Redox investigation of L-A9 peptide and evaluation of its binding with double stranded DNA

Abstract

L-A9 is one of the important short peptides having biomedical applications in relation to its affinity towards HER2 receptors. The peptide has been synthesised using manual solid phase synthesis protocol and the redox property has been investigated using the voltammetric techniques. The oxidation peak of the peptide has shown strong pH dependency, revealed the 1 e and 2 proton transfer process. Electroanalytical method is developed with limit of detection of 1.17 × 10⁻⁷ M. The modulation of the redox property has been monitored to investigate the binding of the peptide with dsDNA, with binding constant of 1.8 × 10⁵ M^− 1. Spectroscopic measurements of the peptide revealed the pH dependent electronic transition and the binding constant with dsDNA of 1.4 × 10⁴ M^− 1. The needle like assembly of L-A9 peptide has been indicated from the SEM measurements with coil kind of morphology of dsDNA has been observed when the L-A9 peptide and dsDNA interacted with each other. The electrochemical and spectroscopic investigations indicated that the acidic functional groups of L-A9 interacts with the major groove of dsDNA through the modifications of 3 hydrogen bonds between the strands, whereas the basic functional groups of L-A9 binds externally with dsDNA by formation of bonds with O atoms of the phosphodiester groups, which has been supported from the molecular docking investigations.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-30000-w.

Introduction

The development of peptides-based drugs and pharmaceuticals have made significant progress in last few decades due to the advancement in the synthesis, purification and analytical methods of their characterisation. Additionally, the improvised delivery strategy of the peptide-based drugs overcomes the challenges of the therapeutic usage of peptides. Insulin has been the most popular and important peptide-based drug dominated the market for over 90 years, which is now replaced by the recombinant insulin^1,2. Smaller sized drugs have the advantages as it is easy to administer which has enhanced patient compliance and penetration^3,4.

Additionally, the short peptides are mostly used due to their biological activities in immune reactions, in brain functions, blood pressure normalization and controlling the vascular tone. Short peptides are present in all tissues, and they are usually treated as “fragments” of functional proteins and having complex functions in biological systems⁵. The short peptides generally considered to have up to 100 amino acids, have the advantage of easy penetration through the cell membranes⁶. Importantly the short peptides can interact with DNA^7–910. The gene expression has strong influence on the controlled combination of DNA-binding proteins, which also has influence of the presence of metals¹¹. Metal ions play important role in modulating the interaction of the peptides with DNA, which influence important biological functions¹². Monoclonal antibodies were considered as the potential magic bullet for cancer treatment because of their antigen specific binding to the neoplastic cells. In the view of challenges posed by their high molecular weight, low penetration, risk of allergic reactions and long half-life¹³, researchers turned to antibody fragments as they retain the specific antigen binding capability while having more tailored half-life and better membrane penetration¹⁴. Antibody fragments offers several simultaneous benefits as they can be perfect drug carriers due to their specific antigen binding while it induces tumour cell apoptosis by interfering with signalling and manipulating immune system. More tailored treatment or imaging can be achieved by small peptide chains which are the principal antigen binding moieties in these antibody fragments. In the field of targeted cancer therapy, these tumour targeting peptides are emerging as appealing choice due to their small molecular weight, cost-effective synthesis, easy modification along with fewer off-target toxicity, high membrane penetration, low immunogenicity, specificity and high action potency^15,16.

In cancer cells, the mutation or alteration in normal functioning gene or proto-oncogenes results in overexpression of its protein product. Receptors found in these overexpressed neoplastic tissues are chosen as targets. In wide varieties of cancer e.g. breast, ovarian, lung, gastric, oropharynx, ovarian, colorectal and bladder, the human epidermal growth factor receptor 2 (HER2), a transmembrane receptor is found to be overexpressed in both primary as well as metastasized organs. Hence, HER2 targeting antibodies or peptides are of importance while developing new cancer drugs¹⁷. In this regard, A9 peptide, 9 amino acid peptide chain, represents a minimal sequence, derived from Herceptin antibody fragment and rationally designed from biomolecular HER2-Herceptin antigen-binding fragment (Fab). L-A9 specifically bind to extracellular domain IV of HER2 receptor^18–22 .

There have been studies relating to the efficacy of A9 in targeted cancer therapy and imaging. When A9 is conjugated to antineoplastic drug like doxorubicin, the cellular internalization and cytotoxicity was improved²³. Diethylenetriamine pentaacetate conjugated A9 peptide radiolabelled with ¹¹¹In has shown nanomolar affinity to HER2 overexpressed cells. However, the problem with ordinary peptides lies in their vulnerability towards enzymatic degradation and subsequent rapid renal clearance resulting in lower bioavailability and lower target accumulation which limits their clinical applicability. Hence, many researchers devise methods such as PEGylation, cyclization, retro-peptide synthesis etc. to confer metabolic stability²⁴.

