Molecular interaction of ctDNA and HSA with sulfadiazine sodium by multispectroscopic methods and molecular modeling

Interactions of sulfadiazine sodium (SD‐Na) with calf thymus DNA (ctDNA) and human serum albumin (HSA) were studied using fluorescence spectroscopy, UV absorption spectroscopy and molecular modeling. The fluorescence experiments showed that the processes were static quenching. The results of UV spectra and molecular modeling of the interaction between SD‐Na and ctDNA indicated that the binding mode might be groove binding. In addition, the interaction of SD‐Na with HSA under simulative physiological conditions was also investigated. The binding constants (K) and the number of binding sites (n) at different temperatures (292, 302, 312 K) were 5.23 × 10³ L/mol, 2.18; 4.50 × 10³ L/mol, 2.35; and 4.08 × 10³ L/mol, 2.47, respectively. Thermodynamic parameters including enthalpy change (ΔH) and entropy change (ΔS) were calculated, the results suggesting that hydrophobic force played a very important role in SD‐Na binding to HSA, which was in good agreement with the molecular modeling study. Moreover, the effect of SD‐Na on the conformation of HSA was analyzed using three‐dimensional fluorescence spectra. Copyright © 2013 John Wiley & Sons, Ltd.     

Protoberberine Alkaloids Berberine,Palmatine, and Coralyne Binding to Poly(dT)⋅(Poly(dA)⋅Poly(dT)) Triplex: Comparative Structural Aspects and Energetics Profiles of the Interaction

The interaction of bioactive protoberberine alkaloids berberine, palmatine, and coralyne with the DNA triplex poly(dT)⋅(poly(dA)⋅poly(dT)) was studied using biophysical and calorimetric techniques. All three alkaloids bound the triplex cooperatively. Berberine and palmatine predominantly stabilized the triplex structure, while coralyne stabilized both triplex and duplex structures as inferred from optical thermal melting profiles. Fluorescence quenching, polarization, and viscometric studies hinted at an intercalative mode of binding for the alkaloids to the triplex, coralyne being more strongly intercalated compared to partial intercalation of berberine and palmatine. The overall affinity of coralyne was two order higher (2.29×10⁷ M ⁻¹) than that of berberine (3.43×10⁵ M ⁻¹) and palmatine (2.34×10⁵ M ⁻¹). Isothermal titration calorimetric studies revealed that the binding to the triplex was favored by negative enthalpy change (ΔH=−3.34 kcal/mol) with favorable entropy contribution (TΔS = 4.07 kcal/mol) for berberine, favored by almost equal negative enthalpy (ΔH =−3.88 kcal/mol) and entropy changes (TΔS = 3.37 kcal/mol) for palmatine, but driven by large enthalpy contributions (ΔH =−25.62 kcal/mol and TΔS =−15.21 kcal/mol) for coralyne. These results provide new insights on the binding of isoquinoline alkaloids to the DNA triplex structure.     

Combined spectroscopic and molecular docking approach to probing binding interactions between lovastatin and calf thymus DNA

The binding interaction of lovastatin with calf thymus DNA (ct‐DNA) was studied using UV/Vis absorption spectroscopy, fluorescence emission spectroscopy, circular dichroism (CD), viscosity measurement and molecular docking methods. The experimental results showed that there was an obvious binding interaction of lovastatin with ct‐DNA and the binding constant (K_b) was 5.60 × 10³ M^–1 at 298 K. In the binding process of lovastatin with ct‐DNA, the enthalpy change (ΔH⁰) and entropy change (ΔS⁰) were –24.9 kJ/mol and –12.0 J/mol/K, respectively, indicating that the main binding interaction forces were van der Waal's force and hydrogen bonding. The molecular docking results suggested that lovastatin preferred to bind on the minor groove of different B‐DNA fragments and the conformation change of lovastatin in the lovastatin–DNA complex was obviously observed, implying that the flexibility of lovastatin molecule plays an important role in the formation of the stable lovastatin–ct‐DNA complex. Copyright © 2015 John Wiley & Sons, Ltd.     

