian, USA) equipped with a Peltiercontrolled thermostatic cell holder at 200−800 nm, and the equilibrium binding constant (Kb) was calculated using standard equations.31,40 Excitation of daunomycin at 480 nm results in intense emission at 558.5 and 592 nm.28,31,40,42,43 Fluorescence spectra were recorded at λem = 520−650 nm at 25 °C for the same samples as that used in absorbance using a Fluorolog-3 spectrofluorimeter (model LS55, make Horiba Jobin Yvon Spex). It may be noted that DNA has relatively poor fluorescence quantum yield, for example, the guanine residue has a quantum yield of 3 × 10−4 for λex = 273 nm and λem = 340 nm.44 Thus, the free G-quadruplex D-[GGGG(TTGGGG)3] and that in bound form do not contribute to fluorescence in the experiments conducted in the emission range λem = 520−650 nm, and the observed fluorescence originates solely from free/bound daunomycin. The fluorescence quenching constant (Ksv), binding constant (Kb), and stoichiometry of the complex (n) were calculated using standard equations.31,40 Continuous variation analysis procedure, that is, Job plot, was used to establish the binding stoichiometry of daunomycin with D-[GGGG(TTGGGG)3] by measuring the fluorescence intensity at λem = 592 nm and λex = 480 nm at 25 °C for different daunomycin−DNA complexes.41 The fluorescence lifetime values were obtained for 7 μM free daunomycin and its complex with D[GGGG(TTGGGG)3] at D/N = 0.3, 0.5, 1.0, 2.0, 3.0, and 4.0 using a Fluoro-Cube system (model Fluoro-Cube, make Horiba Jobin Yvon Spex) operating in time-correlated single photon counting mode equipped with Nano LED (λex = 456 nm, λem = 590 nm).40 The CD spectra were recorded at 200− 600 nm using a spectropolarimeter (model Chirascan, make Applied Photophysics, UK) equipped with Quantum North West TC 125 Peltier unit.40 During titrations, the concentration of D-[GGGG(TTGGGG)3] was kept constant as 10.0 μM, and daunomycin was added progressively at mole equivalent ratios D/N = 0.1−5.0 in 31 experiments. In another set of experiments, daunomycin concentration was kept constant at 400 μM and D-[GGGG(TTGGGG)3] was added stepwise in 11 experiments conducted at D/N = 0.5−4.9. The binding constants (Kb1 and Kb2) for 2:1 stoichiometry of daunomycin to DNA were obtained by giving a user-defined equation written in Origin 2018.40,41,45,46 For NMR measurements, 2.30 mM G-quadruplex DNA D[GGGG(TTGGGG)3] solution in 90% H2O + 10% D2O or 100% D2O solvent was prepared in 10 mM KBPES buffer containing 10 mM K2HPO4, 1 mM EDTA, and 100 mM KCl (pH 7.0).41 1H, 1H−1H NOESY, and phosphorus-31 (31P) NMR spectra were recorded with respect to trimethyl silyl
■
RESULTS AND DISCUSSION Surface Plasmon Resonance. The SPR sensograms (Figure 2A) show that the steady-state response increases with concentration in the range 3.75−960 μM (Figure 2B), confirming thereby that a specific interaction of daunomycin with [D-[GGGG(TTGGGG)3] indeed does take place. The binding isotherms yield affinity constant Kb ≈ 2.1 × 103 M−1 at 25 °C, while the analysis of kinetics of association and dissociation yields Kb ≈ 6.9 × 103 M−1 (Table S1A), giving a direct proof of interaction between two molecules. Absorption. The titrations were monitored at ∼478 nm because daunomycin shows absorption maxima at 290 and 478 nm, whereas DNA absorbs at 260 nm. A 7 μM concentration of daunomycin used is not expected to show effects because of self-association.40−43,47,48 Stepwise addition of D-[GGGG(TTGGGG)3] resulted in hypochromism of ∼37% up to D/ N = 0.18 at 478 nm accompanied by the red shift of Δλmax ≈ 7 nm (selected data shown in Figure 3). The data do not show isosbestic point in the entire range of D/N ratios so that multiple stoichiometric daunomycin−DNA complexes may exist in solution. The plots of absorbance (A) [inset of Figure 3 and 1/A (Figure S1A)] versus D/N show inflection points at D/N ∼0.8 and 2.0 because of a change in slope so that the stoichiometry of the daunomycin−DNA complex is likely to be 1:1 or 2:1.49−52 The Scatchard plots being nonlinear and not fitting into combination of straight lines cannot be used to estimate affinity constant Kb with confidence.52−54 The intrinsic binding constants Kb = 5.6 × 104 and Kb = 6.1 × 105 M−1 at D/N = 0.18−0.33 and 0.33−4.6, respectively,40 may be considered as a rough estimate (Figure S1B) and an average of several complexes. The observed change in 6349
DOI: 10.1021/acsomega.9b00331 ACS Omega 2019, 4, 6347−6359
ACS Omega
Article