Amino acids in small peptides that contain charges at the side chain, have the ability to bind to double stranded DNA (dsDNA), the strong electrostatic binding modify the double helical structure of the dsDNA. It has been reported that the Glu, Asp weaken the H-bonds between DNA strands, whereas amino acids, Arg and Lys, which are basic in nature are known to strengthen the interaction between the strands. The interactions of peptides and proteins with DNA are generally similar, which are essentially driven by the compositions of the amino acids in them. The peptide containing the Asp and Glu, which are negatively charged binds with DNA in major groove of double helix. Through such binding the double helix structure becomes more fragile and results in the separation of strands. These flexible strands of DNA initiate the genetic transcription and replication²⁵. The mechanisms of intermolecular interactions between amino acids and individual nucleic bases are through the hydrogen bonds²⁶. Thus, any probing of such interactions has the essence of the peptide and DNA binding. Therefore, the electrochemical techniques which has the capability to probe the interactions due to the difference in the electrostatic charges could be applied in evaluating the interactions between the peptides and dsDNA. For the redox active peptides, it would open new avenues while the evaluating the electrostatic interactions, groove binding or the intercalative bindings of the peptides with the dsDNA strands. Molecular docking investigation were carried to support the experimental findings on the sites of interactions between the peptide and dsDNA. There are no such investigations has been reported evaluating the interactions between the L-A9 peptides with dsDNA using the electrochemical probing.

The peptide L-A9 that has been used in the investigation is a nine-amino acid peptide which specifically binds to the HER2 receptor at the extracellular domain IV. The A9 sequence essentially is the minimal sequence derived from Herceptin Fab. The dissociation constant has been reasonably good. The bioactive A9 peptide is tagged with the ionic 7-sulfobenzofurazan as the fluorescent tag for the investigation in biological binding and migration^27–29. The design, synthesis, and validation of this peptide was previously described, as well as its application as a radiolabelled tracer for molecular imaging of HER2^18–20,30. Presently, the peptide was synthesized with the synthesis procedure as described in our previous publication^31,32. The electrochemical response of L-A9 peptide over the commercially available electrode is not significant for their investigation and developing method of analytical determination. In order to enhance the electrochemical response, glassy carbon (GC) electrode is modified with gold nano particles (AuNPs) and the AuNPs modified GC electrode has shown enhanced response, which is used to develop method of determination of the L-A9 peptide. The enhanced electrochemical signal was also utilised for the investigation of the interaction between the peptide and dsDNA. Electrochemical measurements are also supported by the UV-Vis spectral measurements and also from the FTIR and Raman measurements. The investigation thus provided comprehensive electrochemical information of the L-A9 peptide, developing the analytical method of its determination and exploring its interactions with dsDNA.

Experimental

Materials

The peptide, A9 was synthesized manually by Fmoc solid-phase peptide synthesis. The protected peptide sequence Gln(Trt)-Asp(OtBu)-Val-Asn(Trt)-Thr(tBu)-Ala-Val-Ala-Trp(Boc)-NH₂ was constructed on NovaSyn TGR resin. Amino acid couplings were performed by activation of Fmoc-amino acids (3 eq.) with O-(7-azabenzotriazol-l-yl)-N,N,N’,N’-tetramethyluroniumhexafluorophosphate (HATU) (3 eq.) and N, N’-diisopropylethylamine (DIPEA) (6 eq.) in dimethylformamide (DMF) followed by gentle rotation for 120 min. Fmoc-deprotection was carried out with 20% piperidine in DMF (v/v, 2 × 10 min). Coupling reactions were monitored by the colorimetric trinitrobenzenesulphonic acid (TNBS) test. The chelator was incorporated at N-terminus by coupling of DOTA-tris (t-Bu) ester (3 eq.) in presence of HATU and DIPEA. The peptide was finally cleaved from the resin with a cocktail mixture of trifluoroacetic acid (TFA)/triisopropylsilane (TIPS)/H₂O (95/2.5/2.5, v/v) wherein protecting groups were also removed. The crude peptide was precipitated by addition of diethyl ether. The precipitate as obtained was dissolved in water and washed with ether for three times. The crude peptide as obtained was purified using semi-preparative HPLC and then lyophilized to obtain white fluffy powder. MALDI-TOF (DOTA-A9); calculated for C₆₀H₉₃N₁₇O₂₁, 1388.48; found, 1388.517. Double-stranded DNA from calf thymus was also sourced from Sigma Aldrich, with 98% purity verified by SDS-PAGE and HPLC. Auric chloride (AuCl₃) with 96% purity and potassium nitrate (KNO₃) with ≥ 99% purity, both obtained from Sigma Aldrich for electrochemical analysis, and their high purity ensured minimal interference during deposition. Throughout the study, ultra-pure Milli-Q water (17.9 MΩ) was used to ensure the highest purity level for all the experimental procedures. Electrochemical measurements were carried out in a phosphate buffer system at pH 7 to maintain consistent conditions throughout the study. The chemical structure of the peptide in shown in Fig. 1.

Fig. 1.

Chemical structure of the L-A9 peptide molecules.

Procedure

The glassy carbon electrode (GCE) is first polished using a cloth sprinkled with 0.05 μm alumina. The electrode is thoroughly cleaned with ultra-pure water post-polishing to ensure complete removal of alumina particles and surface debris before air drying. Electrodeposition of gold nano particles (AuNPs) onto the GCE is carried out by applying − 0.6 V for 200 s under stirring conditions in the electrolyte solution. This modification enhances the sensor’s sensitivity and selectivity for detecting and investigating L-A9 and its interaction with dsDNA in an aqueous solution. The enhanced response from the AuNPs modified electrode over the bare GC electrode is shown from the CV measurements of potassium ferricyanide and the results are shown in Figure S1 of the supporting information. Once the solutions are prepared and stored, the fabrication of the AuNPs modified electrode and the generation of the electrochemical signals of the peptide and analytical determination takes only about 5 min. The GCE was commercially available electrode from CH instruments.

