Effect of the bases flanking an abasic site on the recognition of nucleobase by amiloride

Biochimica et Biophysica Acta 1800 (2010) 599–610

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g e n

Effect of the bases flanking an abasic site on the recognition of nucleobase by amiloride Arivazhagan Rajendran a,1, Chunxia Zhao a,b,2, Burki Rajendar a,3, Viruthachalam Thiagarajan a,b,4, Yusuke Sato a, Seiichi Nishizawa a,b,⁎, Norio Teramae a,b,⁎ a b

Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980-8578, Japan CREST, Japan Science and Technology Agency (JST), Aoba-ku, Sendai 980-8578, Japan

a r t i c l e

i n f o

Article history: Received 28 October 2009 Received in revised form 19 February 2010 Accepted 11 March 2010 Available online 20 March 2010 Keywords: Single nucleotide polymorphism Nucleobase recognition Abasic site Flanking bases Amiloride Molecular modeling TDDFT

a b s t r a c t Background: We explain here the various non-covalent interactions which are responsible for the different binding modes of a small ligand with DNA. Methods: The combination of experimental and theoretical methods was used. Results: The interaction of amiloride with thymine was found to depend on the bases flanking the AP site and different binding modes were observed for different flanking bases. Molecular modeling, absorption studies and binding constant measurements support for the different binding patterns. The flanking base dependent recognition of AP site phosphates was investigated by 31P NMR experiments. The thermodynamics of the ligand–nucleotide interaction was demonstrated by isothermal titration calorimetry. The emission behavior of amiloride was found to depend on the bases flanking the AP site. Amiloride photophysics in the context of AP-site containing DNA is investigated by time-dependent density functional theory. Conclusions: Flanking bases affect the ground and excited electronic states of amiloride when binding to AP site, which causes flanking base-dependent fluorescence signaling. General significance: The various noncovalent interactions have been well characterized for the determination of nucleic acid structure and dynamics, and protein–DNA interactions. However, these are not clear for the DNA–small molecule interactions and we believe that our studies will bring a new insight into such phenomena. © 2010 Elsevier B.V. All rights reserved.

1. Introduction The past few decades have witnessed some promising developments in the field of recognition of nucleobases by small molecules. Recognition of nucleobases is essential for biologically important

Abbreviations: SNP, single nucleotide polymorphism; AP, abasic; DCPC, 3,5diamino-6-chloro-2-pyrazinecarbonitrile; TDDFT, time dependent density functional theory; Spacer C3, Spacer phosphoramidite C3; ITC, isothermal titration calorimetry; CV, cyclic voltammetry; MM, molecular mechanics; HOMO, highest occupied molecular orbital; LUMO, lowest unoccupied molecular orbital; CT, charge transfer ⁎ Corresponding authors. Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980-8578, Japan. Tel.: +81 22 795 6549; fax: +81 22 795 6552. E-mail address: [email protected] (N. Teramae). 1 Present address: Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-Ku, Kyoto 606-8502, Japan. 2 Present address: National Chromatographic R & A Center, Dalian Institute of Chemical Physics, Chinese Academy of Science, 457 Zhongshan Road, Dalian 116023, China. 3 Present address: Department of Chemistry, University of Alabama at Birmingham, 901 14th Street South, Birmingham, AL 35294, USA. 4 Present address: Ecole Normale Supérieure, Laboratoire de Physique Statistique and Département de Biologie, UMR CNRS-ENS 8550, 24 rue Lhomond, 75005 Paris, France. 0304-4165/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2010.03.007

functions such as genome duplication, signal transduction, and protein synthesis [1,2]. Several artificial receptors have been reported which are capable of monomeric complexation with nucleobases, either by multiple hydrogen (H)-bonding or by combination of different modes of interactions [3–5]. The major design rationales have been introduced, all of which are based on the complementary H-bonding in relatively nonpolar organic solvents [6–10]. Later, these interactions have been extended to the complexation in water by utilizing various noncovalent interactions in addition to the multiple H-bonding [11– 16]. Recently, synthetic H-bonding motifs have been applied for the recognition of single nucleotide polymorphism (SNP) for a DNA duplex containing a mismatched base pair [17] or bulged base [18]. In this connection, using synthetic molecules which possess H-bonding surfaces, we have developed a novel method for the detection of SNPs in a DNA duplex reconstituted with an oligodeoxynucleotide containing an abasic (AP) site [19–22]. Among the various experimental techniques, of particular interest to us is the implementation of fluorescence assay for SNPs typing. Upon complexation of the ligand through H-bonding with a specific target base, fluorescence of the ligand is strongly quenched or enhanced (Scheme 1). Regarding the binding modes, as the ligand forms pseudo WatsonCrick base pairs with the interhelical target base, the ligand displays

600

A. Rajendran et al. / Biochimica et Biophysica Acta 1800 (2010) 599–610

2. Experimental section 2.1. Materials and methods

Scheme 1. Schematic illustration of the ligand-based fluorescence detection of SNPs in a DNA duplex reconstituted with an oligonucleotide containing an AP site. Where, N = C or A; N′ = G or T—flanking bases.

various noncovalent interactions inside the duplex. Most important interactions are H-bonding, base-stacking, electrostatic and charge transfer (CT) interactions [23]. The first two interactions are relatively important and play a major role in determining the binding affinity and selectivity of a ligand to a particular target base. It was believed that specific H-bonding and electrostatic interactions are much stronger and contribute dominantly to the stability of nucleic acids and various complexes of biomolecules such as groove binders [24]. H-bonding is highly specific and directional. This type of interaction is responsible for the stabilization of biomacromolecules and its complexes. Electrostatic interaction is especially important in the interaction of charged ligands with nucleic acids. CT interaction does not play essential role in determining the structure and stability of the ligand–nucleic acid complex, but should be properly considered. Stacking interactions are nonspecific and for a long time they were believed to be much weaker than the specific interactions. But later it was realized that stacking interactions are much more important than the specific interactions [25]. In the present study, to probe the effect of flanking bases on the recognition of nucleobase, we chose the ligand amiloride, a diuretic containing a pyrazine ring and a guanidinium group (Fig. 1). Further, investigations were carried out to characterize the various noncovalent interactions inside the duplex DNA. Amiloride was used previously for the selective detection of thymine (T) opposite an AP site in 11-mer duplex DNA [26]. It has three-point H-bonding groups complementary to the nucleobase T and the pyrazine ring can effectively base-stack with the bases flanking the AP site. Further, the positively charged guanidinium group mimics the side chain of the amino acid Arg, which can be useful to probe the electrostatic interactions. Guanine, a purine base, has the lowest ionization potential and thymine, a pyrimidine base, has the highest electron affinity among the nucleobases. Hence, to probe the base-stacking and CT interactions, the DNA with G and T flanking bases (GXG and TXT, where X is the AP site) were chosen for the study. As for the experimental investigation, UV-visible absorption, fluorescence and NMR spectroscopy, isothermal titration calorimetry (ITC) [27,28] and cyclic voltammetry (CV) techniques were used. As a potential combination to these experimental techniques, molecular mechanics and time-dependent density functional theory (TDDFT) calculations were performed. We have previously reported a similar ligand, 3,5-diamino-N-(3aminopropyl)-6-chloropyrazine-2-carboxamide (DCPC-NH2) for the selective detection of thymine [29]. However, the binding constant of DCPC-NH2 to thymine is relatively low when compared to amiloride. Further, the binding mode is independent of the flanking bases when the ligand is DCPC-NH2 while it is dependent in the present case with amiloride. Here, we clearly demonstrated the electronic behavior of amiloride with the help of TDDFT calculations. Moreover, the thermodynamics of the ligand–nucleotide interaction is explained briefly.

