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Interaction of a Cationic Porphyrin and Its Metal Derivatives with G‑Quadruplex DNA Eric Boschi,† Supriya Davis,‡ Scott Taylor,‡ Andrew Butterworth,† Lilyan A. Chirayath,† Vaishali Purohit,† Laura K. Siegel,† Janesha Buenaventura,† Alexandra H. Sheriff,† Rowen Jin,‡ Richard Sheardy,§ Liliya A. Yatsunyk,*,‡,# and Mahrukh Azam*,†,# †

Department of Chemistry, West Chester University of Pennsylvania, West Chester, Pennsylvania 19383, United States Department of Chemistry and Biochemistry, Swarthmore College, 500 College Avenue, Swarthmore, Pennsylvania 19081, United States § Department of Chemistry & Biochemistry, Texas Woman’s University, 324 Ann Stuart Science Center, P.O. Box 425859, Denton, Texas 76204-5859, United States ‡

S Supporting Information *

ABSTRACT: G-quadruplex (GQ) structures formed from guanine-rich sequences are found throughout the genome and are overrepresented in the promoter regions of some oncogenes, at the telomeric ends of eukaryotic chromosomes, and at the 5′-untranslated regions of mRNA. Interaction of small molecule ligands with GQ DNA is an area of great research interest to develop novel anticancer therapeutics and GQ sensors. In this paper we examine the interactions of TMPyP4, its isomer TMPyP2 (containing N-methyl-2-pyridyl substituents, N-Me-2Py) as well as two metal derivatives ZnTMPyP4 and CuTMPyP4 with GQs formed by dT4G4 and dT4G4T in 100 mM K+ or Na+ conditions. The DNA sequences were chosen to elucidate the effect of the 3′-T on the stabilization effect of porphyrins, binding modes, affinities, and stoichiometries determined via circular dichroism melting studies, UV−vis titrations, continuous variation analysis, and fluorescence studies. Our findings demonstrate that the stabilizing abilities of porphyrins are stronger toward (dT4G4)4 as compared to (dT4G4T)4 (ΔTm is 4.4 vs −6.4 for TMPyP4; 12.7 vs 5.7 for TMPyP2; 16.4 vs 12.1 for ZnTMPyP4; and 1.9 vs −8.4 °C for CuTMPyP4) suggesting that the 3′G-tetrad presents at least one of the binding sites. The binding affinity was determined to be moderate (Ka ∼ 106−107 μM−1) with a typical binding stoichiometry of 1:1 or 2:1 porphyrin-to-GQ. In all studies, ZnTMPyP4 emerged as a ligand superior to TMPyP4. Overall, our work contributes to clearer understanding of interactions between porphyrins and GQ DNA.

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mRNA.4−10 Recent biological studies suggest that GQs might have important biological functions, such as chromatin organization, transcription, translation, replication, telomere regulation, genome stability, and DNA repair.1,4,11−16 Many of these functions are directly related to cancer; thus quadruplexes have emerged as a novel therapeutic target for the development of anticancer drugs.11−13 Design and use of novel smallmolecule ligands that can bind to and stabilize, and/or facilitate the formation or interconversion of various types of GQs has become an area of high interest.2,17−28 Small molecules that stabilize GQ DNA generally have a large central aromatic moiety, allowing binding via hydrophobic and π−π interactions. Variations in binding are generally provided by the variations in the size of the aromatic moiety and by the side chains, which are commonly cationic Ncontaining bases (N-melthylpyridine, pyrrolidine, piperidine,

INTRODUCTION G-quadruplex (GQ) structures are formed from guanine-rich DNA sequences as a result of the self-assembly of guanine (G) bases via Hoogsteen hydrogen bonding into G-quartets and subsequent stacking of the G-quartets on top of each other via π−π interactions. Partial negative charges of G’s carbonyl O6 concentrated in the center of the G-quartet are neutralized by monovalent cations, usually K+ or Na+, Figure 1. GQs demonstrate significant diversity in arrangement with regard to folding pattern (parallel, antiparallel, hybrid), repeat number, and inter- or intramolecular bonding (monomolecular, bimolecular, tetramolecular,1,2 and uncommon but possible trimolecular3). The DNA sequence, composition, concentration, guanine base content, environmental conditions, and additives (ligands, PEG, alcohol) play key roles in determining specific types of GQ topology. The DNA sequences with GQ-forming potential are found throughout the genome and are overrepresented in the promoter regions of oncogenes, at the telomeric ends of eukaryotic chromosomes, and at the 5′-untranslated regions of © 2016 American Chemical Society

Received: September 29, 2016 Revised: November 10, 2016 Published: November 10, 2016 12807

DOI: 10.1021/acs.jpcb.6b09827 J. Phys. Chem. B 2016, 120, 12807−12819

Article

The Journal of Physical Chemistry B

Figure 1. G-tetrad composed of four guanines bonded via Hoogsteen hydrogen bonding network constituting the simplest building block of Gquadruplex (top left and space filling model, top middle). The space filling model also contains a central metal ion (K+ or Na+) needed to compensate negative charges on the carbonyl groups. Energy minimized structure of (dT4G4)4 (top right). (Bottom) Starting from the left, structures of TMPyP4, CuTMPyP4, ZnTMPyP4, and TMPyP2 (Note, ZnTMPyP4 contains axial water molecule).