Apparatus

The electrochemical experiments were performed on a CHI 920D electrochemical workstation from CH Instruments, Inc., USA, configured in a three-electrode setup. The working electrode was a glassy carbon electrode (GCE) with a diameter of 3 mm, complemented by a platinum (Pt) wire that functioned as the auxiliary electrode, and a saturated calomel electrode (SCE) served as the reference electrode. The DPV experiments were executed with parameters of pulse amplitude 50 mV width of 0.05 s, a step potential of 10 mV. Electrochemical impedance spectroscopy (EIS) experiments were executed with an amplitude of 10 mV, scanning frequencies from 10 kHz to 0.01 Hz. For spectroscopic analysis, an Ocean Optics DH-2000-BAL served as the light source, alongside a QE 65,000 spectrophotometer with Spectra Suite software. FTIR was carried out using a Bruker Tensor-II model with a diamond crystal ATR setup. Zeta potential measurements were performed with an Anton Paar analyzer litesizer 500, and the morphological changes in peptides induced by dsDNA interaction were imaged using a Nanosurf AFM system controlled by a C-3000 unit. A Labocon biosafety cabinet, equipped with a 0.3 μm HEPA filter, was used to prepare AFM samples, ensuring contamination-free handling. Silanized mica sheets were used as substrates for the AFM scanning. The substrates, loaded with samples were dried under controlled laminar airflow in a biosafety cabinet maintained by a HEPA filter to prevent airborne contamination. The pH of the solutions was monitored and adjusted using an ELICO LI 614 pH analyzer. All electrochemical, spectroscopic, and microscopic analyses were performed under ambient conditions.

Results and discussion

Electrochemical and UV-Vis spectroscopic investigations of A9 peptide

The DPV investigations were carried out for L-A9 peptide at different pH, corresponding plots with variation of concentrations for acidic (pH 3), neutral (pH 7), and alkaline (pH 9) conditions, are shown in Fig. 2. The peptide showed the oxidation peak at around 0.25 V at pH 3. The peak potential is shifted towards less positive potentials with an increase in pH of the solution, indicating the easy oxidation possibility of the peptide at higher pH values. The measured peak potentials are plotted with concentrations of the peptide, and the results are shown in Fig. 2D. The linear fitting of the peak potential vs. the pH of the test solution generated a slope of 25 mV, indicating that for every one-unit change in the proton concentration, the potential is changing double, therefore the reaction involves a two-electron and the one proton transfer process, such analysis has wide spread application in electroanalytical processes of drugs and biomolecules^33–36. The number of electrons transferred in the electrochemical oxidation step is determined from the chronoamperometry experiments and utilisation of ultramicroelectrode. Experiments were carried out at 2 M peptide concentration and the background corrected chronoamperometry plot with the correlation of t^{− 1/2} and the corresponding analysis are shown in Figure S2 of the supporting information^33,37.

Fig. 2.

DPV response of L-A9 peptide at increasing concentrations recorded at (A) pH 3, (B) pH 7, and (C) pH 9, showing concentration-dependent electrochemical responses. (D) The observed linear decrease in peak potential with rising pH suggests a pH-sensitive redox process. The calibration plots (E–G) at pH 3, 7, and 9 with varying peptide concentrations.

The L-A9 peptide has tryptophan at the C-terminal position, it undergoes multistep oxidation process, where in the first step the tryptophan residue undergoes 2 electron and one proton transfer process to generate an intermediate, thereafter it undergoes the chemical change and transfers one proton and a water molecule to generate the hydroxyl group at the tryptophan ring. It has been a well-accepted mechanism of peptide oxidation, present observation on the 2 electron and one proton transfer process also revealed the oxidation of tryptophan at the C-terminal of L-A9 peptide, which generated the electrochemical signal^38,39. The electrochemical oxidation process with two simplified steps over the AuNPs modified electrode surface are shown in the scheme 1.

Scheme 1.

The schematic representation of the electrochemical oxidation process of L-A9 peptide over the AuNPs modified electrode.

In all the pH conditions as in plots A, B, and C, the current response rises steadily as the concentration of L-A9 peptide rises at each pH, indicating the possibility of electrochemical determination of the peptide at all pH values. Corresponding calibration curves (D, E, F) demonstrate a strong linear relationship between current and concentration of peptide for all pH conditions. At pH 3, the linear correlation was analysed and the limit of detection (LOD) of 2.33 × 10⁻⁷ M is observed, the LOD was determined from the signal-to-noise (S/N = 3). Similarly, at pH 7, the DPV plots (B, E) exhibit a linear current response and the LOD value of 1.17 × 10⁻⁷ M is obtained. This enhanced sensitivity at lower and neutral pH is attributed to suitable protonation of the peptide, enhancing electrostatic attraction and promoting efficient electron transfer at the electrode interface. ^40,41. At pH 9, the DPV response (C, F) is characterized by relatively lower current values, with LOD of 3.33 × 10⁻⁷ M. The sensitivity at different pH values are obtained as, 4.12, 12.04 and 3.12 µAcm^− 2/µM at pH 3, 7 and 9 respectively. The observed moderate sensitivity at pH 9 is likely to be due to the retention of some electrostatic interactions, and partial deprotonation of the peptide likely diminishes the efficiency of electron transfer. The electrochemical methods at this pH are more influenced by surface charge effects and variations in electron transfer efficiency. These factors can diminish the method’s sensitivity, as the efficiency of charge transfer between the peptide and electrode may be compromised under such conditions.

