Influence of Green Synthesized Zinc Oxide Nanoparticles on Molecular Interaction and Comparative Binding of Azure Dye with Chymotrypsin: Novel Nano-Conjugate for Cancer Phototherapy.

Till date, different types of conventional drugs have been used to fight tumors. However, they have significant flaws, including their usage being constrained because of their low bioavailability, poor supply, and serious side effects. The modern combination therapy has been viewed as a potent strategy for treating serious illnesses, including cancer-type feared diseases. The nanoparticles are a promising choice for cancer therapeutic and diagnostic applications because of their fascinating optoelectronic and physicochemical features. Among the metallic nanoparticles, Zinc oxide nanoparticles possess interesting physicochemical and anti-cancer characteristics, such as ROS generation, high retention, enhanced permeability etc., making them attractive candidates for the treatment and diagnosis of cancer. Zinc oxide nanoparticles showed anti-cancer property via excessive reactive oxygen species (ROS) production, and by the destruction of mitochondrial membrane. Here, we have synthesized organic/inorganic hybrid nanosystem composed of chymotrypsin protein (Chymo) with AzureC (AzC) conjugated with Zinc oxide nanoparticles (ZnONPs). The conjugation of AzureC with ZnONPs was confirmed by transmission electron microscopy (TEM), zeta potential, and dynamic light scattering (DLS) experiment. The interaction of Chymo with AzC alone and AzC-ZnONPs was investigated, and it was observed that the interaction was enhanced in the presence of ZnONPs, which was concluded by the results obtained from different spectroscopic techniques such as UV-Visible spectroscopy, fluorescence spectroscopy and circular dichroism in combination with molecular docking. UV-Visible spectroscopic studies and the corresponding binding parameters showed that the binding of AzC-ZnONPs complex with Chymo is much higher than that of AzC alone. Moreover, the fluorescence measurement showed enhancement in static quenching during titration of Chymo with AzC-ZnONPs as compared to dye alone. In addition to this, circular dichroism results show that the dye and dye-NPs conjugate do not cause much structural change in α-Chymo. The molecular docking and thermodynamic studies showed the predominance of hydrogen bonding, Van der Waal force, and hydrophobic forces during the interactions. After correlation of all the data, interaction of Chymo with AzC-ZnONPs complex showed strong interaction as compared to dye alone. The moderate binding with chymo without any alteration in the structure makes it desirable for the distribution and pharmacokinetics. In addition, the in vitro cytotoxicity of the AzC-ZnONPs was demonstrated on A-549 adenocarcinoma cell line. Our findings from physiochemical investigations suggested that the chymotrypsin coated AzC conjugated ZnONPs could be used as the novel nanoconjugates for various cancer phototherapies.

Green synthesis and physiochemical characterization of nickel oxide nanoparticles: Interaction studies with Calf thymus DNA.

One of the most promising applications of nanomaterials is that of nanobiosensors, using biomolecules such as nucleic acids as receptors. This study aimed to synthesize nickel oxide nanoparticles (NiO NPs) by an environmentally friendly green synthesis, using the extract of the herb Coriandrum sativum (coriander). The synthesized NPs were characterized using UV-Visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, X-ray photon spectroscopy, field emission scanning electron microscopy coupled with energy dispersive spectroscopy, dynamic light scattering, zeta potential and transmission electron microscopy. All results confirmed the synthesis of pure, spherical, positively charged NiO NPs of around 95 nm in diameter with prominent hydroxyl groups attached to the surface. Furthermore, interaction studies of synthesized NiO NPs with calf thymus DNA (CT DNA) were performed using UV-Visible spectroscopy, UV-thermal melting, circular dichroism, and fluorescence spectroscopy. CT DNA served as a substitute for nucleic acid biosensors. All experimental studies indicated that the NiO NPs bound electrostatically with CT DNA. These studies may facilitate exploring the potential of NiO NP-nucleic acid conjugated materials to be used as nanobiosensors for various applications, especially in pharmacological, epidemiological, and environmental diagnostic applications, and in detection.

Exploring the potential of environment friendly silver nanoparticles for DNA interaction: Physicochemical approach.

Nanosilver, being the most prominent nanoproduct has diverse bio-medical applications and hence the effects associated with their exposure need to be investigated in detail. The interaction of metal nanoparticles with DNA has become a matter of interest, as their effect on structural integrity, synthesis and replication could be explored through it. Present work aims at the facile synthesis and characterization of spherical silver nanoparticles (AgNPs) using Epipremnum aureum leaves extract. Nanoparticles were characterized using UV-Visible spectroscopy, Transmission Electron Microscopy (TEM), High Resolution X-ray Diffraction (HR-XRD) and Dynamic Light Scattering (DLS) studies. The interaction of AgNPs with Calf thymus DNA (CT-DNA) was investigated using different spectroscopic techniques like UV-Visible spectroscopy, UV-thermal melting, Circular Dichroism and fluorescence spectroscopic studies. Fluorescence results suggest van der Waals and H-bonding interactions, which are predominantly responsible for the interaction of AgNPs with CT-DNA. Circular dichroism and thermal melting studies are pointing towards the groove binding of AgNPs to CT-DNA. DNA duplex destabilization was confirmed by the decreased thermal melting temperature of CT-DNA on addition of AgNPs. Present study might open up new vistas for the study of unusual kind of DNA binders, which can destabilize DNA and may further be used for various biomedical applications.