Understanding nucleosomal histone and DNA interactions: a biophysical study

Histones are associated with DNA to form nucleosome essential for chromatin structure and major nuclear processes like gene regulation and expression. Histones consist of H1, H2A, H2B and H3, H4 type proteins. In the present study, combined histones from calf thymus were complexed with ct DNA and their binding affinities were measured fluorimetrically. All the five histones were resolved on SDS page and their binding with DNA was visualized. The values of biding affinities varied with pH and salt concentration. Highest affinity (4.0?×?10⁵ M^?1) was recorded at pH 6.5 in 50 mM phosphate buffer and 1.5?×?10⁴ M^?1 in 2 M NaCl at pH 7.0. The CD spectra support the highest binding affinity with maximum conformational changes at pH 7.0. The time-resolved fluorescence data recorded two life times for histone tyrosine residues at 300 nm emission in phosphate buffer pH 6.5. These life times did not show much change upon binding with DNA in buffer as well as in 2 M NaCl. The isothermal calorimetric studies yielded thermodynamic parameters ΔG, ΔH and ΔS as ?1.6?×?10⁵ cal/mol, ?1.13?×?10³ cal/mol and ?3.80 cal/mol/deg, respectively, evidencing a spontaneous exothermic reaction. The dominant binding forces in building the nucleosome are electrostatic interactions.     

Exploring the binding mode of donepezil with calf thymus DNA using spectroscopic and molecular docking methods

Donepezil (DNP) is one of approved drugs to treat Alzheimer's disease (AD). However, the potential effect of DNP on DNA is still unclear. Therefore, the interaction of DNP with calf thymus DNA (DNA) was studied in vitro using spectroscopic and molecular docking methods. Steady‐state and transient fluorescence experiments showed that there was a clear binding interaction between DNP and DNA, resulting from DNP fluorescence being quenched using DNA. DNP and DNA have one binding site between them, and the binding constant (K_b) was 0.78 × 10⁴ L·mol^?1 at 298 K. In this binding process, hydrophobic force was the main interaction force, because enthalpy change (ΔH) and entropy change (ΔS) of DNP–DNA were 67.92 kJ·mol^?1 and 302.96 J·mol^?1·K^?1, respectively. DNP bound to DNA in a groove‐binding mode, which was verified using a competition displacement study and other typical spectroscopic methods. Fourier transform infrared (FTIR) spectrum results showed that DNP interacted with guanine (G) and cytosine (C) bases of DNA. The molecular docking results further supported the results of spectroscopic experiments, and suggested that both Pi‐Sigma force and Pi‐Alkyl force were the major hydrophobic force functioning between DNP and DNA.     

Evaluation of DNA,BSA binding,and antimicrobial activity of new synthesized neodymium complex containing 29-dimethyl 110-phenanthroline

In order to evaluate biological potential of a novel synthesized complex [Nd(dmp)₂Cl₃.OH₂] where dmp is 29-dimethyl 110-phenanthroline, the DNA-binding, cleavage, BSA binding, and antimicrobial activity properties of the complex are investigated by multispectroscopic techniques study in physiological buffer (pH 7.2).The intrinsic binding constant (K_b) for interaction of Nd(III) complex and FS–DNA is calculated by UV–Vis (K_b = 2.7 ± 0.07 × 10⁵) and fluorescence spectroscopy (K_b = 1.13 ± 0.03 × 10⁵). The Stern–Volmer constant (K_SV), thermodynamic parameters including free energy change (ΔG°), enthalpy change (?H°), and entropy change (?S°), are calculated by fluorescent data and Vant’ Hoff equation. The experimental results show that the complex can bind to FS–DNA and the major binding mode is groove binding. Meanwhile, the interaction of Nd(III) complex with protein, bovine serum albumin (BSA), has also been studied by using absorption and emission spectroscopic tools. The experimental results show that the complex exhibits good binding propensity to BSA. The positive ΔH° and ?S° values indicate that the hydrophobic interaction is main force in the binding of the Nd(III) complex to BSA, and the complex can quench the intrinsic fluorescence of BSA remarkably through a static quenching process. Also, DNA cleavage was investigated by agarose gel electrophoresis that according to the results cleavage of DNA increased with increasing of concentration of the complex. Antimicrobial screening test gives good results in the presence of Nd(III) complex system.     