The amine groups of the peptides are essentially interacting with the AuNPs which resulted in the preconcentration and thus the enhancement in the current response. The interaction pattern of the amine group with the AuNPs should be strong enough for their interactions, however it should foul the electrode to limit the long-term usability of the modified electrode. The is a linear free energy relationship of the amine group between the proton affinity and with the AuNPs. Proticity of the media plays an important role in their relative competitive interactions⁴². Due to the interactions between the peptide and the AuNPs, peptides are considered as good binding ligands for AuNPs^43,44. The zwitterionic type electrostatic interactions are reported between the amino acid and the AuNPs, however the absence of free amino acid directs the limited possibility of such interactions⁴⁵.

The UV-Vis absorption spectra of L-A9 peptide were recorded at pH, 3, 7 and 9, corresponding results are shown in Fig. 3. The spectra are obtained mainly due to the π to π* transition at around 200 nm originated from the conjugated peptide bonds⁴⁶. With an increase in pH of the media, absorption peak position is marginally blue shifted. The absorbance at all the pH conditions were enhanced with addition of L-A9 peptide in the test solution, the linear calibration plots of each pH values are shown along with the corresponding absorption spectra. The analytical methods have been proposed and while comparing the detection sensitivity the LOD parameters at pH 3, 7 and 9 are obtained as 1.87 × 10^{− 7} M, 1.07 × 10^{− 7} M, 1.65 × 10^{− 7} M respectively. The detection limits from the absorption spectral measurements are indicated to be better or comparable with the electrochemical method, interestingly the LOD at neutral pH is better compared to other two pH values even from the spectrochemical measurements.

Fig. 3.

UV–Vis spectral profiles of L-A9 peptide at various concentrations and pH levels at (A) pH 3, (B) pH 7, and (C) pH 9. The corresponding absorbance vs. concentration plots of L-A9 peptide at (D) pH3, (E) pH7, (F) pH9. reveal a linear relationship, confirming consistent peptide behaviour under varied pH conditions.

In summary, the DPV analysis clearly shows that the neutral or mild acidic conditions are optimal for electrochemical determination of L-A9 peptide, offering superior sensitivity at neutral pH. The good sensitivity with variation of the electrochemical response at different pH values indicate the critical role of method selection and pH optimization in achieving sensitive peptide measurements^40,47,48. The pH-dependent changes in absorbance can provide insights chromophores and lead to depict the interactions with dsDNA which influencing UV-Vis spectral features.

The elevated absorbance at neutral and acidic pH likely arises from enhanced protonation of the peptide’s functional groups, which may strengthen electrostatic and hydrogen-bonding interactions, leading to greater absorbance. This behaviour aligns with previous studies showing that peptides exhibit more efficient adsorption and are less susceptible to UV degradation under acidic conditions, a phenomenon attributed to favourable electrostatic and hydrogen-bonding interactions⁴⁹. The findings align with methodologies for regulatory-grade peptide analysis, where neutral pH conditions help reduce ionic interference⁵⁰. In alkaline conditions, partial deprotonation of side chains alters the peptide’s charge distribution and electronic properties, affecting the detection efficiency. These patterns align with the foundational concepts of UV-Visible peptide analysis, emphasizing that deep UV absorption is governed by peptide backbone and aromatic moieties, and is strongly influenced by the peptide’s protonation state and surrounding environment⁵¹. In summary, the UV-Vis absorbance analysis reveals that the neutral pH (pH 7) is optimal for achieving the most sensitive and precise quantification by UV-Vis spectroscopy. However, the analytical determination is possible in other pH 3 and 9 as well. This observation is consistent with earlier studies on the pH-responsive spectral properties of peptides, affirming that careful pH adjustment is critical for achieving analytical precision.^52–54

Electrochemical impedance spectroscopy (EIS) exploration

Electrochemical impedance spectroscopy (EIS) is used for characterizing the interfacial properties and charge transfer dynamics of peptides at electrode surfaces, Fig. 4 illustrates the Nyquist plot and the corresponding impedance circuit used to fit the Nyquist plots for the L-A9 peptide at pH 7. The plots were recorded at three peak potentials: 0.15 V (lower than the peak potential), 0.2 V (at the peak potential), and 0.25 V (higher than the peak potential). The Nyquist plots (plots A–C) display experimental results (black circles) alongside the fitted line (red lines), with the associated equivalent circuits provided as insets.

Fig. 4.

Electrochemical impedance spectra (Nyquist plots) of the L-A9 peptide at pH 7 at varying potentials: (A) at 0.15 V, data are best described by a dual R–CPE circuit as in the inset (B) and (C) at 0.2 V and 0.25 V, respectively, are well-fitted with a simpler single R–CPE model as in the inset of each figure. Experimental data (black circles) are fitted (red curves) at AuNPs modified GC electrode.

