br Corresponding author br E mail address Karin
E-mail address: [email protected] (K. Chumbimuni-Torres).
properties and DNA affinity to these small molecules (Rauf et al., 2005). Unfortunately, these techniques mostly address the issues of structural analysis and binding mechanisms, rather than investigating DNA da-mage and its impact. Electrochemical biosensors, on the other hand, have been efficiently used to monitor the production of DNA damage via small molecule interaction (Lucarelli et al., 2004; Vyskočil et al., 2010). An electrochemical DNA biosensor is preferred due to the highly conducting ability provided by π-stack of nitrogenous bases, versatility to optimize DNA immobilization on an electrode surface, and ability to determine DNA damage induced by drug intercalation or via drug-in-duced oxidative stress (Labuda et al., 2009; Nepali et al., 2014; Arnold et al., 2015; Huang et al., 2016).
Oxidation of electroactive nucleic acids have been used to monitor DNA lesions on modified gold, glassy carbon and mercury electrodes (Paleček et al., 1998; Li et al., 2010; Ibañez et al., 2015). Benvidi et al. employed Au-thiol chemistry to covalently bind a stem-loop (SL)-DNA
Fig. 1. Electrochemical characterization of 7ESTAC01 A) Chemical mechanism of synthesis of 7ESTAC01: Acridine-9-carboxaldehyde with 2-aminothiophene. B) Cyclic voltammogram of 10 mM 7ESTAC01 in mixture of pH 7.2, aqueous phosphate buffer and 20% DMF at a GE, with an Ag/AgCl reference electrode (RE), and the scan rate 0.1 V s−1, E = potential, V = volt, A = ampere. Cyclic voltammogram of 10 mM 9-aminoacridine (B, inner graph*). (*) Red line represents the first scan. The green line represents the second scan with the potential range adjusted only to get the peak already registered on the first scan. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
probe and 6-mercapto-1-hexanol (MCH) to form self-assembled mono-layer on gold surfaces (Benvidi et al., 2015). MCH played a significant role in the overall optimized response of the DNA biosensor by avoiding non-specific DNA adsorption and adjusting the interfacial EIPA transfer on the electrode (Mills et al., 2017; McEwen et al., 2009). A Stem-Loop DNA (SL-DNA) structure offers higher thermodynamic sta-bility when compared to a linear DNA structure. That stability could be explained by the presence of the hairpin loop with a reduced negative charge, which reduces the non-specific binding at the loop without compromising the first binding on the stem (Nguyen and Wilson, 2009).
The hybrid drug, a combination of two pharmacophores, has the potential to improve binding affinity, selectivity, and synergic activity towards nucleic acids (Goodell et al., 2006; Cholewiński et al., 2011; Nepali et al., 2014; Harbinder et al., 2017). Recent research has also investigated the amplification of oxidative stress in relation to DNA damage caused by sulfur, thiophene, triazole and acridine moieties (Brett et al., 2003; Pontinha et al., 2013; Sazhnikov et al., 2013; Noh et al., 2015; Deng et al., 2017). Acridine derivatives are highly inter-esting chemotherapeutic agents, which are linked to different phar-macophores in order to modify reactivity. Modifications of substituent groups in acridine derivatives have been found to further enhance the anti-cancer drug efficacy (Putic et al., 2010; Lafayette et al., 2013).
Here, we developed a highly sensitive electrochemical biosensor based on an SL-DNA probe that can detect DNA damage via hybrid drug, 7ESTAC01 interaction. 7ESTAC01 is composed of two anti-cancer pharmacophores, acridine, and thiophene. A complementary DNA strand (cDNA) was introduced to hybridize the SL-DNA probe to form a double-stranded DNA (dsDNA) biosensor. The optimization of SL-DNA/ GE and dsDNA/GE modified electrode for sensitive detection of DNA damage was assessed by DPV and cyclic voltammetry (CV).
The present work involves two sections. First, we investigated the electrochemical oxidation of SL-DNA probe and dsDNA on the surface of the gold electrode induced by the presence of 7ESTAC01 to detect DNA damage by DPV. Second, the interaction of 7ESTAC01 with DNA was analyzed via UV–Vis absorption spectroscopy, in-silico dynamic simulations, and molecular docking.
2. Experimental section
2.1. Chemicals and reagents
All solutions were prepared with Milli-Q water using a Siemens
PURELAB Ultra system (Lowell, USA). The immobilization buffer (IB) contains 50 mM Sodium Phosphate (Monobasic/Dibasic), 250 mM NaCl at pH 7.4. Hybridization buffer (HB) contains 50 mM Tris-HCl, 25 mM NaCl, 50 mM MgCl2 at pH 7.4. Stock solutions of 1 mM 7ESTAC01 were prepared in HB and were evaluated in acetate solution at different concentrations. 1.0 M Acetate buffer solution at pH 4.2 was used for the DNA oxidation in presence of the 7ESTAC01. The Tris-HCl buffer was used for UV–Vis measurements and contains 50 mM NaCl, 5 mM, Tris-HCl at pH 7.2. The pH was adjusted with either NaOH or HCl solution. The calf thymus DNA (ctDNA) was prepared from dissolution of 12 mg mL−1 in acetate buffer (pH 4.2; 1.0 M). This stock solution was kept at 8 °C for 24 h and stirred at certain intervals to ensure homo-geneity of the final DNA solution. Trizma hydrochloride (Tris-HCl), tris (2-carboxyethyl) phosphine hydrochloride (TCEP), sodium phosphate dibasic (Na2HPO4), sodium phosphate monobasic dihydrate (NaH2PO4·2H2O), 6-mercapto-1-hexanol (MCH) and magnesium chloride (MgCl2) were purchased from Sigma-Aldrich. Sodium chloride (NaCl), potassium chloride (KCl), sodium hydroxide (NaOH) and sul-furic acid (H2SO4) were obtained from Fisher Scientific (Pittsburgh, USA).