Amiloride was purchased from Sigma-Aldrich Chemicals Co. (Milwaukee, WI). DNA samples were custom synthesized and purified using HPLC by Nihon Gene Research Laboratories Inc. (Sendai, Japan). For the synthesis of AP site containing DNA duplexes, a propyl residue (Spacer phosphoramidite C3, Spacer C3) was utilized. The DNAs were 11-mer and 23-mer oligonucleotides with sequences 5'-G TTG NTN TGG A-3'/5'-T CCA N′XN′ CAA C-3' and 5'-G TGT GCG TTG NTN TGG ACG CAG A-3'/5'-T CTG CGT CCA N′XN′ CAA CGC ACA C-3', (X = Spacer C3, N = C or A, N' = G or T), respectively. Water was deionized (≥18.0 MΩ cm of specific resistance) by a Milli-Q system (Millipore Corp., Bedford, MA). The other reagents were commercially available analytical grade and used without further purification. All measurements were performed in 10 mM sodium cacodylate solution (pH 7.0) containing 100 mM NaCl and 1 mM EDTA. Concentrations of DNAs were determined by the molar extinction coefficient at 260 nm calculated according to the literature [30]. Before any experiment, the DNA samples were annealed as follows: heated at 75 °C for 10 min, gradually cooled down to 5 °C (3 °C/min), and finally the solution temperature was raised again to 20 °C (1.5 °C/min). All the DNA sequences are given in the 5'-3'/5'-3' order throughout the article.

2.2. Electronic spectra Absorption and emission spectra were measured at 5 °C on a JASCO, V-570 double beam UV/VIS/NIR spectrophotometer and FP6500 spectrofluorometer (Japan Spectroscopic Co. Ltd., Tokyo, Japan), respectively. Each spectrophotometer was equipped with a thermoelectrically temperature-controlled cell holder. For the fluorescence measurements, 1 µM of the ligand was titrated with 0–5 µM of DNA. The ligand was excited at 380.5 nm. For the absorption measurements, 10 µM of the ligand was titrated with 0-10 µM of DNA. The optical path length used was 10 mm. To avoid the volume effect on the spectroscopic experiments, a constant total volume of 250 µl of the samples was taken for the measurements. 2.3. ITC experiments ITC measurements were carried out at 10 °C using a VP-ITC microcalorimeter (MicroCal Inc., Northampton, MA). In a typical experiment, 10 µM ligand solution (1.42 ml) was titrated with 150 µM DNA solution (25 injections of 8 µl each), using 250 µl syringe rotating at 300 r.p.m. The peaks produced during titration were converted to heat output per injection by integration and correction for the cell volume and sample concentration. The thermodynamic parameters were derived from the data by non-linear least-squares regression analysis of a function based on the binding of a ligand to a macromolecule assuming the law of mass action and 1:1 binding stoichiometry [31]. The origin software was used for data acquisition and analysis. 2.4.

31

P NMR

31 P NMR spectra were recorded at 10 °C on a JEOL, ECA600SL spectrometer with standard pulse sequences operating at 594.171 MHz. H3PO4 (0 ppm) was used as an external reference. The measurement conditions were as follows: spectral width 60680 Hz, acquisition time 0.5 s, delay of 2 s, 5000 scans per spectrum, pulse width 50 °, WALTZ decoupling mode. The concentrations of DNA and the ligand were 150 µM and 0-400 µM, respectively.

A. Rajendran et al. / Biochimica et Biophysica Acta 1800 (2010) 599–610

601

Fig. 1. Line drawing of the structures of amiloride, DCPC and possible H-bonding modes between amiloride and T target base.

2.5. CV experiment Electrochemical measurement was performed using an electrochemical analyzer (ALS model 660A, BAS, Tokyo, Japan). An Ag/AgCl (sat. KCl) electrode and a platinum wire were used as reference and counter electrodes, respectively. Glassy carbon electrode (1 mm in diameter) was purchased from BAS and used as a working electrode. A solution of 50 µM amiloride, 100 mM NaCl, 1 mM EDTA and 10 mM sodium cacodylate buffer (pH 7.0) was taken for the experiment. 2.6. Selection of theoretical models Models of 23-mer oligodeoxynucleotides and amiloride were constructed using Maestro 7.0. To construct the AP site opposite the target base of T in a 23-mer duplex, the complementary adenine (A) base and the sugar unit were removed and a propyl residue (Spacer C3) was inserted between two phosphate moieties in the backbone. Models for TDDFT [32] calculations were constructed from the molecular mechanics (MM) optimized geometries of the complex of ligand–23-mer DNA with an AP site, removing the entire sugarphosphate backbone and the bases except the flanking bases to AP site. The bases terminated with hydrogens at the N1 (pyrimidines) or N9 (purines) position as appropriate. This is justified given that the transitions of interest take place between molecular orbitals (MOs) that are localized on the ligand and the flanking bases. Each base was optimized separately at the B3LYP/6-311 + G(d) level, maintaining the double helical parameters of the B-DNA. 2.7. Molecular modeling Molecular modeling was carried out using MacroModel Ver. 9.0 [33,34]. The MM optimizations were performed with Amber* force field and GB/SA solvation model for water (with constant dielectric treatment for the electrostatic part) together with the default cut-off

criterions, except the gradient convergence threshold which had been set to be 0.005. Since the binding model is quite straight forward and follows the 1:1 binding isotherm, the docking processes were done by manually inserting the ligand into the DNA duplex. The interaction energies of amiloride–DNA complexes were estimated by calculating the difference between their total energies and the sum of lowest energies found for the optimized structures of DNA and amiloride. The negative of the interaction energy was taken to be the binding energy [29,35]. EI = ET – ðsum of the individual energyÞ EB = –EI Where EI is the interaction energy, ET is the total energy of the DNA–ligand complex and EB is the binding energy. 2.8. TDDFT calculations The vertical excitation energies and oscillator strengths were computed using time dependent approach as implemented in Gaussian03 (G03) program [36]. Predicted lowest energy singlet– singlet transitions of amiloride were calculated for the ground state geometry. Calculations were carried out with the B3LYP exchangecorrelation functional, which has been shown to provide accurate values for low-lying valence transition energies for the molecule flanked with one or two nucleobases [37,38]. All reported results were obtained by using the triple-ζ 6-311+G(d) basis set. In order to include the bulk solvent effects, calculations were performed in water, by means of the continuum solvation C-PCM implementation [39]. The MOs rendered using the program Molden [40] (contour value = 0.02). The H-bonded and base-stacked systems used for TDDFT calculations, are denoted as T ≡ amiloride and GamilorideG/ T amilorideT, respectively; where ≡ represents the three-points hydrogen bonding between the ligand and target base (T) and superscript G's and T's represent the flanking bases.