morpholine, guanidine, 1-methylpiperazine, and N,N-diethylethylenediamine, and many others). Quadruplex binders are currently designated into 23 main classes, one of which is porphyrins.2 The quadruplex sequence and topology, salt concentration and type, buffer conditions and additives play important roles in dictating the specific modes of ligand binding to GQ DNA.1,29−31 One of the most utilized ligands in the quadruplex field is 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4, Figure 1). TMPyP4 is an excellent but modestly selective GQ stabilizer16,20,32−35,30,36−38 capable of inducing the formation, and facilitating the interconversion of different forms of Gquadruplexes in vitro.39−42 In biological experiments, TMPyP4 and its derivatives have been shown to target GQs and to display antitumor activity35,43 by inhibiting telomerase44−47 and by down-regulating the expression of the MYC32 and KRAS48 oncogenes.49,50 Though it is well established that TMPyP4 interacts with GQ DNA, the mode of binding is still a question of controversy. X-ray51 and NMR17 structures of TMPyP4 bound to parallel GQs indicate loop binding and end-stacking, respectively. Spectroscopic and calorimetric experiments as well as calculation results suggest a variety of binding modes, such as intercalation9,38,50,52−54 between adjacent G-tetrads, or at a G− A interface,37 sandwich type (vide infra), end-stacking37,38,50 groove binding,37,38 and backbone binding.55 Understanding how porphyrins bind to GQ DNA will aid in the design of highly efficient future anticancer therapeutics. A large body of recent work suggests that end-stacking is the most common binding mode in ligand−GQ complexes. To test the validity of this suggestion we designed experiments around two DNA sequences, dT4G4T and dT4G4T, both of which form tetramolecular parallel GQ structures in both Na+ and K+ buffers.56−60 The GQ formed by dT4G4 has one open G-tetrad,

while in dT4G4T this tetrad is blocked by 3′T nucleotides. dT4G4 has been shown to form a tetramer structure in Na+ solution, with higher order structures formed in K+ containing solutions.57 In the T-tract, T nucleotides directly adjacent to Gquartets interact strongly with guanines, stabilizing the overall GQ, while the rest of T tract does not form any detectable bonding network and is unstructured causing destabilization of the DNA.56,61 Previous studies suggest that TMPyP4 interacts with GQ formed by dT4G4 by both intercalation into the GQ core and end-stacking at the 3′G-quartet face.54,62 The latter mode of binding, if indeed present, will be weakened or abolished when dT4G4T is used instead. In this work we studied the interactions of TMPyP4 and three of its derivatives, ZnTMPyP4, CuTMPyP4, and TMPyP2, with GQs formed by dT4G4 and dT4G4T. CuTMPyP4 forms a square planar complex and is very similar in geometry and size to metal-free TMPyP4. Unlike TMPyP4, it binds only weakly to parallel29,63 and antiparallel quadruplexes. ZnTMPyP4 with one axial water ligand adopts square pyramidal geometry and binds to GQ by outside binding/end-stacking mode.42,55 In another study, ZnTMpyP4 was shown to induce and stabilize GQ formation from the d(TTAGGG) sequence in K+.64 Finally, TMPyP2, is a positional isomer of TMPyP4 where Nmethyl groups occupy the ortho-position on the pyridyl relative to its connection to the porphyrin core. Steric crowding results in nonflat geometry and therefore the inability of TMPyP2 to intercalate into the DNA. Due to high barrier to rotation of its N-MePy groups, TMPyP2 exists as a mixture of eight rotamers, one of which, with four N-methyl groups pointing in the same direction, should be capable of end-stacking. In spite of the difference in geometry, both TMPyP4 and TMPyP2 displayed similar binding affinities and stabilization (ΔT ∼ 9 °C) of human telomeric DNA in Na+ and K+, yet they have 12808

DOI: 10.1021/acs.jpcb.6b09827 J. Phys. Chem. B 2016, 120, 12807−12819

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The Journal of Physical Chemistry B

DNA and the specific porphyrin (ultimately on the binding ratio) the titrations reached completion at 2−10 GQ/P ratios. Titration data were used to obtain bathochromic shift, Δλ, and hypochromicity, %H. Δλ is the difference in the position of Soret maximum between free and bound porphyrin, and %H is calculated according to the following formula: ε − εb H= f × 100% εf

contrasting inhibitory activities toward telomerase (TMPyP4 is 10-fold more potent) as well as preference in binding sites (external stacking for TMPyP4 and binding outside a TTA loop for TMPyP2).47 Therefore, the mode of binding, most likely, defines the biological activity of a GQ ligand. The binding modes, binding affinities, and stoichiometries of porphyrin binding to GQ DNA were determined from UV−vis titrations and continuous variation analysis, as well as fluorescence studies. The ability of porphyrins to stabilize GQ was determined based on circular dichroism melting studies. Our findings suggest that indeed the 3′ end of GQ presents at least one of the binding sites where porphyrin is either end-stacked onto an individual GQ structure or being sandwiched between two quadruplexes.

where εf and εb represent the extinction coefficients of free and bound porphyrins at their respective Soret maxima. In addition, titration data were used to construct Scatchard plots (data not reported) of r/Cf versus r, which allows determination of binding parameters according to the following equation:

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MATERIALS AND METHODS Materials. 5,10,15,20-Tetrakis(N-methyl-4-pyridyl)porphyrin, TMPyP4, was purchased as tosylate salt and 5,10,15,20-tetrakis(N-methyl-2-pyridyl)porphyrin, TMPyP2, was purchased as chloride salt from Frontier Scientific (Logan, UT). Both porphyrins were used without further purification. ZnTMPyP4 and CuTMPyP4 were synthesized according to previously published literature procedures65 using a 20-fold molar excess of CuCl2 × 2H2O and ZnCl2, respectively. The purity of prepared ZnTMPyP4 and CuTMPyP4 was confirmed by 1H NMR spectroscopy. Porphyrin stock solutions were prepared in water at 10−20 mM concentration and stored in the dark. The porphyrin concentration was determined on the basis of the following extinction coefficients: ε424 = 2.26 × 105 for TMPyP4, ε414 = 1.82 × 105 M−1 cm−1 for TMPyP2, ε437 = 2.04 × 105 M−1 cm−1 for ZnTMPyP4, and ε424 = 2.31 × 105 M−1 cm−1 for CuTMPyP4.65 All porphyrin solutions were protected from direct sunlight or other light exposure. Oligonucleotides were purchased from either Midland Certified Reagent Co. or Integrated DNA Technologies, dissolved in water to ∼0.6 mM concentration and stored at −80 °C. Samples were diluted with either KPi buffer (10 mM KPi, pH 7.0, 100 mM KCl) or NaPi buffer (10 mM NaPi, pH 7.0, 100 mM NaCl), annealed at 92 °C for 10 min, slowly cooled to room temperature, and stored at 4 °C for over 48 h before use. Note, a long equilibration time is required for complete folding of each dT4G4 and dT4G4T into tetrastranded quadruplex structure due to tetramolecularity of the folding process. DNA concentration was determined using extinction coefficients of 73.6 and 82.1 mM−1 cm−1 for dT4G4 and dT4G4T, respectively.66 The concentration of GQ DNA is 4 times that of the corresponding oligonucleotide. UV−Vis Titration Experiments. Absorption spectra in the 350−700 nm range were recorded at 25.0 ± 0.1 °C on a Cary300 spectrophotometer (Varian) fitted with a constant temperature accessory unit. All titration experiments were performed in either KPi or NaPi buffer in 1 cm methacrylate cuvettes to minimize porphyrin adsorption to the cuvette walls. A solution of fixed concentration of porphyrin (1−5 μM) was titrated by stepwise addition of stock DNA solution (50−350 μM per quadruplex) to the final GQ/P ratio of 2−10. The concentration of porphyrin was kept constant throughout the titration by including porphyrin in a DNA stock solution. After each addition, the resulting solution was incubated for 2 min and UV−vis absorbance was recorded. A titration was considered complete when the spectra collected after three additions of DNA were superimposable. Depending on the

r /Cf = K (n − r )

where K is the equilibrium binding constant, n represents the number of ligand molecules bound per GQ DNA, and r is a binding ratio that equals to Cb/[DNA]. The fraction of bound porphyrin, α at each intermediate titration position was calculated according to the formula: α=

A free − A A free − Abound

where Afree and Abound are the absorbances of free and fully bound porphyrins at λmax of free porphyrin. The concentration of bound porphyrin was calculated from the total porphyrin concentration, Ct as Ct × α, and the concentration of free porphyrin, Cf, was calculated as Cf = Ct − Cb. Additionally, the titration data were analyzed using singular value decomposition added (SVD) analysis67 and the Direct Fit method in a manner described in our earlier work.64 SVD analysis suggested that in most cases the binding is a simple two-state process. Data at two wavelengths, maxima for free and bound porphyrin, were fit in most cases. For complexes with low values of the red shift parameter, the absorbance difference between maxima for free and bound porphyrins were fit using the Direct Fit method, instead. We tried a variety of binding models where the complex stoichiometry was varied. Each binding model, however, assumed equal and independent binding sites. Binding model with the lowest stoichiometry and the best value of fitting parameters was deemed the most probable. All data manipulations were performed in Origin 8.1 and GraphPad Prism 4.0. Continuous Variation Analysis (Job Plot). The Job plot method provides model-independent values of binding stoichiometries68 that were used to evaluate and to compare to the data obtained from the Direct Fit (see above). Porphyrin and DNA solutions of equal concentration were prepared in KPi or NaPi buffers. Two sets of titrations were completed. First, 800 μL of 4 μM porphyrin were placed in two 1 cm cuvettes, one to be used as a reference. Aliquots (25−100 μL) of 4 μM GQ solution in the same buffer were added to the sample cell, and an equal volume of buffer was added to the reference cell. It is essential to keep the concentration of porphyrin and GQ DNA equal in this experiment. Each sample was mixed thoroughly and incubated for 2−5 min to allow complete equilibration. In the second set of titrations, 800 μL of 4 μM GQ solution was placed in a sample compartment and 800 μL of buffer was placed in a reference cell. Both cells were titrated with 4 μM porphyrin solution. Difference spectra were collected in the 350−700 nm range at 25 °C. Job plots were 12809