Characterization of the binding of neomycin/paromomycin sulfate with DNA using acridine orange as fluorescence probe and molecular docking technique

The binding of neomycin sulfate (NS)/paromomycin sulfate (PS) with DNA was investigated by fluorescence quenching using acridine orange (AO) as a fluorescence probe. Fluorescence lifetime, FT-IR, circular dichroism (CD), relative viscosity, ionic strength, DNA melting temperature, and molecular docking were performed to explore the binding mechanism. The binding constant of NS/PS and DNA was 6.70 × 10³/1.44 × 10³ L mol^?1 at 291 K. The values of ΔH^θ, ΔS^θ, and ΔG^θ suggested that van der Waals force or hydrogen bond might be the main binding force between NS/PS and DNA. The results of Stern–Volmer plots and fluorescence lifetime measurements all revealed that NS/PS quenching the fluorescence of DNA–AO was static in nature. FT-IR indicated that the interaction between DNA and NS/PS did occur. The relative viscosity and melting temperature of DNA were almost unchanged when NS/PS was introduced to the solution. The fluorescence intensity of NS/PS–DNA–AO was decreased with the increase in the ionic strength. For CD spectra of DNA, the intensity of positive band at nearly 275 nm was decreased and that of negative band at nearly 245 nm was increased with the increase in the concentration of NS/PS. The binding constant of NS/PS with double-stranded DNA (dsDNA) was larger than that of NS/PS with single-stranded DNA (ssDNA). From these studies, the binding mode of NS/PS with DNA was evaluated to be groove binding. The results of molecular docking further indicated that NS/PS could enter into the minor groove in the A–T rich region of DNA.     

Molecular aspects on adriamycin interaction with hmga1 regulatory region and its inhibitory effect on HMGA1 expression in human cervical cancer

High mobility group A1 (HMGA1), a non-histone chromosomal protein, is highly expressed in a wide range of human cancers including cervical, breast, and prostate cancers. Therefore, hmga1 gene is considered as an attractive potential target for anticancer drugs. We have chosen 27 bp DNA sequence from a regulatory region of hmga1 promoter and studied its interaction with adriamycin (ADM) and in vitro expression of HMGA1 in the presence of ADM in HeLa cell line. A variety of biophysical techniques were employed to understand the characteristics of [DNA–ADM] complex. Spectrophotometric titration data, DNA denaturation profiles, and quenching of fluorescence of ADM in the presence of DNA demonstrated a strong complexation between DNA and ADM with a high binding affinity (Ka) of 1.3 × 10⁶ M^?1 and a stoichiometry of 1:3 (drug:nucleotide). The energetics of binding obtained from isothermal titration calorimetry and differential scanning calorimetry suggest the binding to be exothermic and enthalpy (?H, ?6.7 ± 2.4 kcal M^?1) and entropy (TΔS, 18.5 ± 6.4 kcal M^?1) driven (20°C), which is typical of intercalative mode of binding. Further, results on decreased expression (by ~70%) of HMGA1 both at mRNA and protein levels in association with the observed cell death (by ~75%) in HeLa cell line, clearly confirm that ADM does target hmga1; however, the effect of ADM on genes other than hmga1 either directly or via hmga1-mediated pathways cannot be ruled out in the observed cytotoxicity. Therefore, hmga1 in general and particularly the regulatory region is a promising target for therapeutic strategy in combating cancer.     

Studies on the interaction between vincamine and human serum albumin: a spectroscopic approach

The interaction between vincamine (VCM) and human serum albumin (HSA) has been studied using a fluorescence quenching technique in combination with UV/vis absorption spectroscopy, Fourier transform infrared (FT–IR) spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling under conditions similar to human physiological conditions. VCM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding constants were calculated from the fluorescence data. Thermodynamic analysis by Van't Hoff equation revealed enthalpy change (ΔH) and entropy change (ΔS) were ?4.57 kJ/mol and 76.26 J/mol/K, respectively, which indicated that the binding process was spontaneous and the hydrophobic interaction was the predominant force. The distance r between the donor (HSA) and acceptor (VCM) was obtained according to the Förster's theory of non‐radiative energy transfer and found to be 4.41 nm. Metal ions, viz., Na⁺, K⁺, Li⁺, Ni²⁺, Ca²⁺, Zn²⁺ and Al³⁺ were found to influence binding of the drug to protein. The 3D fluorescence, FT–IR and CD spectral results revealed changes in the secondary structure of the protein upon interaction with VCM. Furthermore, molecular modeling indicated that VCM could bind to the subdomain IIA (site I) of HSA. Copyright © 2013 John Wiley & Sons, Ltd.     