602

A. Rajendran et al. / Biochimica et Biophysica Acta 1800 (2010) 599–610

3. Results and discussion 3.1. Emission spectra The effect of bases flanking the AP site on the interaction of amiloride with T target base was examined by fluorescence measurements. Fig. 2 shows the fluorescence emission spectra of amiloride in the absence and presence of 23-mer AP site-containing DNA duplex with two different flanking bases (GXG and TXT, where X is the AP site). Interestingly, the fluorescence response of amiloride strongly depends on the bases flanking the AP site. When guanine bases were flanked with the AP site (GXG), fluorescence of amiloride at 415 nm was quenched and the emission peak became broader upon addition of the DNA duplex (Fig. 2A), while its fluorescence was enhanced and the peak was narrowed when the flanking bases were T (TXT, Fig. 2B). Further, the emission maximum of amiloride was red shifted about 5 nm upon adding one equivalent of the DNA duplex having T flanking bases at the AP site, whereas no noticeable shift was observed for the DNA duplex containing an AP site with G flanking bases. The variation in the emission properties of amiloride depending on the flanking bases may be due to either different ground–state interactions or excited-state properties. Before discussing the quenching mechanism, the binding constants of amiloride for both the flanking bases were determined by nonlinear regression analysis of fluorescence titration curves based on 1 : 1 binding isotherm model. The calculated binding constants of amiloride to T target-containing DNA were 24.3 × 106 and 4.1 × 106 M-1 for G and T flanking bases, respectively. Surprisingly, amiloride exhibited stronger binding constant when it basestacked with G and the binding constant for G-stacking was about 6 times higher than T-stacking. This explains that the flanking bases have strong influence on the emissive property and the binding affinity of amiloride with T. The higher binding affinity with G-stacking is either due to stronger stacking interaction by the extended π orbitals of G or due to the different ground-state interaction (for instance, different binding modes) of amiloride with T-target base. 3.2. UV-visible spectral trends The effect of flanking bases on the ground-state interactions of amiloride with DNA was studied by UV-visible absorption measurements. Any ground state interaction between amiloride and DNA is

expected to perturb the π-π* transition of the former. The changes in the absorption spectrum of amiloride in the presence of T targetcontaining DNA with both the flanking bases were monitored and are given in Fig. 3. Irrespective of the flanking bases, the absorbance of the absorption spectrum centered at 362 nm decreased upon addition of DNA and a strong red shift was also observed. The observed red shift in the absorption spectrum of amiloride was 12.5 nm by the addition of one-equivalent of DNA with G flanking bases, while it was 9.5 nm with T flanking. This result indicates that the ground-state interactions of amiloride with DNA of different flanking bases, are different. This in turn may influence the binding constants for G and T flanking bases. 3.3. ITC experiments The thermodynamics of the interaction of amiloride with thymine base opposite the AP site were examined by ITC experiments. The resulting ITC curves and the plots of heat evolved per mole of amiloride added against the molar ratio of amiloride to DNA, are given in Fig. 4. The thermodynamic parameters are given in Table 1. Irrespective of the bases flanking the AP site, the addition of DNA duplex into the amiloride solution caused large exothermic heat of complexation while the heat of dilution for the DNA duplex was almost negligible. Non-linear least square analysis of the titration curve revealed that the association constant Ka = 2.7 × 106 M−1 for the DNA with G flanking bases. As listed in Table 1, the ligand–nucleotide interaction was enthalpically driven. Whereas, for the DNA with T flanking bases, the association constant Ka was found to be 1.1 × 106 M −1 . Like the previous case, the complexation was found to be enthalpy driven. Irrespective of the flanking bases, the thermodynamic feasibility of complexation is apparent from the negative values of the free energy change associated with the complex formation. However, the enthalpy term for the T flanking bases, became destabilizing to about 0.7 kcal/mol compared to that for the G flanking. This was partially compensated by the less negative value (about 0.2 kcal/mol) of entropy, which lead to the lower value of ΔGº to about 0.5 kcal/mol for the T flanking bases than G. From this observation, it is clear that the ligand is relatively more stable inside the AP site when the flanking bases are G. The observed nature of the thermodynamic profile was clearly different from that of groove binding [28], but rather similar to intercalation of drugs [41]. Thus, it is highly likely that the binding event of amiloride does take place at the AP site in the DNA duplex,

Fig. 2. Steady-state fluorescence spectra of amiloride in the absence and presence of increasing amounts of 23-mer DNA duplexes of the sequence (A) CTC/GXG and (B) ATA/TXT. [DNA] = 0–5 µM; [amiloride] = 1 µM; [NaCl] = 100 mM; [EDTA] = 1 mM; [sodium cacodylate] = 10 mM; pH = 7; temperature = 5 °C; λexc. = 380.5 nm.

A. Rajendran et al. / Biochimica et Biophysica Acta 1800 (2010) 599–610

603

Fig. 3. Absorption spectra of amiloride in the absence and presence of increasing amounts of (A) CTC/GXG and (B) ATA/TXT. [DNA] = 0, 2, 4, 6, 8, and 10 µM; [amiloride] = 10 µM; [NaCl] = 100 mM; [EDTA] = 1 mM; [sodium cacodylate] = 10 mM; pH = 7; temperature = 5 °C; optical path length = 10 mm.

and the AP site-based binding pocket effectively allows the formation of molecular interactions including the stacking and H-bonding for the complexation of the ligand with target T base. The association constant measured by ITC experiment for amiloride stacked with G was about 2.5 times higher than that for the T-stacking. The same trend was also observed in the binding constants determined by fluorescence measurements. In general, it is expected that the stacking of a ligand with both 5' and 3' G-bases leads to an additional stabilization of the complex due to the extended π orbitals of G. This in turn will lead to the stronger binding affinity for G than T flanking. But, we have found that the binding constant was almost comparable (5.04 × 105 M−1 and 3.81 × 105 M−1 for G and T flanking bases respectively) with both the flanking bases when we used 3,5-diamino-6-chloro-2-pyrazinecarbonitrile (DCPC, Fig. 1) as a ligand. DCPC has a pyrazine moiety similarly to amiloride but it lacks

the guanidinium side chain of amiloride. Hence, it seems that the reason for the increased binding constant is certainly not the stronger base-stacking effect of G. The observed higher binding constant may be due to different ground-state interactions of amiloride with T target base. Because, the palindromic alignment of the hydrogen bonding surface of the ligand can lead to different binding modes with the target base. Moreover, the guanidinium side chain of the ligand may show different ground-state interactions with the phosphate group. The details will be explained later in this article. 3.4.

31

P NMR experiments

To investigate the recognition of AP site phosphate by amiloride guanidinium side chain, 31P NMR experiments were carried out. The DNAs with G and T bases flanking the AP site were used for the

Fig. 4. ITC curves for the binding of amiloride to DNA duplexes with (A) 23-mer CTC/GXG sequence and (B) 23-mer ATA/TXT sequence. [DNA] = 150 µM; [amiloride] = 10 µM; [sodium cacodylate] = 10 mM; [NaCl] = 100 mM; [EDTA] = 1 mM; pH = 7; temperature = 10 °C.