DOI: 10.1021/acs.jpcb.6b09827 J. Phys. Chem. B 2016, 120, 12807−12819

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The Journal of Physical Chemistry B

min−1. Using a different temperature change rate will inevitably lead to a different value of T1/2. Although T1/2 is not a thermodynamic parameter, it is very useful for comparison between complexes in this study for which the data were collected under identical conditions.

constructed by plotting the difference in the absorbance values at various wavelengths versus mole fraction of porphyrin, X=

no. of moles of porphyrin no. of moles of porphyrin + no. of moles of GQ

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The values of the mole fraction at maxima or minima were used to obtain the stoichiometry of porphyrin binding to GQ DNA. Fluorescence Emission Spectroscopy. Fluorescence emission spectra were measured at room temperature on a Cary Eclipse 1.1 Fluorescence spectrometer using a 1 cm path-length quartz cuvette. Fluorescence spectra were acquired in either KPi or NaPi buffers for 2.5 μM porphyrin alone or in the presence of 50 μM (dT4G4T)4 and (dT4G4)4 (per strand, 5-fold excess GQ vs porphyrin). Four scans were collected using an integration time of 0.5 s, an excitation slit width of 10 nm, and an emission slit width of 5 nm. The emission spectra were collected from 600 to 900 nm. Excitation wavelengths were 433 nm for TMPyP4, 433 nm for CuTMPyP4, 419 nm for TMPyP2, and 437 nm for ZnTMPyP4. Thermal Denaturation Studies Monitored by Circular Dichroism (CD). For thermal denaturation studies the stocks of annealed oligonucleotides were diluted to ∼5.5 μM (per GQ) with 10 mM NaPi, pH 7.0, 50 mM NaCl buffer. Note the concentration of NaCl was lowered from 100 mM in our titration studies to 50 mM to decrease DNA thermal stability into the range observable by CD. Porphyrins were added to ∼10 μM final concentration ([GQ]:[P] ≈ 1:2). Solutions were equilibrated at 3−5 °C for at least 1 h before melting (or in some cases overnight) and CD wavelength scans between 220 and 500 nm were collected in a 1 cm cell on Aviv 62DS and on Aviv 410 CD spectrometer equipped with a Peltier heating unit. This was followed by the melting of the GQ-porphyrin complexes monitored at 265 nm. The accuracy of the external temperature probe was ±0.2 °C, the heating rate was set at 0.5 °C min−1, the equilibration time was 1 min (resulting in the overall temperature increase rate of 0.33 °C min−1), the temperature range was from 3 to 95 °C and then back from 95 to 3 °C, the response averaging time was 8 s, and the bandwidth was 10 nm. Melting and cooling curves were not superimposable. CD wavelength scans in the UV region were collected before and after melting, where “after melting” data were usually collected on the sample incubated for 24 h at 4 °C after melting was completed. The analysis of the melting data was conducted in the following way. Two baselines that correspond to folded and unfolded conformations were determined using linear fits. Then the data were normalized using the following forY −Y mula:FU = Y F− Y , where FU is fraction of unfolded GQ DNA F

RESULTS DNA oligonucleotides dT4G4 and dT4G4T form stable tetramolecular parallel G-quadruplex structures in KPi and NaPi buffers with the maximum and minimum on their CD spectra at 264 and 242 nm, respectively, according to our data (Supporting Information Figure S1) and previous work.57 Here we set out to perform thorough characterization of binding between four porphyrins, TMPyP4, TMPyP2, CuTMPyP4, and ZnTMPyP4, and two parallel GQ DNA, (dT4G4)4 and (dT4G4T)4. Our goal is to clarify the binding modes of porphyrins to GQ DNA, chemical features of ligands (e.g., presence of a central metal, position of N-methyl group on pyridyl) and DNA (unobstructed 3′G-tetrad vs the presence of thymine at the 3′ face), and the nature of the monovalent ion (K+ vs Na+) that affect binding. Also, we wanted to determine if any of the spectroscopic signatures (red shift, hypochromicity, and fluorescence enhancement) can be correlated with the binding mode. Porphyrin Binding to GQ DNA via UV−Vis Titrations. Representative absorption spectra for titration of TMPyP4, TMPyP2, and Cu- and ZnTMPyP4 with (dT4G4T)4 in KPi buffer are shown in Figure 2 and in NaPi buffer are provided in

U

at a given temperature, YF is the CD absorbance (in mdeg) of completely folded DNA (obtained from the linear fit), YU is the CD absorbance (in mdeg) of unfolded DNA, and Y is the CD absorbance of the DNA sample, all at the same temperature. The temperature for which FU = 0.5 is the half transition temperature, T1/2, that corresponds to the temperature of the sample with an equal amount of folded and unfolded GQ DNA. Due to the irreversible nature of tetramolecular GQ melting, thermodynamic parameters of unfolding were not extracted from the melting data; also the melting temperature is reported as the temperature of half transition, T1/2, and not a true (thermodynamic) melting temperature. The absolute value of T1/2 reported here is only valid for reported melting parameters, specifically a temperature change rate of 0.33 °C