At 0.15 V, before the peak point (Cf. Figure 4A), the peptide’s impedance profile is most accurately modelled by a complex equivalent circuit composed of two resistors (R1, R2) and two constant phase elements (CPE1, CPE2) in both series and parallel arrangements. This arrangement suggests the involvement of multiple interfacial phenomena, including adsorption and distributed capacitance, which are frequently encountered in peptide-coated electrodes at lower potentials. The observed circuit complexity aligns with previous findings, where peptide or protein coatings introduce heterogeneous charge transfer and double-layer phenomena at the electrode interface⁵⁵. When the potential is increased to 0.2 V at the redox peak point (Cf. Figure 4B), the impedance response becomes less complex, with the experimental data fitting a circuit composed of a single resistor (R1) in series with a parallel resistor (R2) and a constant phase element (CPE1). This simplification indicates a shift in the predominant interfacial electron transfer processes, potentially resulting from peptide conformational changes or the emergence of a more homogeneous charge transfer process. This observation is in agreement with previous studies, where the applied potential regulates the arrangement and electron transfer dynamics of peptide layers on electrodes⁵⁶. At 0.25 V, higher than the peak potential (cf. Figure 4C), the impedance response mirrors that observed at 0.2 V, as evidenced by the continued use of the same equivalent circuit (R1 in series with a parallel R2–CPE1 branch). The circuit models across these potentials is consistent with prior findings, showing that peptide films exhibit stable, potential-dependent electrochemical characteristics at elevated potentials⁵⁷.

Overall, the impedance spectra of the L-A9 peptide at different potentials demonstrate a transition from intricate, multi-component interfacial phenomena at low potentials to a more straightforward, stable charge transfer regime over the AuNPs modified electrode surface at elevated potentials suitable for the redox response. Such results align with well-documented studies in the field of electrochemical analysis of biomolecular surfaces and reinforce the importance of EIS and equivalent circuit analysis in deciphering the dynamic processes at peptide-modified electrode interfaces. The impedance measurements and the related RC circuit analysis indicated that at acidic pH two equivalent circuit are to be introduced to simulate the Nyquist plot, at pH 3 the functional groups of peptides remain in the protonated form therefore the possibility of interactions and adsorption over the AuNPs modified electrode surface is relatively strong, while the at neutral or higher pH values, the functional groups at peptides tend to be deprotonated, thus the adsorptive interactions between the AuNPs and peptide might not be strong enough and the Nyquist plots are fitted with simple RC circuit and the requirements of additional RC circuit for the fitting is not essential. The Nyquist plots of the peptide at three applied potentials over bare GC electrode are shown in Figure S3 of the supporting information, indicated higher charge resistance values.

Visualizing of Peptide–DNA binding

After the electrochemical and spectroscopic investigation of the peptide, its interaction with dsDNA has been investigated using electrochemical and the UV-Vis spectroscopic measurements. The differential pulse voltammetry (DPV) results depicted in Fig. 5 (A–C) comprehensively characterize the binding between L-A9 peptide and dsDNA at pH 3, 7, and 9. Across all conditions, increasing the dsDNA concentrations lead to systematic shifts in both peak current and potential, pointing to the gradual formation of peptide–DNA complexes. The peak positions are shifted towards more positive potentials indicating the complexation between the dsDNA with peptide. Notably, the extent of these electrochemical shifts and the overall analytical significance of these shifts are pH-dependent. The binding constant K_b of the peptide and dsDNA has been determined from the relation as below in Eq. 1.

Fig. 5.

DPV response of L-A9 peptide (0.2 µM) in the presence of varying concentrations of dsDNA at (A) pH 3, (B) pH 7, and (C) pH 9.

1

The K_b values were calculated at all the three pH values and obtained as 2.59 × 10⁴, 1.85 × 10⁵, 4.59 × 10⁴ respectively at pH 3, 7 and 9 respectively. The binding constant is observed to be highest at pH 7, indicating enhanced binding property of the peptide with dsDNA.

The electrochemical stability of the AuNPs modified electrode has been assessed over a period of one month by measuring the current response for 6 µM concentration of the L-A9 peptide and the results are shown in Figure S4 of the supporting information. The electrodes remain stable for the period of about one month. The electrode is required to be gently washed using deionised water after everyday use and stored in inert atmosphere.

The UV-Vis absorbance spectra depicted in Fig. 6 (A–C) reveal the interactions between the A9 peptide and dsDNA at pH 3, pH 7, and pH 9. At each pH level, a progressive increase in absorbance is detected as the concentration of dsDNA rises, especially near 220 nm, a region indicative of π→π* transitions in peptides. The observed absorbance enhancement indicates the interaction between peptide-and dsDNA. Interactions patters were somewhat different across different pH values indicating the molecular interactions were pH-dependent in nature.

Fig. 6.

UV-Vis absorbance spectroscopy plot of A9 PEPTIDE with variable concentration of dsDNA at (A) pH3, (B) pH7, (C) pH9, corresponding double reciprocal plots of 1/(A_t−A₀) versus 1/[dsDNA], where linearity confirms a defined binding interaction at (D) pH 3, (E) pH 7, and (F) pH 9.

The double reciprocal plot as shown in Eq. 2 is constructed for all the pH conditions and plotted in Fig. 6(D–F) for pH 3, 7 and 9 respectively.

2

At pH 3 the plot generated the linear relation and from intercept and slope of the linear fitting the formation constant is obtained as K_b = 1.4 × 10⁴ M^− 1.

At pH 7 (Fig. 6B, E), similar exercise was carried out and the stability constant was obtained as 4.1 × 10⁴ M^− 1. At pH 9 (Fig. 6C, F), the stability constant was obtained as 2.6 × 10⁴ M^− 1.

Overall, the UV-Vis results indicate that the most robust peptide-DNA binding is observed at pH 7. These findings stress the importance of binding affinity and analytical considerations when optimizing peptide-DNA assays^58–61.