(86−93%) may be attributed to electron transfer by the proximity of daunomycin to guanines. Similar results have been reported on binding of doxorubicin/daunomycin to the 21-/22-mer human telomeric DNA sequence.28,29,31 The fluorescent decay of daunomycin was monoexponential with a lifetime of τ = 1.03 ns (Figure S2D). The decay for several complexes at D/N = 0.3, 0.5, 1.0−4.0 remained monoexponential with a lifetime of τ = 0.97−1.00 ns (Figure S2D), which may be due to equal lifetime for free and bound daunomycin or alternately for both complexes that are nonemissive.28 The plot of difference in fluorescence intensity as a function of mole fraction of daunomycin shows inflection points at ∼0.48 and 0.67 (Figure 4B), yielding stoichiometries of 1:1 and 2:1 of the daunomycin-D-[GGGG(TTGGGG)3] complex,40,57 which is consistent with titration studies by absorbance and fluorescence. A scatter in data at higher values of mole fraction of daunomycin is observed. Circular Dichroism. The CD spectra of D-[GGGG(TTGGGG)3] in the presence of 100 mM K+ show (Figure 5A) a well-defined positive band at 265 nm, indicating stacking arrangements of tetrads that are characteristic of a parallelstranded G-quadruplex DNA having stacking of bases with the same glycosidic bond angle (i.e., either anti or syn) and a positive band at 290 nm that is characteristic of antiparallelstranded G-quadruplex DNA showing stacking of guanosines with different glycosidic bond angles (i.e., anti and syn).61−65 Parallel and antiparallel forms of G-quadruplex show a negative and positive CD band centered around 238−240 and 240−245 nm, respectively.61−67 Thus, the observed negative band at 240 nm and the dominant positive band at 265 nm (Figure 5A) show that the sequence D-[GGGG(TTGGGG)3] forms a mixture of 3 + 1 hybrid as well as parallel quadruplexes in the presence of 100 mM K+ and the population of latter arrangement prevails.61 The formation of G-quadruplex is influenced by the K+ concentration,61,62,68 and we therefore recorded CD spectra in water and buffer containing 0 (no additional salt), 25, 150, and 200 mM K+ (Figure 5A). It is found that in the absence of any additional K+ ions, there are two positive CD bands of comparatively same intensities at 265 and 290 nm, that is, identical to the CD spectra61 of the 3 + 1 hybrid structure of the 24-mer G-quadruplex DNA sequence, [D-(T2G4)4] determined by NMR in Na+-rich solution.69 On adding 25 mM K+, the 265 nm peak intensity is found to be more intense
Figure 3. Absorption spectra of 7 μM free daunomycin and its complex with increasing concentration of D-[GGGG(TTGGGG)3] in 10 mM phosphate buffer containing 100 mM KCl at some selected daunomycin (D) to nucleic acid (N) ratios, D/N, at 25 °C. The inset shows plot of absorbance (A) as a function of D/N ratio at 478 nm at 25 °C.
absorbance accompanied by a minor red shift indicates that classical intercalation is not a preferred mode of binding.28,29,54−58 The daunomycin molecule may however interact with DNA on binding externally at ends of DNA or along grooves in the backbone of DNA. The alteration in absorbance may be due to interaction of π-electron cloud of daunomycin and G-quadruplex DNA. Similar results have earlier been reported on binding of doxorubicin/daunomycin with 21-/22mer human telomeric DNA quadruplex in K+- and Na+-rich aqueous solutions28,29,31 Fluorescence. Addition of D-[GGGG(TTGGGG)3] to daunomycin results in quenching of fluorescence intensity up to 99% at D/N = 0.18 with a red shift Δλem = 0.5−1 nm in both the emission maxima (selected data shown in Figure 4A), indicating strong interaction. A plot of F (inset of Figure 4A) and 1/F (Figure S2A) versus D/N shows inflection at D/N ≈ 1.0 and 2.0.40,54,57,59,60 The Stern−Volmer plot showing F/F0 versus [DNA] (Figure S2B) and a plot of log[F−F0]/F versus log [DNA] (Figure S2C), respectively, give40,57 KSV and Kb in the range 1.5−4.9 × 105 M−1. The bimolecular quenching constant, Kq ≈ 4 × 1014 M−1 s−1 using Ksv = 4.0 × 105 M−1 and lifetime τ = 1.0 ns, is much greater than the collision constant of biomolecules and small molecules.40,57 Presence of static quenching owing to ground-state interactions is also evident from the accompanied changes in absorbance and no change in fluorescence lifetime (discussed later). Efficient quenching
Figure 4. (A) Fluorescence emission intensity of 7 μM free daunomycin and its complex with increasing concentration of D-[GGGG(TTGGGG)3] in 10 mM phosphate buffer containing 100 mM KCl at some selected daunomycin (D) to nucleic acid (N) ratios, D/N, using λex = 480 nm at 25 °C. The inset shows the plot of fluorescence intensity (F) as a function of D/N ratio at 592 nm at 25 °C and (B) Job plot for binding of daunomycin to D-[GGGG(TTGGGG)3] using fluorescence. The total concentration of daunomycin and D-[GGGG(TTGGGG)3] was kept fixed as 4 μM in 10 mM phosphate buffer (KBPES) containing 100 mM KCl. 6350
DOI: 10.1021/acsomega.9b00331 ACS Omega 2019, 4, 6347−6359
ACS Omega
Article
Figure 5. (A) CD spectra of free 10 μM D-[GGGG(TTGGGG)3] in water and KBPES buffer containing 0, 25, 100, 150, and 200 mM KCl; (B) CD spectra of free 10 μM D-[GGGG(TTGGGG)3] and its complex with increasing concentration of daunomycin at some selected daunomycin (D) to nucleic acid (N) ratios, D/N, in 10 mM phosphate buffer with 100 mM KCl at 25 °C. The inset shows the plot of CD (mdeg) at 265 nm as a function of D/N ratio at 25 °C; CD spectra of titration of daunomycin into 10 μM solution of D-[GGGG(TTGGGG)3] in KBPES buffer containing (C) 0 mM KCl; (D) 25 mM KCl; (E) 200 mM KCl at 25 °C; and (F) CD spectra of 400 μM daunomycin in free form and its complex with the increasing concentration of D-[GGGG(TTGGGG)3] at some selected D/N ratios in 10 mM phosphate buffer with 100 mM KCl at 25 °C. The inset shows the plot of CD (mdeg) at 458 nm as a function of D/N ratio.