Figure 2. (Left) Representative UV−vis titrations for indicated porphyrins with (dT4G4T)4 in KPi buffer at 25 °C. (Right) Best fit (solid line) to the titration data (solid squares) monitored at the specified wavelength. The [binding sites] is in μM. The dashed lines represent 95% confidence interval. The binding model which yielded the best fit expressed as Ligand:GQ is specified on each graph. 12810

DOI: 10.1021/acs.jpcb.6b09827 J. Phys. Chem. B 2016, 120, 12807−12819

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Figure 3. Graphical representation of the average Δλ and %H for all porphyrins, quadruplexes, and salts: (left) red shift, Δλ; (right) %H values. The dotted bars represent (dT4G4)4, and the solid bars represent data for (dT4G4T)4. It is interesting to note that in K+ buffer the presence of 3′T in (dT4G4T)4 GQ caused statistically significant increase in Δλ as compared to (dT4G4)4 GQ by 2.0, 1.1, 2.7, and 0.6 nm for TMPyP4, CuTMPyP4, ZnTMPyP4, and TMPyP2, respectively. Such an increase is not observed in the Na+ buffer. No clear trends can be detected for %H as a function of DNA sequence; at the same time, %H seems to be independent of the buffer composition. Both Δλ and %H are the highest for TMPyP4 and ZnTMPyP4.

Table 1. Binding Parameters for Porphyrin−GQ Complexes in KPi Buffera titration data porphyrin

λmax, nm

ε for porphyrin λmax × 10−5, M−1 cm−1

Job plot P:GQ

binding model from the direct fit

(dT4G4)4

TMPyP4 CuTMPyP4 ZnTMPyP4 TMPyP2

424 424 437 414

2.26 2.31 2.04 1.82

2:1 1:1b 2:1 1:1

(dT4G4T)4

TMPyP4 CuTMPyP4 ZnTMPyP4 TMPyP2

424 424 437 414

2.26 2.31 2.04 1.82

2:1 1:1c 2:1 1:1

3:1 2:1 2:1 1:1 2:1 3:1 2:1 2:1 1:1 2:1

Ka, μM−1 3.7 1.4 16 2.0 0.7 3.0 1.1 11 6.3 1.3

± ± ± ± ± ± ± ± ± ±

0.9 0.6 6 1.0 0.1 1.5 0.7 2 0.8 0.3

Δλ, nm

ε at complex λmax × 10−5, M−1 cm−1

%H

13.4 7.1 11.5 5.7

± ± ± ±

0.7 0.7 0.2 0.6

35 22 32 23

± ± ± ±

1 1 3 4

1.46 1.81 1.38 1.30

± ± ± ±

0.03 0.03 0.08 0.06

15.4 8.3 14.2 6.3

± ± ± ±

0.3 0.1 0.3 0.6

36 26 32 22

± ± ± ±

1 2 1 2

1.45 1.70 1.38 1.44

± ± ± ±

0.05 0.04 0.02 0.06

a

The uncertainty in each value represents the standard deviation of at least two trials. Binding constants for higher P:GQ ratios are reported for cases where increasing the binding ratio lead to some improvement in the fitting parameters (e.g.,TMPyP2 case). bAt high CuTMPyP4 saturation 4:1 complexes are also observed. cAt low porphyrin concentration 1:3 or 1:2 CuTMPyP4:GQ complexes are also detected; at high porphyrin concentrations, 3:1 CuTMPyP4:GQ complexes are also detected.

Table 2. Binding Parameters for Porphyrin−GQ Complexes in KPi Buffera titration data

(dT4G4)4

(dT4G4T)4

porphyrin

λmax, nm

ε for porphyrin λmax × 10−5, M−1 cm−1

Job plot P:GQ

binding model from the direct fit

TMPyP4 CuTMPyP4

424 424

2.26 2.31

1:1 1:1, 3:1

ZnTMPyP4 TMPyP2 TMPyP4 CuTMPyP4 ZnTMPyP4 TMPyP2

437 414 424 424 437 414

2.04 1.82 2.26 2.31 2.04 1.82

1:1, 2:1 1:1, 1:2 1:1 1:1 2:1 1:1

2:1b 1:1 2:1 2:1 1:1 3:1 2:1 2:1 1:1

Ka, μM−1

Δλ, nm

%H

ε at complex λmax × 10−5, M−1 cm−1

± ± ± ± ± ± ± ± ±

15 ± 2 7.2 ± 0.10

37 ± 3 18 ± 2

1.43 ± 0.05 1.89 ± 0.05

1.8 3.0 0.9 3.0 1.9 3.3 2.0 5.5 7.1

0.5 1.0 0.2 1.5 0.5 0.3 0.1 1.2 2.3

12.4 5.3 14.4 7.2 12.8 5.3

± ± ± ± ± ±

0.2 0.6 0.4 0.3 0.3 0.3

30 22 35.4 21 33 19.4

± ± ± ± ± ±

1 1 0.1 3 2 0.9

1.43 1.43 1.45 1.82 1.37 1.48

± ± ± ± ± ±

0.03 0.02 0.01 0.08 0.04 0.01

a

The uncertainty in each value represents the standard deviation of at least two trials. Binding constants for higher P:GQ ratios are reported for cases where increasing the binding ratio lead to some improvement in the fitting parameters (e.g., CuTMPyP4 case). bKa for the 3:1 binding model is 0.8 ± 0.2 μM−1. These data is provided for ease of comparison with the data for other buffers and GQ DNA for which the 3:1 binding model provides the best fit.