FTIR and Raman spectroscopic Elucidation of L-A9 Peptide–dsDNA interactions

Figure 7 displays FT-IR and Raman spectra for the L-A9 peptide, dsDNA, and their complexes, providing insight into their structural properties and mutual interactions. The FT-IR spectrum of the L-A9 peptide (plot 7 A) shows prominent amide I bands at 1630 and 1660 cm⁻¹⁻¹, reflecting C = O stretching vibrations from the peptide backbone—a region sensitive to secondary structure motifs such as α-helix and β-sheet⁶². A broad absorption ranges from 2800 to 3500 cm⁻¹, with a characteristic dip at 2355 cm⁻¹, is typically assigned to N–H and O–H stretching vibrations, while the 2355 cm⁻¹ signal often results from atmospheric CO₂.

In the FT-IR spectrum of dsDNA (plot 7 B), a prominent absorption at 1250 cm⁻¹ is observed, characteristic of the asymmetric stretching of the phosphate (PO₄³⁻) groups in the DNA backbone. The spectrum also shows a peak at 1597 cm⁻¹ is attributed to the vibrational modes of the nucleobase rings⁶³. Additional features at 1750 and 2100 cm⁻¹ correspond to overtones or combination bands, and the broad absorption near 2678 cm⁻¹ can be ascribed to O–H stretching vibrations from water molecules or DNA hydration.

The FT-IR spectrum of the L-A9 peptide-dsDNA complex (plot 7 C) reveals several spectral modifications that become apparent relative to the individual spectra. New or shifted peaks at 1070, 479, 1637, 1978, 2137, 2300, 3300, and 3400 cm⁻¹ are observed. the 1070 cm⁻¹ band is associated with symmetric phosphate stretching, while the 1479 and 1637 cm⁻¹ bands reflect alterations in the structural environments of both the peptide and DNA backbones, potentially arising from hydrogen bonding or electrostatic interactions. The broadening and shift of the absorption bands between 3300 and 3400 cm⁻¹ further support the formation of new hydrogen bonds in the complex, as these bands correspond to N–H and O–H stretching vibrations that are sensitive to molecular interactions.

The Raman spectrum of the L-A9 peptide (plot 7D) exhibits prominent bands at 480, 875, 992, 1070, 1320, 1416, and 1620 cm⁻¹. The 992 cm⁻¹ peak is typically linked to the ring breathing mode of phenylalanine, while the peaks at 1070 and 1320 cm⁻¹ correspond to C–N stretching and CH₂ wagging vibrations. The 1620 cm⁻¹ band corresponds to the amide I vibration reflects the secondary structure of the peptide backbone.

In Fig. 7E, the dsDNA Raman spectrum reveals characteristic peaks at 450, 805, 875, 986, 1080, 1306, 1411, 1500, and 1633 cm⁻¹. Notably, the 1080 cm⁻¹ signal reflects the symmetric stretching of phosphate groups (PO₄⁻), and the 1633 cm⁻¹ band corresponds to vibrational modes related to nucleobase ring stretching and base stacking⁶⁴. The Raman spectrum for the L-A9 peptide-dsDNA complex (plot 7 F) reveals several new or shifted bands at 494, 790, 863, 1000, 1073, 1324, 1500, and 1638 cm⁻¹. The 494 cm⁻¹ band may indicate skeletal deformations arising from adduct between L-A9 and dsDNA. The appearance of peaks at 790 and 863 cm⁻¹, as well as the shift in the phosphate stretching band from 1080 cm⁻¹ (in dsDNA) to 1073 cm⁻¹ (in the complex), suggests direct peptide–DNA phosphate backbone interactions. The 1324 and 1500 cm⁻¹ bands reflect changes in the nucleobase environment, while the shift of the band from 1633 cm⁻¹ in free DNA to 1638 cm⁻¹ in the complex indicates altered base stacking or hydrogen bonding^64,65. To summarize, both FT-IR and Raman analyses reveal significant spectral shifts and additional peaks in the L-A9 peptide-dsDNA complex relative to the free peptide and dsDNA. These spectral modifications serve as strong evidence of specific L-A9-dsDNA interactions, such as hydrogen bonding and electrostatic association. The observed modifications in the amide and phosphate vibrational regions point to conformational adjustments in both the peptide and dsDNA upon complex formation, supporting previous reports on the structural effects of peptide-DNA binding^62–64.

Fig. 7.

FT-IR spectra of (A) L-A9 peptide at pH 7, (B) dsDNA at pH 7, and (C) L-A9 peptide in the presence of dsDNA at pH 7. Raman spectra of (D) L-A9 peptide, (E) dsDNA, and (F) L-A9 peptide–dsDNA complex.