604

A. Rajendran et al. / Biochimica et Biophysica Acta 1800 (2010) 599–610

Table 1 The effect of base-stacking on the thermodynamic parameters of amiloride–T interaction. Flanking sequence

Ka (×106 M−1)

n

GXG TXT

2.7 (± 0.38) 1.1 (± 0.07)

1.3 (± 0.02) 1.3 (± 0.01)

kcal/mol ΔG°

ΔH°

TΔS°

−8.3 −7.8

−14.1 (± 0.22) −13.4 (± 0.13)

−5.8 −5.6

Ka = association constant; n = stoichiometry; [DNA duplex] = 150 µM; [amiloride] = 10 µM; [NaCl] = 100 mM ; [EDTA] = 1 mM ; [sodium cacodylate] = 10 mM; pH = 7; temperature=10 °C.

studies. The experiments were carried out by using 150 µM of DNA duplexes in the absence and presence of various concentrations of amiloride. The results are summarized in Fig. 5 and Table 2. As shown in Fig. 5A, the phosphate groups around the AP site flanked with G gave two characteristic signals at 0.747 (α) and 0.408 ppm (β) which are farther shifted to downfield compared to signals of normal phosphate groups. The addition of 75 µM of amiloride caused the downfield shift for the signals of both the phosphate groups and such a shift was more pronounced upon increasing the ligand concentration. The observed downfield shifts in the presence of 400 µM of amiloride were 0.162 and 0.108 ppm for α and β phosphates, respectively. This indicates clearly that the guanidinium group is strongly interacting with both the phosphate groups around the AP site, when the flanking bases are G at both 5' and 3' sides. The guanidinium group is known to recognize the phosphate [42] and hence the interaction is expected to take place through the Hbonding. The observed trends in Fig. 5A are quite different from that observed for the DNA with T flanking bases (Fig. 5B). In this case, the phosphate signals of the AP site were found at 0.560 and 0.440 ppm for α and β phosphates, respectively. The presence of 75 µM amiloride caused the downfield shift only for the α phosphate group and such a shift was more pronounced upon increasing the ligand concentration. The observed downfield shift in the presence of 400 µM ligand was 0.160 ppm which is nearly the same as the shift observed for the G flanking bases. Hence, it seems that the α phosphate-guanidinium interaction is independent of the bases flanking the AP site. Interestingly, the β phosphate signal was not affected by the presence of 75 µM of the ligand, while an upfield shift was observed at higher concentrations. The observed upfield shift in the presence of 400 µM of amiloride was 0.100 ppm. This observation indicates that the guanidinium group of amiloride has no H-bonding interaction with the β phosphate group, when the ligand base-stacked with T at the AP site. The 31P NMR experiments reveal that different H-bonding interactions take place between the guanidinium group and the AP site phosphates for different flanking bases. This could be the possible reason for the observed higher binding constant with G flanking bases and the different absorption spectral trends.

3.5. Molecular modeling Though the detailed information on the recognition of the AP site phosphates by the guanidinium moiety was obtained from the spectroscopic experiments and binding constant measurements, the recognition of the T target base by amiloride was not clear. As shown in Fig. 1, amiloride can adopt two different binding modes with the T target because of the palindromic alignment of the H-bonding surface of T and amiloride. In the absence of flanking bases, the B3LYP/6-311 +G(d) calculated energy difference between the two binding modes was found to be 0.01 kcal/mol, which has no chemical significance. However, the flanking bases may have influence on the binding motif of amiloride with the target base.

Fig. 5. 31P NMR spectra of AP site-containing DNA duplexes (5'-GTTGNTNTGGA-3'/5'TCCAMXMCAAC-3', X = AP site) of sequence (A) CTC/GXG and (B) ATA/TXT. The line drawing of the AP site is given in (C). (A), (B), (C) and (D) are, respectively, in the presence of 0, 75, 200 and 400 µM of amiloride; [DNA] = 150 µM; [NaCl] = 100 mM; [EDTA] = 1 mM; [sodium cacodylate] = 10 mM; pH = 7; temperature = 10 °C. 10% of D2O was added to lock the signals.

A. Rajendran et al. / Biochimica et Biophysica Acta 1800 (2010) 599–610 Table 2 The 31P NMR signals (in ppm) of AP site phosphates in the absence and presence of amiloride. Concentration of amiloride (µM) 0 75 200 400

31

P NMR signals (ppm)

GXG

TXT

α

β

α

β

0.747 0.839 0.920 0.909

0.408 0.480 0.520 0.516

0.560 0.618 0.720 0.720

0.440 0.440 0.395 0.340

α and β are 5' and 3' side phosphates around AP site; [DNA duplex] = 150 µM; [NaCl] = 100 mM; [EDTA] = 1 mM; [sodium cacodylate] = 10 mM; pH = 7; [D2O] = 10%; temperature = 10 °C.

Force field calculations were carried out to ascertain the detailed structural information and the binding mode of amiloride inside the AP site with different flanking bases. The pre-optimized amiloride was manually inserted into the optimized 23-mer duplexes containing G and T flanking bases and further optimizations were carried out on the assembly of the DNA–ligand complexes. Both the binding modes were considered for the molecular modeling. The optimized geometries of the 1:1 complex of amiloride with T target base for both the flanking bases are given in Fig. 6. Several features are immediately notable. In both cases, amiloride binds to T via three-points H-bonding along the Watson–Crick edge. As we

605

expected, different binding patterns were observed with different flanking bases. It preferred the binding model (Ha) when the flanking bases were G and the guanidinium moiety of amiloride remain in the major groove (Fig. 6A), while it prefers the binding model (Hb) when it flanked with T and the guanidinium moiety was located in the minor groove as shown in Fig. 6B. The various interactions of amiloride observed inside the AP site with G flanking bases were: (i) three-points H-bonding with target T through the binding mode (Ha); (ii) being the phosphate receptor, the guanidinium moiety makes strong H-bonding with phosphate groups (both α and β) around the AP site; (iii) the H-bonding between N–H of guanidinium moiety and the N7 of 3'-G flanking base; and (iv) apart from the specific H-bonding, the non-specific stacking interaction between the pyrazine ring of amiloride and the flanking G bases. Combination of these specific and non-specific interactions makes binding more stable at the major groove side. The noncovalent interactions observed when the ligand basestacked with T were: (i) specific three-points H-bonding with the target T base through the binding mode (Hb); (ii) the H-bonding interaction between the guanidinium moiety and the α phosphate group; (iii) the non-specific stacking interaction between the pyrazine ring of amiloride and the flanking T bases; and (iv) the methyl-CH/π (3'-T methyl-CH/π of amiloride pyrazine ring) interaction (Fig. 7A). In this case, the guanidinium moiety-β phosphate interaction was not observed. This will lead to relatively weaker binding of the ligand. This could be the reason for the lower binding

Fig. 6. (A) The optimized structures of the complexes between amiloride and T target with GXG (A) and TXT (B) sequence. The H-bonding lengths are given in Å unit. 23-mer oligodeoxynucleotides were considered for MM calculations. For clarity only parts of the optimized structures are given.