extent of electronic overlap between porphyrin core and bases in the G-quadruplex. Although the values of %H and Δλ for porphyrin−dsDNA complexes are clearly assigned to a specific binding mode (e.g., intercalation, outside binding and/or endstacking),65 this correlation is much less clear for porphyrins

Supporting Information Figure S2. In each case, the titration is accompanied by the decrease in the intensity (hypochromicity, %H) and the red shift (Δλ) of the Soret band, summarized in the bar graph of Figure 3, and in Tables 1 and 2 for KPi and NaPi buffers, respectively. %H and Δλ are indicative of the 12811

DOI: 10.1021/acs.jpcb.6b09827 J. Phys. Chem. B 2016, 120, 12807−12819

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The Journal of Physical Chemistry B bound to GQ DNA. The values of %H are high for TMPyP4 (35−37%) and ZnTMPyP4 (30−33%) and are significantly lower for CuTMPyP4 (18−26%) and TMPyP2 (19−23%), regardless of DNA or buffer. Similarly, the values of Δλ are higher for TMPyP4 (13.4−15.4 nm) and ZnTMPyP4 (11.5− 14.2 nm, and are much lower for CuTMPyP4 (7.1−8.3 nm) and TMPyP2 (5.3−6.3 nm). For both %H and Δλ, the porphyrins can be placed in the following order: TMPyP4 > ZnTMPyP4 > CuTMPyP4 > TMPyP2. Large magnitudes of % H and Δλ observed for TMPyP4 and ZnTMPyP4 suggest strong excitonic coupling between π−π* transition of the porphyrin ring and the aromatic system of G-tetrad or DNA bases.69 Interestingly, for a given porphyrin, the values of %H and Δλ do not seem to be sensitive to the nature of monovalent cations or GQ DNA. The only consistent difference is that Δλ is higher (although not by a large margin) for every porphyrin interacting with (dT4G4T)4 in KPi buffer as compared to other conditions. The large difference in spectroscopic parameters between TMPyP4 and CuTMPyP4 is surprising in view of similarities of their structures and their binding to dsDNA,65 but consistent with previous GQ studies.29,63,70 Overall, the results suggest that either %H and Δλ are not sensitive reporters of the specif ic binding mode or that binding of a given porphyrin is independent of monovalent cation and the presence of 3′-T. UV−vis titrations at Soret maxima for free and bound porphyrins (or their difference for porphyrins with small Δλ) were fit assuming simple two-state system with equal and independent binding sites (Direct Fit), which was justified in most (but not all) cases by the presence of a clear isosbestic point. In the cases where an isosbestic point was not clear (CuTMPyP4 and some cases of ZnTMPyP4) using a two-state model is an oversimplification, because the binding events are not equal and independent. In those cases the obtained binding constants represent all binding events combined and thus should be used only as estimates, and not as accurate values. The resulting binding curves are shown in Figures 2 and S2, and binding constants are presented in Tables 1 and 2. Various binding ratios were tested and models with 1:1 and 2:1 P:GQ worked the best, except in the case of TMPyP4 for which the 3:1 binding model yielded the best results. Binding stoichiometry was confirmed experimentally in a modelindependent method of continuous variation analysis, also known as Job Plot.68 The representative data are shown in Figure 4 and in Supporting Information Figures S3−S5. The stoichiometry ratios obtained via Job Plot experiments (Tables 1 and 2) agree or are lower than those obtained via Direct Fit. This is most likely because the Job Plot method detects the major (strongest) binding event, whereas titrations characterize the binding in general, including strong, weak, and nonspecific interactions. TMPyP4 displays the highest stoichiometry among all porphyrins studied, binding with 2:1 P:GQ stoichiometry in KPi and 1:1 in NaPi, according to Job Plot; but with 3:1 stoichiometry according to Direct Fit in all conditions but (dT4G4)4 in NaPi, where Direct Fit yielded 2:1 stoichiometry. The high stoichiometry of binding is not unprecedented for TMPyP4 as this porphyrin was shown to bind four variants of human telomeric G-quadruplex with 4:1 stoichiometry.55,71 Interestingly, for ZnTMPyP4, the stoichiometry values from Direct Fit generally match those from Job Plot experiments. Independent of the buffer, ZnTMPyP4 binds to both GQ DNAs with 2:1 binding stoichiometry. However, in many cases

Figure 4. Representative Job plots for (A) TMPyP4, (B) CuTMPyP4, (C) ZnTMPyP4, and (D) TMPyP2 in complex with (dT4G4T)4 in KPi buffer at 25 °C. Porphyrin and GQ DNA concentrations were at 4.0 μM. Job plots were constructed by plotting the difference in the absorbance values at a specified wavelength vs mole fraction of porphyrin.