Investigation on the interaction of letrozole with herring sperm DNA through spectroscopic and modeling methods

The interaction of letrozole, an efficient and safe aromatase inhibitor, with herring sperm DNA (hsDNA) was investigated in vitro through spectroscopy analysis and molecular modeling to elucidate the binding mechanism of anticancer drugs and DNA. The binding constant and the number of binding sites were 2.13 × 10⁴ M^?1 and 1.09, respectively, at 298 K. Thermodynamic parameters (ΔG, ΔH and ΔS) exhibited negative values, which indicated that binding was spontaneous and Van der Waals forces and hydrogen bond were the main interaction forces. Fourier transform infrared spectroscopy and other spectroscopy analysis methods illustrated that letrozole could intercalate into the phosphate backbone of hsDNA and interact with the nitrogenous bases. Consistent with the experimental findings, molecular modeling results demonstrated that the interaction was dominated by intercalation and hydrogen bonding. Copyright © 2015 John Wiley & Sons, Ltd.     

Fluorescent bovine serum albumin interacting with the antitussive quencher dextromethorphan: a spectroscopic insight

The interaction of dextromethorphan hydrobromide (DXM) with bovine serum albumin (BSA) is studied by using fluorescence spectra, UV–vis absorption, synchronous fluorescence spectra (SFS), 3D fluorescence spectra, Fourier transform infrared (FTIR) spectroscopy and circular dichroism under simulated physiological conditions. DXM effectively quenched the intrinsic fluorescence of BSA. Values of the binding constant, K_A, are 7.159 × 10³, 9.398 × 10³ and 16.101 × 10³ L/mol; the number of binding sites, n, and the corresponding thermodynamic parameters ΔG°, ΔH° and ΔS° between DXM and BSA were calculated at different temperatures. The interaction between DXM and BSA occurs through dynamic quenching and the effect of DXM on the conformation of BSA was analyzed using SFS. The average binding distance, r, between the donor (BSA) and acceptor (DXM) was determined based on Förster's theory. The results of fluorescence spectra, UV–vis absorption spectra and SFS show that the secondary structure of the protein has been changed in the presence of DXM. Copyright © 2015 John Wiley & Sons, Ltd.     

The binding characteristics of the interaction between 3-(2-cyanoethyl) cytosine and human serum albumin

The binding characteristics of the interaction between 3-(2-cyanoethyl) cytosine (CECT) and human serum albumin (HSA) were investigated using fluorescence, UV absorption spectroscopic and molecular modeling techniques under simulative physiological conditions. The intrinsic fluorescence intensity of HSA was decreased with the addition of CECT. The fluorescence data handled by Stern–Volmer equation proved that the quenching mechanism of the interaction between CECT and HSA was a static quenching procedure. The binding constants evaluated utilizing the Lineweaver–Burk equation at 17, 27 and 37?°C, were 2.340?×?10⁴, 2.093?×?10⁴ and 1.899?×?10⁴?L?mol^?1, respectively. The thermodynamic parameters were calculated according to van’t Hoff equations. Negative enthalpy (ΔH) and positive entropy (ΔS) values indicated that both hydrogen bond and hydrophobic force played a major role in the binding process of CECT to HSA, which was consistent with the results of the molecular modeling study. In addition, the effect of other ions on the binding constant of CECT-HSA was examined.     

Multivariate spectrochemical analysis of interactions of three common Isatin derivatives to calf thymus DNA in vitro

Interactions of Isatin and its derivatives, Isatin-3-isonicotinylhydrazone (IINH) and Isatin-β-thiosemicarbazone (IBT), with calf thymus DNA (ctDNA) have been investigated to delineate pharmaceutical-physicochemical properties using UV–Vis/fluorescence/circular dichroism (CD) spectroscopy, viscosity measurements, and multivariate chemometrics. IINH and IBT molecules intercalate between base pairs of DNA, hypochromism in UV absorptions, increase in the CD positive band, sharp increase in specific viscosity, and the displacement of the methylene blue and Neutral Red dye in complexes with ctDNA, by the IINH and IBT molecules, respectively. The observed intrinsic binding constants (K_{b[IBT–ctDNA]?}=?1.03 × 10⁵ and K_{b[IINH–ctDNA]?}=?1.09 × 10⁵ L mol^?1) were roughly comparable to other intercalators. In contrast, Isatin binds with ctDNA via groove mode (K_{b[Isatin–ctDNA]?}=?7.32 × 10⁴ L mol^?1) without any significant enhancement in ctDNA viscosity. The fluorescence quenching of Isatin by ctDNA was observed as static. CD spectra indicated that Isatin effectively absorbs into grooves of ctDNA, leading to transition from B to C form. Thermodynamic parameters like enthalpy changes (?H < 0) and entropy changes (?S > 0) were calculated according to Van’t Hoff’s equation, indicating the spontaneous interactions. The common soft/hard chemometric methods were used not only to resolve pure concentration and spectral profiles of components using the acquired spectra but also to calculate Stern–Volmer quenching constants, binding stoichiometry, apparent binding constants (K_a), binding constants (K_b), and thermodynamic parameters. The K_b values for Isatin, IINH, and IBT were calculated as 9.18 × 10³, 1.53 × 10⁵, and 2.45 × 10⁴ L mol^?1, respectively. The results obtained from experimental-spectroscopic analyses showed acceptable agreement with chemometric outlines.     