606

A. Rajendran et al. / Biochimica et Biophysica Acta 1800 (2010) 599–610

Fig. 7. The various noncovalent interactions of amiloride at the AP site in a DNA duplex: (A) twin CH/π interaction; (B) stair-like binding motif; (C) cation/methyl CH repulsion. In structure (C), amiloride is in the (Ha) binding mode which is not a preferred one with T flanking bases. It is just given to explain that the repulsive interaction drives the ligand to the (Hb) binding mode.

constant observed with TXT sequence. Molecular modeling results are consistent with the trends obtained by NMR experiments. The observed difference in red shift in the absorption spectra with different flanking bases is possibly due to the different binding modes between target nucleobase–ligand–AP site phosphates.

Now, the immediate question is why the binding mode (Ha) is favorable with G base-stacking and (Hb) mode with the T-stacking? The reason is quite simple; in case of G flanking bases, the H-bonding between the N7 of 3'-G and the N-H group of the guanidinium moiety (Fig. 7B) will increase the binding constant. Further, the guanidinium

A. Rajendran et al. / Biochimica et Biophysica Acta 1800 (2010) 599–610

cation can be stabilized in the major groove by means of the cation/π interaction [43] (guanidinium cation/π orbital of flanking G, Fig. 7B), which will lead to the stronger binding in the major groove. Where as with T flanking bases there is no such H-bonding interaction with flanking bases. Also, the guanidinium cation and 3'-T methyl-CH will have repulsion in the major groove (Fig. 7C). This will lead to the binding mode (Hb) with T flanking bases. Moreover, in the binding mode (Hb), the interaction of 3'-T methyl-CH/π of amiloride pyrazine ring will stabilize the binding, i.e. the methyl group of the 3'-T in the major groove increases polarisability [44], allowing for more favorable van der Waals interactions with the pyrazine ring of amiloride. This CH/π interaction is obvious because amiloride replaces a nucleobase at an AP site and the optimum distance between the methyl-CH and the π cloud of pyrazine ring is anticipated. Further, as the 5'amilorideT-3' (amiloride flanked with T at the 3' side) is accompanied by 5'-AT-3' in the complementary strand, the interaction is duplicated, thus forming a twin CH/π interaction at the AP site, as shown in Fig. 7A. The interesting finding here is that it is the 3' base which is responsible for the stabilization (3'-G) or the repulsion (3'-T) of the guanidinium moiety in the major groove, which will lead to different binding patterns of amiloride with the same target base. To confirm this further, the force field calculations were carried out for 6 different combinations of flanking bases and the calculated binding energies for both major and minor groove binding modes are given in Table 3. As it can be seen from the Table 3, amiloride prefers the binding mode (Hb) when 3'-flanking base is T, while it prefers the binding mode (Ha) with the other flanking bases. Moreover, the binding pattern seems to be independent of the 5'-flanking base and even the 5'-T has poor repulsion on the guanidinium moiety. The results obtained here are in good agreement with the results by Viswamitra et al [45,46]. They determined the crystal structure of a deoxytetranucleotide (pATAT) and found that T is stacked with the preceding adenine base, however, there is no interaction of T with A next to it. Klug et al. [47] followed the finding and they found that the methyl group of T in an A-T step to place itself over the five-member ring of the purine, whereas in the T-A step there was no such interaction. The 3'-T methyl-CH/π interaction and its consequences are further explained by Umezawa et al. [48,49]. At protein–DNA interfaces, two successive nucleobases in a BDNA and a protein side chain with a positive charge (Arg and Lys) or a partially charged group (Gln and Asn) are often found to form simultaneously three different pairwise interactions; (i) base-stacking, (ii) H-bonding, and (iii) cation/π. They are called stair motifs as they have a stair-like shape, with H-bond forming the horizontal part of the stair, and the cation/π interaction makes the vertical part. These stair motifs have previously been identified from the crystal structures of protein/double-stranded DNA complexes [43,50,51]. The best example for the formation of the stair motif is observed between the positively charged guanidinium side chain of the amino acid Arg and two successive guanines in a DNA sequence. Here in our case, the formation of the stair-like motif is found between the

Table 3 Binding energies (kcal/mol) of different modes of binding of amiloride with different flanking base sequences. Flanking sequence

EB (Ha)

EB (Hb)

ΔEB

TXT AXT TXA TXG CXC GXG

40.65 42.69 43.48 40.78 40.63 46.09

43.53 45.91 43.37 40.82 38.63 43.94

−2.88 −3.22 0.11 −0.04 2.00 2.15

ΔEB = EB(Ha) − EB(Hb). (Ha) and (Hb) are the binding motifs as given in Fig. 1.

607

guanidinium cation of amiloride and G flanking bases (Fig. 7B), which in turn is expected to stabilize the ligand binding at the major groove. 3.6. Fluorescence quenching mechanism Next, we have focused our investigation on the fluorescence response of amiloride when it stacked with different bases. Guanine and cytosine are very polar bases. Therefore, the GC base pair is also very polar. Thymine has smaller dipole moment and adenine is even less polar. As a result, the whole AT base pair is much less polar than the GC one [25]. Hence, the ligand may experience different polarity with different flanking bases, which in turn may affect its fluorescence emission. But the observed red shift in the absorption spectra of amiloride with DNA of different flanking bases is clear indicative that the polarity is not a reason for the trends observed in the emission spectra. Since the singlet state energies of the DNA bases are at least 1.34 eV greater than that of amiloride, the quenching through an energy transfer mechanism is of course, out of question. There may be a possibility for the conformational change in DNA upon binding of amiloride, which may exert different electronic behavior. But no such conformational change was observed in the circular dichroic spectra of DNA with G and T flanking bases. TDDFT calculations were carried out to study the excited-state properties of amiloride, on the H-bonded and base-stacked complexes. We adopt here, a supermolecule approach, because the electronic properties of the H-bonded and base-stacked complexes are neither sum nor perturbation of the monomer properties. Similar approach was used previously for various systems [37,38,52]. 3.6.1. T ≡ amiloride TDDFT calculation on amiloride, assigned the lowest excited singlet state has π–π* character and the excitation energy was found to be 343 nm. Table 4 shows the transition energies and oscillator strengths predicted at the B3LYP/6-311+G(d) level for the complex in which amiloride is H-bonded to thymine. Here, both the H-bonded complexes, structures (Ha) and (Hb), were considered for the calculations and the transition energies and oscillator strengths predicted for both the systems were almost same (shown in Fig. 8A and B). The calculations predict that the first excited-state of complexes in which amiloride is H-bonded to thymine is of π–π* character and the orbitals involved in the transitions were centered completely on amiloride and resemble those involved in the first transition of amiloride itself. The wavelength was slightly, 3–4 nm, red shifted compared to that of free amiloride and the oscillator

Table 4 Experimental absorption maximum of amiloride in the absence and presence of DNA duplexes and wavelengths and oscillator strengths calculated at the TDB3LYP/6-311 +G(d) level of the H-bonded and base-stacked complexes. Systems

Experiment Amiloride Amiloride-CTC/GXG Amiloride-ATA/TXT TDDFT Amiloride T ≡ amiloride (Ha) T ≡ amiloride (Hb) T amilorideT G amilorideG

Relevant to absorption process λ (nm)

f

Assignment

362.0 374.5 371.5

– – –

π–π* (amiloride) – –

343 347 346 349 350 391

0.415 0.402 0.410 0.305 0.238 0.021

π–π* (amiloride) π–π* (amiloride like) π–π* (amiloride like) π–π* (amiloride like) π–π* (amiloride like) CT (amiloride-G)

(Ha) and (Hb) are the structures as given in Fig. 1; f—oscillator strength; CT—charge transfer transition.