the molar ratio is below 0.67 (suggestive of a clear 2:1 binding event). For ZnTMPyP4 binding to (dT4G4)4 in NaPi buffer two binding events could be distinguished: binding of the first porphyrin molecule to GQ followed by binding of the second porphyrin molecule, leading to an overall 2:1 binding stoichiometry (Figure S2). According to Job Plot data, CuTMPyP4 binds with 1:1 stoichiometry to both GQ DNA in both buffers; in most cases a second binding even can be distinguished with 3:1 or 4:1 CuTMPyP4:GQ binding. For CuTMPyP4 Direct Fit worked best when 2:1 binding was used. Finally, for TMPyP2, the binding model is 1:1 irrespective of buffer and GQ DNA. Just like for ZnTMPyP4, the binding ratios obtained from Job Plot, in general, matched those obtained from the Direct Fit of the titration data. It is possible that in the cases of ZnTMPyP4 and TMPyP2 a single binding event happens during titration of the porphyrin with GQ DNA, whereas for TMPyP4 and CuTMPyP4, multiple binding events of differing strength occur. To sum up, the stoichiometry (and most likely mode of binding as reflected by %H and Δλ) for CuTMPyP4 and TMPyP2 does not depend strongly on the nature of monovalent cation (K+ vs Na+) or the presence of 3′T. It is only for TMPyP4 and ZnTMPyP4 that the binding stoichiometry decreases when K+ is replaced with Na+. The values of binding affinities, Ka, obtained using Direct Fit are presented in Tables 1 and 2 for KPi and NaPi, respectively. Unlike %H and Δλ, the Ka values show greater variation with GQ or buffer type. Overall, ZnTMPyP4 binds the strongest and CuTMPyP4 binds the weakest. Specifically, ZnTMPyP4 displays Ka of 11−16 μM−1 in KPi buffer and 3−5.5 μM−1 in NaPi buffer. At the same time, CuTMPyP4 displays Ka of 0.9− 2.0 μM−1 irrespective of buffer or GQ DNA (for 2:1 model). TMPyP4 displays Ka of 3.0−3.7 μM−1 (for 3:1 model) for binding under all conditions, except in the case of (dT4G4)4 in NaPi buffer for which Ka is 1.8 μM−1 and the stoichiometry is 12812

DOI: 10.1021/acs.jpcb.6b09827 J. Phys. Chem. B 2016, 120, 12807−12819

Article

The Journal of Physical Chemistry B

Figure 5. Representative fluorescence emission spectra for (A) TMPyP4, (B) CuTMPyP4, (C) ZnTMPyP4, and (D) TMPyP2 with GQ DNA in KPi and in NaPi buffers at 25 °C. Note the difference in scale between the spectra in A−D. The porphyrin concentration was ∼2.5 μM and [GQ] was ∼12.5 μM. Free porphyrin spectra are represented by solid lines, in the presence of (dT4G4)4 GQ by dashed lines, and in the presence of (dT4G4T)4 GQ by dotted lines. NaPi data are in black and KPi data are in red.

both GQs to TMPyP2 in both buffers lowers the intensity of its emission spectrum. Finally, binding of GQ DNA to ZnTMPyP4 does not change the shape of its partially resolved double peak (631 and 671 nm) emission spectrum but lowers its intensity under all condition studied. Stability of GQ−Porphyrin Complexes via CD Melting Studies. To investigate the ability of porphyrins to stabilize GQ structures, melting studies were performed by monitoring G-quadruplex unfolding via CD spectroscopy, Figure 6. Both (dT4G4)4 and (dT4G4T)4, are extremely stable in K+ buffer; thus the melting was performed in 10 mM NaPi buffer supplemented with 50 mM NaCl, where well-defined melting profiles were observed earlier.54,57 Under this condition, the half-transition temperature, T1/2, for (dT4G4)4 and (dT4G4T)4 is 35.0 ± 0.8 and 50.9 ± 0.8 °C, respectively. Thus, the presence of the 3′T stabilizes parallel GQ by 15.9 °C, in agreement with the previous report.57 Addition of 2 equiv of CuTMPyP4, TMPyP4, TMPyP2, and ZnTMPyP4 to (dT4G4)4 stabilizes its structure by 1.9, 4.4, 12.7, and 16.4 °C, respectively. Though the stabilization caused by CuTMPyP4 and TMPyP4 is seemingly modest, the presence of both porphyrins resulted in a substantially sharper melting transition as compared to the melting of free (dT4G4)4, Figure 6A, suggesting an increase in enthalpy of unfolding upon ligand binding. On the contrary, only ZnTMPyP4 and surprisingly TMPyP2 stabilize the (dT4G4T)4 quadruplex by 12.2 and 5.8 °C, respectively. Both CuTMPyP4 and TMPyP4 destabilize (dT4G4T)4 by −6.4 and −8.4 °C, respectively, possibly due to the conformational selection of ssDNA over GQ structure. Alternatively, the porphyrins might intercalate into the (dT4G4T)4 quadruplex between G and T bases as was suggested by Szalai earlier,29 destabilizing the important