Surface charge landscapes using zeta potential analysis of L-A9 peptide and DsDNA assemblies

Figure 8 presents the zeta potential analysis of the L-A9 peptide, providing important insights into the pH-dependent surface charge characteristics of the peptide and its interaction with dsDNA. Figure 8. A illustrates zeta potential measurements of the peptide at various pH values, specifically at pH 3, 5, 7, 8, and 10. At lower pH levels (pH 3 and 5), the peptide exhibits a positive zeta potential of 7 to 10 mV, consistent with the protonation state of basic side chains, including lysine and arginine, which hold onto their positive charge in such environments and contribute to the peptide’s overall surface charge⁶⁶. As the pH rises to 7, the zeta potential drops to 1.5 mV, reflecting a state near the isoelectric point where surface charges are nearly balanced and the net charge is minimal⁶⁷.The zeta potential reaches as low as 0.3 mV at pH 8, likely due to enhanced deprotonation of basic amino acids and greater exposure of acidic moieties, and reduced protonation of basic residues. This outcome is anticipated because, at high pH, most basic amino acids have lost their positive charges. At the same time, acidic side chains retain their negative charge, resulting in a net negative charge on the peptide. Such pH-dependent reversals in surface charge are widely reported in protein and peptide chemistry and are crucial for influencing peptide interactions and solubility under varying conditions⁶⁶.

Fig. 8.

(A) Zeta potential profiles of L-A9 peptide at different pH values (pH 3, 5, 7, 8, and 10), demonstrating variations in net surface charge with changes in protonation state. (B) Zeta potential comparison of L-A9 peptide, dsDNA, and L-A9-dsDNA.

As expected, at pH 10, the zeta potential climbs to negative side at -9 mV. While acidic residues like glutamate and aspartate are fully deprotonated and negatively charged, some basic amino acids may remain partially protonated, resulting in a net negative zeta potential at pH 10^66,67.

Figure 8B presents the zeta potential profiles for dsDNA, L-A9 peptide, and their complex at neutral pH 7. The dsDNA alone has a low zeta potential of -19 mV, and the L-A9 peptide alone exhibits a zeta potential of 1.5 mV. When the peptide and DNA form a complex, the zeta potential drops to -1 mV, due to the strong charge compensation resulting from their interaction. This pronounced decrease in zeta potential reflects the electrostatic binding and partial masking of charges between the cationic peptide and anionic DNA, resulting in a complex that approaches charge neutrality^68,69. The addition of dsDNA results in a pronounced decrease in zeta potential, supporting the presence of electrostatic binding and partial charge neutralization, which are critical for bioanalytical applications^68,70. These insights are vital for the strategic design and refinement of peptide-based drugs systems, as managing surface charge and affinity is fundamental to their biological application.

The peptide-DNA interactions generate the basis of several biological activity through the modes of bindings and their relative binding strengths. Presence of metal cations has shown strong influence in the Simulation has shown that Mg²⁺ ions contribute to the formation of the peptide − DNA complex formation. The negative charged phosphate groups of DNA are somewhat masked in presence of positively charged metal cation, which results in the modification of the interaction pattern⁷¹. Previous investigation indicated that Glu, in an untwisted shape, generally binds with the major groove of dsDNA due to which the three H-bonds between the strands are modified which essentially destabilises the DNA double helix. Similarly the Lys, in an untwisted shape, binds with external side of the dsDNA by forming two bonds with O atoms of neighbouring phosphodiester groups, such bonding strengthens the DNA helix²⁵. The L-A9 peptide has both acidic and basic functional groups, the acidic functional groups interacts with the major groove of dsDNA through the modifications of 3 hydrogen bonds between the strands, whereas the basic functional groups of L-A9 binds externally with dsDNA by formation of bonds with O atoms of the phosphodiester groups⁷². The Raman spectra measurements has indicated the possibility of both these two types of interactions between dsDNA and L-A9 peptide.

Topographical measurements using AFM and SEM

The AFM morphology of the of the peptide and dsDNA were carried out along with that the surface morphology of the AuNPs are also shown in Fig. 9. The AFM micrograph revealed that the AuNPs exhibit a near-spherical shape and are uniformly dispersed over the surface of the GCE. The average diameter of the AuNPs was estimated to be ~ 85 nm. The peptide film was generated over mica substrates, displayed a uniform surface with moderate roughness, as shown by a root mean square roughness (Rq) of 16.68 nm and an arithmetic mean height (Sa) of 76.44 nm. The maximum vertical distance (Ry) observed was 277.8 nm, with the most prominent peak (Sp) at 18.77 nm and the deepest valley (Sv) at 2.87 nm, signifying the existence of some marked but not extreme surface irregularities. Conversely, the AFM image of the L-A9 PEPTIDE-dsDNA Figure (9 C) sample displays a markedly more granular and uneven surface, with numerous sharp peaks and deep valleys distributed densely across the sample. Notably, the average roughness parameters (R_q = 11.99 nm, S_a = 10.64 nm) are reduced after dsDNA binding, implying a smoother average surface. Nevertheless, both the maximum peak (S_p = 82.13 nm) and valley (S_v = 36.16 nm) heights are substantially elevated, along with a reduction in total height (R_y = 63.0 nm), indicate that dsDNA incorporation leads to the formation of sharper, and more distinct nanostructures, likely reflecting self-assembly or aggregation within the L-A9 -dsDNA. These observations align with earlier studies, which have demonstrated that dsDNA interactions with peptide or protein surfaces can induce substantial reorganization of the surface, increase nanoscale heterogeneity, and create novel domains^73,74. In Fig. 9, the SEM images provide a comparative view of L-9 A peptide morphology in its native state and after binding to dsDNA. The dsDNA modified mica substrate has shown the dsDNA threads spread all along the mica surface, (Fig. 9D), whereas the L-A9 modified mica sheets are characterised with agglomerated deposits with occasional needle type morphology. While the L-A9 and dsDNA modified substrate has shown relatively thicker dsDNA threads (Fig. 9E and F) compared to the bare dsDNA, the occasional needle type of morphology remain visible across the mica sheet. ^75,76,77.