608

A. Rajendran et al. / Biochimica et Biophysica Acta 1800 (2010) 599–610

Fig. 8. Orbitals involved in the first transition of the complexes in which amiloride is Hbonded or base-stacked with T, as predicted by TDB3LYP/6-311 + G(d). Both the Hbonding models (Ha) and (Hb) were considered for the calculations. Oscillator strengths are shown in square brackets.

strength for the transition from the ground-state to the first excitedstate was scarcely affected from that of the transition of amiloride alone. Hence, it is concluded that the fluorescence emission of amiloride is not affected by the H-bonding to target base T. 3.6.2. TamilorideT Results obtained for the complex in which amiloride is sandwiched with T, as it would appear in the double-stranded B-DNA, are summarized in Fig. 8C. There was only one transition (S0 → S1) in the relevant spectral region. This absorption was dominated by the highest occupied molecular orbital (HOMO) with electron density mainly on amiloride to lowest unoccupied molecular orbital (LUMO, with electron density exclusively on amiloride) transition and was similar to that of amiloride alone. The wavelength for this absorption was about 6 nm red shifted as compared to the π–π* transition of amiloride. Because of the stacking interaction, the oscillator strength of this transition was decreased about 27% when compared to S0 → S1 transition (π–π*) of isolated amiloride. The same trend was also observed in the experimental absorption spectra. In the absence of any quenching process, the fluorescence emission of amiloride was hardly affected by T flanking bases. 3.6.3. GamilorideG TDDFT results acquired for the complex of amiloride base-stacked with two G are summarized in Table 4. Jablonski diagram explaining the possible transitions in the relevant spectral region, and orbital amplitudes for this system are given in Fig. 9. As shown in Fig. 9, the ground state (S0) to the second excited state (S2) vertical transition possesses stronger oscillator strength. This absorption, dominated by the HOMO-2 → LUMO transition, was similar to that of amiloride alone, and should produce similar fluorescence emission. Here,

Fig. 9. Singlet excited-state transitions of the system GamilorideG as determined by TDB3LYP/6-311 + G(d). Jablonski diagram for the system is given. Oscillator strengths are shown in square brackets as are the dominant one-electron contributions for the respective transitions. Orbital amplitudes for the HOMO-2, HOMO-1, HOMO and LUMO are given. The widths of the lines denoting the transitions reflect the relative oscillator strengths.

stacking caused the molecular orbital involved in the fluorescence transition to spread over more than one base. As a consequence, the oscillator strength of S0 → S2 transition was decreased about 43% when compared to S0 → S1 transition of isolated amiloride. Further, this transition was about 7 nm red shifted with respect to the π–π* transition of amiloride. Strikingly, there was a low-lying S1 state in the supermolecule and the transition from S0 → S1 was very weak. This transition had contributions from HOMO and HOMO-1 (with electron density mainly on flanking G) → LUMO (with electron density solely on amiloride). This transition will result in a “hole” transfer from amiloride to both the G, which define this as a CT state. As a result, the intensity of amiloride-like fluorescence from S2 → S0 transition would be significantly reduced. Although TDDFT method can be used to predict the accurate energies for valence states (such as local π–π* states), the method seriously underestimates the energies of CT states [53]. Hence, the energy of the S1 state needs to be recalculated with different method. Note that the problem with the TDDFT is the position of the charge transfer state (whether it is low-lying state or not). But, still the CT process could be possible in the excited-state which in turn may influence the amiloride fluorescence. The same conclusion was previously drawn by the combination of TDDFT and configuration interaction singles (CIS) calculations for the fluorescence quenching of 2-aminopurine in a duplex DNA [53]. TDDFT results reveal that the first excited state, the fluorescent state, of H-bonded and base-stacked complexes containing amiloride and thymine, is just the first excited state of amiloride alone. The ligand is being rendered more inaccessible to water molecules inside the AP site. As a consequence its fluorescence could be enhanced by the reduction of radiationless decay upon binding to DNA duplexes

A. Rajendran et al. / Biochimica et Biophysica Acta 1800 (2010) 599–610

having T flanking bases. However, the same cannot be said for the structure in which amiloride is base-stacked with G. In this case, its fluorescence could be quenched dynamically by means of internal conversion because of the charge transfer interactions. The theoretical calculations agree well with the experimental trends in the emission spectra. 3.7. Cyclic voltammetry To confirm the thermodynamic feasibility of “hole” transfer mechanism for the fluorescence quenching of amiloride with G flanking bases, the cyclic voltammetry experiment was carried out. The reduction potential (Ered) of amiloride in neutral solution was found to be Ered = −1.19 V vs. Ag/AgCl. The λemi of amiloride is 415 nm. Using the equation E*red = Ered + ΔE0–0, the excited-state reduction potential (E*red) for amiloride was calculated, to be 1.80 V vs. Ag/AgCl. The oxidation potentials of guanine and thymine were reported to be 1.27 and 1.89 V vs. Ag/AgCl respectively [54]. The excited-state reduction potential of amiloride is higher than that of the oxidation potential of guanine, while it is lower than the oxidation potential of thymine. Hence, the interaction between amiloride and G may involve redox activity, while the redox interaction with T will not be favorable. Further, the standard free energy change calculated using Rehm–Weller equation [55] is negative for guanine. Hence the redox interaction is thermodynamically feasible. Though the “hole” transfer quenching mechanism in the excited state is possible for the quenching of amiloride fluorescence, there is an additional possibility for the reduction of its emission intensity. As it was observed in the absorption, NMR spectral studies and molecular modeling, there is a ground state interaction between the guanidinium group of amiloride and the phosphate backbone at the AP site. This interaction may change the absorptivity of amiloride and can affect its emission intensity. Due to the limitations in the computation, the phosphate interaction is omitted during the TDDFT calculations. In order to find out whether the quenching is mainly caused by the guanidinium–phosphate interaction in the ground state or only due to the “hole” transfer in the excited state, the results are compared with DCPC, a similar molecule which lacks the guanidinium group of amiloride. The fluorescence behavior of DCPC in DNA is similar to that of amiloride. Its fluorescence is quenched with G flanking bases while it is enhanced with T flanking [22]. Hence, it could be the “hole” transfer which mainly dominates the quenching process of the emission of amiloride. 4. Conclusions We investigated the effect of flanking bases on the simultaneous recognition of nucleobase and the phosphates around AP site. From the combined experimental and theoretical investigation, it was found that the flanking bases have strong influence on the ground and excited-state interactions between nucleobase–ligand–AP site phosphates. From UV-visible spectral trends and binding constant measurements, it was observed that the ground state interactions of amiloride with DNA of different flanking bases are different. The effect of base-stacking on the recognition of the phosphates around AP site was investigated by 31P NMR experiments. Different H-bonding patterns between the guanidinium group and AP site phosphate were observed with different stacking environment. The thermodynamic feasibility of the complex formation of the ligand–nucleotide was investigated by ITC measurements and the results suggest that the ligand is relatively more stable inside the AP site when the flanking bases were G. The ground state interactions of amiloride were well characterized by molecular modeling. Because of the palindromic alignment, amiloride adopts the binding mode (Ha) with G flanking bases and the guanidinium moiety remains in the major groove. While it prefers the (Hb) mode with T flanking and the