2:1. TMPyP2 displays preference for (dT4G4T)4 GQ, binding to it with a Ka of 6.3−7.1 μM−1 as compared to (dT4G4)4 GQ to which it binds with Ka of 1.9−2.6 μM−1 (for 1:1 model); the binding of TMPyP2 to GQ DNA does not seem to depend on the monovalent cation. All titration data were also analyzed using Scatchard analysis (data not included).72 In many (but not all) cases, Scatchard plots were linear, yielding stoichiometries and binding constants similar to those obtained via the Direct Fit. Overall, with parameters from UV−vis titrations (e.g., Ka, %H, Δλ), the general trends of porphyrins’ behavior toward the two GQs in KPi and NaPi buffers cannot be established. However, it is very clear that the presence of 3′thymines (T) does not significantly alter the porphyrins’ binding modes and affinities. Interaction between Porphyrins and GQ DNA Using Fluorescence Emission Spectroscopy. Fluorescence spectroscopy can be used to probe the interaction of porphyrins with DNA because the local environment of porphyrins is sensitive to the presence of the binding partner. We have examined the interaction of all four porphyrins with both (dT4G4)4 and (dT4G4T)4 in the KPi and in NaPi buffers, Figure 5. Upon binding to GQ DNA, TMPyP4 displays substantial changes in the emission spectra as compared to other porphyrins. Its broad spectrum splits into two peaks at 657 and 722 nm and their intensities increase significantly in KPi buffer. The (dT4G4)4 GQ leads to higher fluorescence enhancement as compared to (dT4G4T)4. Free CuTMPyP4 does not have an emission profile in the range 600−900 nm. When bound to GQ DNA, CuTMPyP4 displays emission enhancement with two broad peaks at 560 and 805 nm. This enhancement is independent of the salt type and DNA sequence. Free TMPyP2 in solution displays a split peak due to restricted rotation of its peripheral substituents. Binding of 12813

DOI: 10.1021/acs.jpcb.6b09827 J. Phys. Chem. B 2016, 120, 12807−12819

Article

The Journal of Physical Chemistry B

Figure 7. CD melting (●) and annealing (○) curves for (dT4G4T)4 in 10 mM sodium phosphate, pH 7.0, 50 mM NaCl buffer. The temperature increase rate was 0.5 °C min−1 and the equilibration time was 1 min, which resulted in an overall temperature change rate of 0.33 °C min−1. The melting and cooling curves are not superimposable, confirming that the folding/unfolding process is not reversible.

porphyrins interact more favorably with (dT 4 G 4 ) 4 vs (dT4G4T)4. The last statement might suggest that the presence of unobstructed 3′G-tetrad is of great importance for the interactions between porphyrins and GQ DNA.

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DISCUSSION To investigate the details of GQ binding to TMPyP4 and its derivatives (and planar aromatic ligands in general), two DNA sequences, dT4G4 and dT4G4T, that differ by the presence of 3′T nucleotide were selected. They represent G-rich DNA sequences found in Oxytricha nova and form all-parallel tetramolecular GQ structures in Na+ and K+ buffers.56,57 Association of four strands of dT4G4 results in the structure with one open G-tetrad, whereas the same structure composed of dT4G4T strands will have the G-tetrad face obstructed by 3′T nucleotides. If ligand binding involves primarily endstacking, open 3′G-tetrad of (dT4G4)4 should be more accessible than any other G-tetrad in either (dT4G4)4 or in (dT4G4T)4 and differences in binding behavior of porphyrins toward these two GQs should be observed. On the contrary, if porphyrins bind to grooves, intercalate, or end-stack between G-tetrad and T bases, minimal changes in the binding behavior should be observed. Of course, binding behavior also depends on porphyrin’s central metal coordination geometry and peripheral groups, resulting in a combination of a variety of binding modes which are further complicated by the nonspecific binding (loop binding is not possible due to the nature of the DNA sequences). Therefore, the observed effect could be less pronounced than expected and harder to interpret. Effect of 3′T-Nucleotide on GQ Stability Alone and in the Presence of Porphyrins. Addition of 3′T nucleotide to the dT4G4 DNA increased the stability of the GQ by 15.9 °C in 10 mM NaPi buffer supplemented with 50 mM NaCl. This finding is in agreement with the original report where near 20 °C stabilization was observed for the same GQ DNA sequences in the presence of 200 mM Na+ buffer at low strand concentration.57 Addition of 3′T to the fluorescently labeled dR-FAM-TGGGG oligonucleotide increased its melting temperature by 8−12 °C, depending on the heating rate.73 It is suggested that the 3′T base stabilizes GQ by a strong π−π stacking interaction with the terminal quartet, which masks its exposure to solvent.61 This suggestion is supported by the

Figure 6. Thermal denaturation of (A) ∼5.5 μM (dT4G4)4 and (B) ∼5.5 μM (dT4G4T)4 alone and in the presence of TMPyP4, TMPyP2, ZnTMPyP4, and CuTMPyP4 ([P]/[GQ] ≈ 2) monitored by CD spectroscopy at 265 nm. FU represents the fraction of unfolded GQ. Buffer: 10 mM sodium phosphate, pH 7.0, 50 mM NaCl.

interaction between the terminal 3′G-tetrad and nearest Ts leading to an overall destabilization of the (dT4G4T)4 structure. Melting data allowed us to assess qualitatively the kinetics of GQ folding. Heating and cooling curves as well as pre- and postmelt CD scans were collected and were not superimposable, as seen in Figure 7. Even with a slow temperature change rate of 0.33 °C min−1, no signs of refolding of (dT4G4T)4 either by itself or in the presence of any porphyrins were seen. Similarly, (dT4G4)4 did not refold when being cooled alone, but ∼30% refolding was observed upon cooling in the presence of porphyrins. Postmelt samples of the DNA alone stored for 24 h at 4 °C refolded only by