Synthesis and biological evaluation of a new dysprosium(III) complex containing 2,9-dimethyl 1,10-phenanthroline

The binding of [Dy(dmp)₂Cl₃(OH₂)], where dmp is 2,9-dimethyl 1,10-phenanthroline, with Fish salmon DNA (FS-DNA) is investigated by absorption and emission spectroscopy, quenching studies, salt dependent, and gel electrophoresis. The binding constant (K_b) of the interaction is calculated as (1.27 ± .05) × 10⁵ M^?1 from absorption spectral titration data. The Stern–Volmer constant (K_SV), thermodynamic parameters involves ΔG°, ?H°, and ?S° are calculated by fluorescent data and Van’t Hoff equation. The thermodynamic studies show that the reaction for the binding of the complex with FS-DNA is endothermic and entropically driven (ΔS° > 0, ΔH° > 0). The effect of the complex concentration on FS-DNA cleavage reactions is also investigated by gel electrophoresis. Furthermore, the Dy(III) complex has been screened for its antibacterial activity. The experimental results suggest that the Dy(III) complex binds significantly to FS-DNA by hydrophobic groove binding mode and the complex has more efficient antibacterial activity compared to its metal salt.     

Multi-spectroscopic and voltammetric evidences for binding,conformational changes of bovine serum albumin with thiamine

The interaction between thiamine hydrochloride (TA) and bovine serum albumin (BSA) was investigated by fluorescence, FTIR, UV–vis spectroscopic and cyclic voltammetric techniques under optimised physiological condition. The fluorescence intensity of BSA is gradually decreased upon addition of TA due to the formation of a BSA–TA complex. The binding parameters were evaluated and their behaviour at different temperatures was analysed. The quenching constants (K_sv) obtained were 2.6 × 10⁴, 2.2 × 10⁴ and 2.0 × 10⁴ L mol^?1 at 288, 298 and 308 K, respectively. The binding mechanism was static-type quenching. The values of ΔH° and ΔS° were found to be 26.87 kJ mol^?1 and 21.3 J K^?1 mol^?1, and indicated that electrostatic interaction was the principal intermolecular force. The changes in the secondary structure of BSA upon interaction with TA were confirmed by synchronous and 3-D spectral results. Site probe studies reveal that TA is located in site I of BSA. The effects of some common metal ions on binding of BSA–TA complex were also investigated.     

Titanium dioxide nanoparticles preferentially bind in subdomains IB,IIA of HSA and minor groove of DNA

Titanium dioxide nanoparticles (TiO₂-NPs) interaction with human serum albumin (HSA) and DNA was studied by UV–visible spectroscopy, spectrofluorescence, circular dichroism (CD), and transmission electron microscopy (TEM) to analyze the binding parameters and protein corona formation. TEM revealed protein corona formation on TiO₂-NPs surface due to adsorption of HSA. Intrinsic fluorescence quenching data suggested significant binding of TiO₂-NPs (avg. size 14.0 nm) with HSA. The Stern–Volmer constant (K_sv) was determined to be 7.6 × 10² M^?1 (r² = 0.98), whereas the binding constant (K_a) and number of binding sites (n) were assessed to be 5.82 × 10² M^?1 and 0.97, respectively. Synchronous fluorescence revealed an apparent decrease in fluorescence intensity with a red shift of 2 nm at Δλ = 15 nm and Δλ = 60 nm. UV–visible analysis also provided the binding constant values for TiO₂-NPs–HSA and TiO₂-NPs-DNA complexes as 2.8 × 10² M^?1 and 5.4 × 10³ M^?1. The CD data demonstrated loss in α-helicity of HSA and transformation into β-sheet, suggesting structural alterations by TiO₂-NPs. The docking analysis of TiO₂-NPs with HSA revealed its preferential binding with aromatic and non-aromatic amino acids in subdomain IIA and IB hydrophobic cavity of HSA. Also, the TiO₂-NPs docking revealed the selective binding with A-T bases in minor groove of DNA.     