609

guanidinium moiety lies in the minor groove. Moreover, it is the 3' base which is responsible for the stabilization or the repulsion of the guanidinium moiety in the major groove, which in turn leads to different binding patterns of amiloride with the same target base. Not only the molecular level interactions, but also the electronic properties of amiloride are strongly dependent on the bases flanking the AP site. Its fluorescence is enhanced when it base-stacked with T, while it is quenched with G. In order to characterize the excited state properties of amiloride, TDDFT calculations were carried out on the H-bonded and base-stacked complexes. The results suggest that H-bonding and base-stacking of T do not have a major influence on the fluorescence properties of amiloride; indeed, the fluorescence transition predicted for the complexes is just that of isolated amiloride. In contrast, base-stacking with G is predicted to alter the nature of the amiloride emission. In this case, the fluorescence could be quenched dynamically by means of internal conversion because of the CT state. In the absence of any quenching process, the expected reason for the fluorescence enhancement with T flanking bases is the reduction in radiationless decay upon binding to the DNA duplex. The various noncovalent interactions (such as H-bonding, basestacking, electrostatic, CT, CH/π, and cation/π interactions, and CH/ cation repulsion) have been well characterized for the determination of nucleic acid structure and dynamics, and protein–DNA interactions. However, these are not clear for the DNA–small molecule interactions and hence we believe that our studies will bring a new insight into such phenomena.

Acknowledgments This work was partly supported by Grants-in-Aid for Scientific Research (A) and (B) (No. 17205009 and No. 18350039, respectively), and for the GCOE Project “Molecular Complex Chemistry” from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Gaussian03 calculations were carried out using the resources at Information Synergy Center, Tohoku University. References [1] J.D. Watson, T.A. Baker, S.P. Bell, A. Gann, M. Levine, R. Losick, Molecular biology of the gene, 5th ed.Benjamin-Cummings, San Francisco, 2003. [2] B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, P. Walter, Molecular biology of the cell, 4th ed.Garland Science, New York, 2002. [3] S. Sivakova, S.J. Rowan, Nucleobases as supramolecular motifs, Chem. Soc. Rev. 34 (2005) 9–21. [4] S. Aoki, E. Kimura, Zinc–nucleic acid interaction, Chem. Rev. 104 (2004) 769–788. [5] G. Cooke, V.M. Rotello, Methods of modulating hydrogen bonded interactions in synthetic host–guest systems, Chem. Soc. Rev. 31 (2002) 275–286. [6] K.S. Jeong, J. Rebek Jr, Molecular recognition: hydrogen bonding and aromatic stacking converge to bind cytosine derivatives, J. Am. Chem. Soc. 110 (1988) 3327–3328. [7] S. Goswami, D.V. Engen, A.D. Hamilton, Nucleotide base recognition: a macrocyclic receptor for adenine employing hydrogen bonding and aromatic stacking interactions, J. Am. Chem. Soc. 111 (1989) 3425–3426. [8] H. Ogoshi, H. Hatakeyama, J. Kotani, A. Kawashima, Y. Kuroda, New mode of porphyrin complexation with nucleobase, J. Am. Chem. Soc. 113 (1991) 8181–8183. [9] M.M. Conn, G. Deslongchamps, J. de. Mendoza, J. Jr. Rebek, Convergent functional groups. 13. High-affinity complexation of adenosine derivatives within induced binding pockets, J. Am. Chem. Soc. 115 (1993) 3548–3557. [10] T.K. Park, J. Schroeder, J. Rebek Jr., Convergent functional groups XI. Selective binding of guanosine derivatives, Tetrahedron 47 (1991) 2507–2518. [11] W.S. Yeo, J.I. Hong, Thiouronium-thymine conjugate as a new carrier for selective transport of 5′-AMP, Tetrahedron Lett. 39 (1998) 3769–3772. [12] S.E. Schneider, S.N. O'Neil, E.V. Anslyn, Coupling rational design with libraries leads to the production of an ATP selective chemosensor, J. Am. Chem. Soc. 122 (2000) 542–543. [13] M.W. Hosseini, A.J. Blacker, J.M. Lehn, Multiple molecular recognition and catalysis. A multifunctional anion receptor bearing an anion binding site, an intercalating group, and a catalytic site for nucleotide binding and hydrolysis, J. Am. Chem. Soc. 112 (1990) 3896–3904. [14] J.Y. Kwon, N.J. Singh, H.N. Kim, S.K. Kim, K.S. Kim, J. Yoon, Fluorescent GTP-sensing in aqueous solution of physiological pH, J. Am. Chem. Soc. 126 (2004) 8892–8893.