The plot of CD signal at 265 nm (inset of Figure 5A) shows inflection at D/N = 1.0 and 2.0, suggesting stoichiometries of the complex as ∼1:1 and 2:1,71 and the absence of an isoelliptic point confirms the presence of multiple stoichiometries of complexes. Nonlinear curve fitting of the plot of magnitude of CD at 265 and 243 nm as a function of daunomycin concentration (Figure S3A,B) using standard equations40,45,46 yields different affinity constants, Kb = 3.9 × 106 and 4.0 × 105 M−1, respectively, which may be due to the presence of several conformations of the complex, which contribute differently at different wavelengths. We also carried out titrations of daunomycin with G-quadruplex DNA comprising 0, 25, and 200 mM K+ (Figure 5C−E) and found that the affinity constant at 0−25 mM K+ is ∼1.4 × 106 M−1 as compared to the corresponding value of 4.0 × 106 M−1 at 100−200 mM K+ (Figure S3 C−H and Table S1B). Apparently, the binding to 3 + 1 hybrid structure (at 0 mM K+) has weaker affinity than the parallel G-quadruplex structure stabilized by high concentrations (100−200 mM) of K+. In order to examine the existence of induced CD bands, DNA was added to 400 μM daunomycin (Figure 5F) in steps. The bisignate CD band characteristic of the self-associated dimeric form of daunomycin, having positive and negative
than the corresponding 295 nm peak, which indicates the presence of a parallel quadruplex.61 Further stepwise increase in K+ concentration from 25 to 200 mM shows that the positive 260 nm CD band dominates, and the spectra are characteristic of a mixture of 3 + 1 hybrid and parallel quadruplexes and that a significant population of latter arrangement prevails.61 Thus, K+ promotes the formation of parallel quadruplex, as has also been observed earlier for other G-quadruplex DNA sequences.61,62,68 Further, the relative population of two structures remains unchanged at concentration >150 mM K+ reaching an equilibrium with respect to relative populations of two conformations. Upon addition of daunomycin to 10 μM D-[GGGG(TTGGGG)3] up to D/N = 5.0 in the presence of 100 mM K+, the magnitude of 290, 265, and 240 nm bands decreases significantly by 60, 63, and 67%, respectively, which perhaps reflects change in relative population of two G-quadruplex conformations. The binding is accompanied by a minor red shift ∼1−3 nm (Figure 5B), which rules out classical intercalation and is indicative of external binding.70 Groove binding has been shown to induce changes in magnitude of 265 and 243 nm bands, whereas end stacking does not show any significant change.70 Accordingly, both end stacking and external groove binding may occur in present investigations. 6351
DOI: 10.1021/acsomega.9b00331 ACS Omega 2019, 4, 6347−6359
ACS Omega
Article
peaks at 460 and 540 nm, respectively, and crossover point at 500 nm, observed at high concentrations of daunomycin,72 disappeared on interaction with DNA. This demonstrates that daunomycin binds as monomer. A small positive band at ∼460−480 nm was observed (Figure 5F), which increased slightly in magnitude with the D/N ratio (inset of Figure 5B). The presence of a small positive induced CD band has earlier been attributed to binding of ligands at grooves or end stacking with bases of DNA.28,29,49,70,71,73−76 On binding of adriamycin to human telomeric DNA 22-mer sequence in K+-rich solution, end stacking was inferred28 on the basis of the observed red shift of 25−30 nm in both the absorption maxima and 350 nm CD band of adriamycin. We did not observe significant shift in wavelength maxima of absorption (∼7 nm) and emission (