Combined multispectroscopic and molecular docking investigation on the interaction between strictosamide and human serum albumin

The interaction between strictosamide (STM) and human serum albumin (HSA) was investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, three‐dimensional fluorescence spectroscopy, ultraviolet‐visible absorption spectroscopy, circular dichroism spectroscopy and molecular modeling under physiological pH 7.4. STM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding site number n and apparent binding constant K_a were determined at different temperatures by fluorescence quenching. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated as ?3.01 kJ/mol and 77.75 J/mol per K, respectively, which suggested that the hydrophobic force played major roles in stabilizing the HSA–STM complex. The distance r between donor and acceptor was obtained to be 4.10 nm according to Förster's theory. After the addition of STM, the synchronous fluorescence and three‐dimensional fluorescence spectral results showed that the hydrophobicity of amino acid residues increased and the circular dichroism spectral results showed that the α‐helix content of HSA decreased (from 61.48% to 57.73%). These revealed that the microenvironment and conformation of HSA were changed in the binding reaction. Furthermore, the study of molecular modeling indicated that STM could bind to site I of HSA and the hydrophobic interaction was the major acting force, which was in agreement with the binding mode study. Copyright © 2013 John Wiley & Sons, Ltd.     

Fluorescence spectrometric study on the interaction of tamibarotene with bovine serum albumin

The interaction between tamibarotene and bovine serum albumin (BSA) was studied using fluorescence quenching technique and ultraviolet–visible spectrophotometry. The results of experiments showed that tamibarotene could strongly quench the intrinsic fluorescence of BSA by a dynamic quenching mechanism. The apparent binding constant, number of binding site and corresponding thermodynamic parameters at different temperatures were calculated respectively, and the main interaction force between tamibarotene and BSA was proved to be hydrophobic force. Synchronous fluorescence spectra showed that tamibarotene changed the molecular conformation of BSA. When BSA concentration was 1.00 × 10^?6 mol L^?1, the quenched fluorescence ΔF had a good linear relationship with the concentration of tamibarotene in the range 1.00 × 10^?6 to 12.00 × 10^?6 mol L^?1 with the detection limit of 6.52 × 10^?7 mol L^?1. Copyright © 2010 John Wiley & Sons, Ltd.     

Recognition of a Flipped Base in a Hairpinloop DNA by a Small Peptide

Two tiny hairpin DNAs, CORE (dAGGCTTCGGCCT) and AP2 (dAGGCTXCGGCCT; X: abasic nucleotide), fold into almost the same tetraloop hairpin structure with one exception, that is, the sixth thymine (T6) of CORE is exposed to the solvent water (Kawakami, J. et al., Chem. Lett. 2001, 258–259). In the present study, we selected small peptides that bind to CORE or AP2 from a combinatorial pentapeptide library with 2.5 × 10⁶ variants. On the basis of the structural information, the selected peptide sequences should indicate the essential qualifications for recognition of the hairpin loop DNA with and without a flipped base. In the selected DNA binding peptides, aromatic amino acids such as histidine for CORE and glutamine/aspartic acid for AP2 were found to be abundant amino acids. This amino acid preference suggests that CORE-binding peptides use π–π stacking to recognize the target while hydrogen bonding is dominant for AP2-binding peptides. To investigate the binding properties of the selected peptide to the target, surface plasmon resonance was used. The binding constant of the interaction between CORE and a CORE-binding peptide (HWHHE) was about 1.1 × 10⁶ M^?1 at 25°C and the resulting binding free energy change at 25°C (ΔG°₂₅) was ?8.2 kcal mol^?1. The binding of the peptide to AP2 was also analyzed and the resulting binding constant and ΔG°₂₅ were about 4.2 × 10⁴ M^?1 and ?6.3 kcal mol^?1, respectively. The difference in the binding free energy changes (ΔΔG°₂₅) of 1.9 kcal mol^?1 was comparable to the values reported in other systems and was considered a consequence of the loss of π–π stacking. Moreover, the stabilization effect by stacking affected the dissociation step as well as the association step. Our results suggest that the existence of an aromatic ring (T6 base) produces new dominant interactions between peptides and nucleic acids, although hydrogen bonding is the preferable mode of interaction in the absence of the flipping base. These findings regarding CORE and AP2 recognition are expected to give useful information in the design of novel artificial DNA binding peptides.