Fig. 9.

Atomic Force Microscopy (AFM) images showing: (A) AuNPs-modified glassy carbon electrode surface, (B) L-A9 peptide deposited on mica sheet, (C) L-A9 peptide with dsDNA on mica, showing topographical variation due to interaction, (D) SEM micrographs of dsDNA and (E, F) SEM morphological alterations upon interaction with dsDNA at different resolutions.

Molecular docking investigation

The peptide structures were prepared and energy minimized using the ACD Chem Sketch suite (ACD/Chem Sketch for Academic and Personal Use: ACD/Labs.com, 2022). The DNA coordinates were obtained from Protein Data Bank (PDB) having PDB ID 1bna⁷⁸. The DNA and peptide were prepared for docking after adding hydrogens and charges using Auto Dock Tools 4.2.⁷⁹ The docking was performed using a grid box of 32 Å × 32 Å × 37 Å, spanning the dsDNA surface. Molecular docking studies were performed using AutoDock Vina 1.2 with its scoring functions.^80,81 The best ten peptide poses were sorted after docking based on their binding free energies with the receptor and were analysed. The structural analysis and figures were made using open-source PyMOL (The PyMOL Molecular Graphics System, version 2.3.4. Schrödinger, LLC) and Chimera 1.16.⁸² The best four docked peptides are shown in Fig. 10 (A) to (D). The hydrogen bonds are shown as green lines. The peptide binds in an extended conformation having two major orientations and wraps around DNA along the major grove. The binding free energies are similar at - -32.2 kJ/mol with structures (A) and (B) of Fig. 10. Two more structures of binding free energies of 32.8 and 32.3 kJ/mol are also shown in Fig. 10 (C) and (D) respectively. The peptide terminals are names as terminal 1 where it ended towards indole ring, the other terminal is named as terminal 2. Two most stable conformations with highest binding free energy are generated with the indole ring positioned both upward and in downward positions as in Fig. 10 (A) and (B) respectively. In conformation (A) the indole ring contended in terminal 1 of the peptide is upwardly directed and not directly bound to the DNA and the amide group in other terminal makes hydrogen bonds through its amine hydrogen and also through the acidic hydrogen present at the carboxylic acid group. Whereas in conformation (B) the indole is placed downward and the terminal 2 is upward, the hydrogen bonding is done through the oxygen atoms of L-A9 peptide with DNA stands. In conformation (C) the indole ring in placed upward, and bound through the -NH hydrogen of the indole ring of the peptide. Hydrogen bonding through the amine hydrogen and carbonyl oxygen are also seen at the terminal 2 of the peptide. In conformation (D), which resulted with lowest binding free energy has terminal 1 of the peptide downwardly placed and the indole ring is twisted without any binding with DNA. The terminal 2 is placed upward and the hydrogen bonding is done through the -NH- hydrogen from the chain of the peptide and hydrohen at the amine group. The experimental finding on binding through both acidic and basic functional groups of the L-A9 with major groves of dsDNA, which is supported from the molecular docking investigations with indicating the detailed hydrogen bonding interactions from the acidic and basic functional groups of L-A9 peptide.

Fig. 10.

The interactions pattern generated with best binding free energies between dsDNA and the L-A9 peptide.

Conclusion

To summarize, the electrochemical analysis of the L-A9 peptide’s interactions with dsDNA on gold-modified electrodes has offered substantial insights into its binding dynamics and potential uses in various applications. DPV analysis revealed significant shifts in peak current and potential, with a limit of detection of 1.17 × 10^− 4 M at pH 7 for the L-A9 peptide–dsDNA complex the binding constant is obtained as 1.8 × 10⁵ M^− 1, indicating a robust interaction and its promise as a biorecognition element. The UV-Vis absorbance study at 215 nm revealed a pronounced increase (or shift) in absorbance upon complex formation, and the formation constant at pH 7 was determined as 4.1 × 10⁴ M⁻¹ for the L-A9 peptide–dsDNA complex. FTIR and Raman spectroscopy confirmed conformational changes, with distinct shifts at 1750 cm⁻¹ in FTIR and at 1638 cm⁻¹ in Raman, indicating that complex formation leads to significant alterations in the peptide’s secondary structure. The binding interactions between DNA and L-A9 peptide are indicated from the molecular docking. The findings demonstrate the L-A9 peptide’s utility in both drug delivery systems and its effectiveness as a biorecognition element in biosensors. This research ultimately emphasizes the quantitative performance of the L-A9 peptide in biomedicine, setting the stage for subsequent research that may further examine its interactions in diverse biological contexts and enhance its use in therapeutic and diagnostic applications.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Preeti Dwivedi: Experimental work, data analysis, writing of the draftSudipa Manna: Technical help during experimentsDrishty Satpati: Synthesis and characterization of L-A9 PeptideAmi Das: Molecular Docking investigationSuresh Shendage: Project AdministrationAshis Kumar Satpati: Conceptualization, Data analysis, Funding arrangements, Final Manuscript preparation.

Funding

Open access funding provided by Department of Atomic Energy. The project is fully funded by Bhabha Atomic Research Centre, Government of India.

Data availability

The data used in the manuscript will be made available on request. The molecular docking was carried out using the openly available resources which are indicated in the relevant section, however any further data related to any finding of the manuscript will be made available on request.

Declarations

Competing interests

The authors declare no competing interests.