610

A. Rajendran et al. / Biochimica et Biophysica Acta 1800 (2010) 599–610

[15] F. Mancin, J. Chin, An artificial guanine that binds cytidine through the cooperative interaction of metal coordination and hydrogen bonding, J. Am. Chem. Soc. 124 (2002) 10946–10947. [16] S.M. Butterfield, M.M. Sweeney, M.L. Waters, The recognition of nucleotides with model ß-Hairpin receptors: investigation of critical contacts and nucleotide selectivity, J. Org. Chem. 70 (2005) 1105–1114. [17] K. Nakatani, S. Sando, I. Saito, Scanning of guanine-guanine mismatches in DNA by synthetic ligands using surface plasmon resonance, Nat. Biotechnol. 19 (2001) 51–55. [18] K. Nakatani, S. Sando, I. Saito, Recognition of a single guanine bulge by 2acylamino-1, 8-naphthyridine, J. Am. Chem. Soc. 122 (2000) 2172–2177. [19] K. Yoshimoto, S. Nishizawa, M. Minagawa, N. Teramae, Use of abasic sitecontaining DNA strands for nucleobase recognition in water, J. Am. Chem. Soc. 125 (2003) 8982–8983. [20] K. Yoshimoto, C.Y. Xu, S. Nishizawa, T. Haga, H. Satake, N. Teramae, Fluorescence detection of guanine-adenine transition by a hydrogen bond forming small compound, Chem. Commun. 24 (2003) 2960–2961. [21] B. Rajendar, A. Rajendran, Y. Sato, S. Nishizawa, N. Teramae, Effect of methyl substitution in a ligand on the selectivity and binding affinity for a nucleobase: a case study with isoxanthopterin and its derivatives, Bioorg. Med. Chem. 17 (2009) 351–359. [22] C. Zhao, A. Rajendran, Q. Dai, S. Nishizawa, N. Teramae, A pyrazine-based fluorescence-enhancing ligand with a high selectivity for thymine in AP sitecontaining DNA duplexes, Anal. Sci. 24 (2008) 693–695. [23] K. Muller-Dethlefs, P. Hobza, Noncovalent interactions: a challenge for experiment and theory, Chem. Rev. 100 (2000) 143–168. [24] P.B. Dervan, B.S. Edelson, Recognition of the DNA minor groove by pyrroleimidazole polyamides, Curr. Opin. Struct. Biol. 13 (2003) 284–299. [25] P. Hobza, J. Sponer, Structure, energetics, and dynamics of the nucleic acid base pairs: nonempirical ab initio calculations, Chem. Rev. 99 (1999) 3247–3276. [26] C. Zhao, Q. Dai, T. Seino, Y.Y. Cui, S. Nishizawa, N. Teramae, Strong and selective binding of amiloride to thymine base opposite AP sites in DNA duplexes: simultaneous binding to DNA phosphate backbone, Chem. Commun. 11 (2006) 1185–1187. [27] Y. Liang, Applications of isothermal titration calorimetry in protein folding and molecular recognition, J. Iranian Chem. Soc. 3 (2006) 209–219. [28] J. Wu, F. Du, P. Zhang, I.A. Khan, J. Chen, Y. Liang, Thermodynamics of the interaction of aluminum ions with DNA: implications for the biological function of aluminum, J. Inorg. Biochem. 99 (2005) 1145–1154. [29] A. Rajendran, V. Thiagarajan, B. Rajendar, S. Nishizawa, N. Teramae, Simultaneous recognition of nucleobase and sites of DNA damage: effect of tethered cation on the binding affinity, Biochim. Biophys. Acta 1790 (2009) 95–100. [30] J.D. Puglisi, I. Jr Tinoco, Absorbance melting curves of RNA, Methods Enzymol. 180 (1989) 304–325. [31] T. Wiseman, S. Williston, J.F. Brandts, L.N. Lin, Rapid measurement of binding constants and heats of binding using a new titration calorimeter, Anal. Biochem. 179 (1989) 131–137. [32] M.E. Casida, in: D.P. Chong (Ed.), In recent advances in density functional methods, 1, World Scientific, Singapore, 1995. [33] MacroModel 9.0, Schrödinger, Inc., L. L. C., (www.schrodinger.com) New York, NY, USA, 2005. [34] F. Mohamadi, N.G.J. Richards, W.C. Guida, R. Liskamp, M. Lipton, C. Caufield, G. Chang, T. Hendrickson, W.C. Still, Macromodel—an integrated software system for modeling organic and bioorganic molecules using molecular mechanics, J. Comput. Chem. 11 (1990) 440–467. [35] A. Rajendran, B.U. Nair, Unprecedented dual binding behaviour of acridine group of dye: a combined experimental and theoretical investigation for the development of anticancer chemotherapeutic agents, Biochim. Biophys. Acta 1760 (2006) 1794–1801. [36] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T.

[37] [38] [39]

[40]

[41]

[42]

[43]

[44] [45]

[46]

[47]

[48] [49] [50]

[51] [52]

[53]

[54]

[55]

Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian, J.B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg, V.G. Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M.W. Wong, C. Gonzalez, J.A. Pople, Gaussian 03, Revision D.01, Gaussian, Inc., Wallingford CT, 2004. J.M. Jean, K.B. Hall, 2-Aminopurine fluorescence quenching and lifetimes: role of base stacking, Proc. Natl. Acad. Sci. USA 98 (2001) 37–41. J.M. Jean, K.B. Hall, 2-Aminopurine electronic structure and fluorescence properties in DNA, Biochemistry 41 (2002) 13152–13161. V. Barone, M. Cossi, Quantum calculation of molecular energies and energy gradients in solution by a conductor solvent model, J. Phys. Chem. A 102 (1998) 1995–2001. G. Schaftenaar, J.H. Noordik, Molden: a pre- and post-processing program for molecular and electronic structures, J. Comput.-Aided Mol. Des. 14 (2000) 123–134. H.P. Jr, J. Fumero Hopkins, W.D. Wilson, Temperature dependence of enthalpy changes for ehtidium and propidium binding to DNA: effect of alkylamine chains, Biopolymers 29 (1990) 449–459. S. Nishizawa, Y. Kato, N. Teramae, Fluorescence sensing of anions via intramolecular excimer formation in a pyrophosphate-induced self-assembly of a pyrenefunctionalized guanidinium receptor, J. Am. Chem. Soc. 121 (1999) 9463–9464. C. Biot, R. Wintjens, M. Rooman, Stair motifs at protein–DNA interfaces: nonadditivity of H-bond, stacking, and cation-π interactions, J. Am. Chem. Soc. 126 (2004) 6220–6221. S. Wang, E.T. Kool, Origins of the large differences in stability of DNA and RNA helixes: C-5 methyl and 2′-hydroxyl effects, Biochemistry 34 (1995) 4125–4132. M.A. Viswamitra, O. Kennard, P.G. Jones, G.M. Sheldrick, S.A. Salisbury, L. Falvello, Z. Shakked, DNA double helical fragment at atomic resolution, Nature 273 (1978) 687–688. M.A. Viswamitra, Z. Shakked, P.G. Jones, G.M. Sheldrick, S.A. Salisbury, O. Kennard, Structure of the deoxytetranucleotide d-pApTpApT and a sequence-dependent model for poly (dA-dT), Biopolymers 21 (1982) 513–533. A. Klug, A. Jack, M.A. Viswamitra, O. Kennard, Z. Shakked, T.A. Steitz, A hypothesis on a specific sequence-dependent conformation of DNA and its relation to the binding of the lac-repressor protein, J. Mol. Biol. 131 (1979) 669–680. M. Nishio, M. Hirota, U. Umezawa, The CH/π interaction, Wiley-VCH, New York, 1998. A comprehensive literature list regarding the CH/π interaction, http://www.tim. hi-ho.ne.jp/dionisio. R. Wintjens, C. Biot, M. Rooman, J. Lievin, Basis set and electron correlation effects on ab initio calculations of cation-π/H-bond stair motifs, J. Phys. Chem. A 107 (2003) 6249–6258. M. Rooman, J. Lievin, E. Buisine, R. Wintjens, Cation-π/H-bond stair motifs at protein–DNA interfaces, J. Mol. Biol. 319 (2002) 67–76. G. Ricciardi, A. Rosa, S.J.A. van Gisbergen, E.J. Baerends, A density functional study of the optical spectra and nonlinear optical properties of heteroleptic tetrapyrrole sandwich complexes: the porphyrinato–porphyrazinato–zirconium(IV) complex as a case study, J. Phys. Chem. A 104 (2000) 635–643. S.J.O. Hardman, K.C. Thompson, Influence of base stacking and hydrogen bonding on the fluorescence of 2-aminopurine and pyrrolocytosine in nucleic acids, Biochemistry 45 (2006) 9145–9155. C.A.M. Seidel, A. Schulz, M.H.M. Sauer, Nucleobase-specific quenching of fluorescent dyes. 1. Nucleobase one-electron redox potentials and their correlation with static and dynamic quenching efficiencies, J. Phys. Chem. 100 (1996) 5541–5553. R.A. Bissell, A.P. de Silva, H.Q.N. Gunaratne, P.L.M. Lynch, G.E.M. Maguire, C.P. McCoy, K.R.A.S. Sandanayake, Fluorescent PET (photoinduced electron transfer) sensors, Topics in Current Chemistry, 168, Springer, Berlin, 1993, pp. 223–264.