Presence of divalent cation is not mandatory for the formation of intramolecular purine-motif triplex containing human c-jun protooncogene target.

Modulation of endogenous gene function, through sequence-specific recognition of double helical DNA via oligonucleotide-directed triplex formation, is a promising approach. Compared to the formation of pyrimidine motif triplexes, which require relatively low pH, purine motif appears to be the most gifted for their stability under physiological conditions. Our previous work has demonstrated formation of magnesium-ion dependent highly stable intermolecular triplexes using a purine third strand of varied lengths, at the purine•pyrimidine (Pu•Py) targets of SIV/HIV-2 (vpx) genes (Svinarchuk, F., Monnot, M., Merle, A., Malvy, C., and Fermandjian, S. (1995) Nucleic Acids Res. 23, 3831-3836). Herein, we show that a designed intramolecular version of the 11-bp core sequence of the said targets, which also constitutes an integral, short, and symmetrical segment (G(2)AG(5)AG(2))•(C(2)TC(5)TC(2)) of human c-jun protooncogene forms a stable triplex, even in the absence of magnesium. The sequence d-C(2)TC(5)TC(2)T(5)G(2)AG(5)AG(2)T(5)G(2)AG(5)AG(2) (I-Pu) folds back twice onto itself to form an intramolecular triple helix via a double hairpin formation. The design ensures that the orientation of the intact third strand is antiparallel with respect to the oligopurine strand of the duplex. The triple helix formation has been revealed by non-denaturating gel assays, UV-thermal denaturation, and circular dichroism (CD) spectroscopy. The monophasic melting curve, recorded in the presence of sodium, represented the dissociation of intramolecular triplex to single strand in one step; however, the addition of magnesium bestowed thermal stability to the triplex. Formation of intramolecular triple helix at neutral pH in sodium, with or without magnesium cations, was also confirmed by gel electrophoresis. The triplex, mediated by sodium alone, destabilizes in the presence of 5'-C(2)TC(5)TC(2)-3', an oligonucleotide complementary to the 3'-oligopurine segments of I-Pu, whereas in the presence of magnesium the triplex remained impervious. CD spectra showed the signatures of triplex structure with A-like DNA conformation. We suggest that the possible formation of pH and magnesium-independent purine-motif triplexes at genomic Pu•Py sequences may be pertinent to gene regulation.

Possibility of an antiparallel (tetramer) quadruplex exhibited by the double repeat of the human telomere.

Under physiological concentrations of Na+ and K+, human telomeric DNA can self-associate into G-quadruplexes. On the basis of circular dichroism, gel electrophoresis, gel filtration, and UV-melting experiments, we report here that the double repeat of human telomere (d-TTAGGGTTAGGG; HUM2) forms parallel as well as antiparallel quadruplexes in the presence of K+, whereas Na+ facilitates only the antiparallel form. Here, the gel techniques and CD studies have proved to be complementary in detecting the molecularity and pattern of strand orientation. By correlating the gel and CD experiments, the antiparallel G-quadruplex was identified as a tetrameric species, whereas the parallel G-quadruplex was found to be dimeric. Both structural species were separated through gel filtration, which when run on native polyacrylamide gel electrphoresis (PAGE), confirmed their molecularity. UV-melting profiles also confirm the presence of two biphasic and one monophasic structural species in the presence of K+ and Na+, respectively. Though our observation is consistent with the recent NMR report (Phan, A. T., and Patel, D. J. (2003) J. Am. Chem. Soc. 125, 15021-15027), it seems to differ in terms of the molecularity of the antiparallel quadruplex. A model is proposed for an antiparallel tetrameric quadruplex, showing the possibility of Watson-Crick hydrogen bonds between intervening bases on antiparallel strands. This article expands the known structural motifs of DNA quadruplexes. To the best of our knowledge, four-stranded antiparallel quadruplexes have not been characterized to date. On the basis of the model, we hypothesize a possible mechanism for telomere-telomere association involving their G-overhangs, during certain stages of the cell cycle. The knowledge of peculiar geometries of the G-quadruplexes may also have implications for its specific recognition by ligands.

Calorimetric unfolding of the bimolecular and i-motif complexes of the human telomere complementary strand, d(C(3)TA(2))(4).

A combination of spectroscopic and calorimetric techniques is used to determine the unfolding thermodynamics of the complexes formed by the complementary sequence of the human telomere, d(C(3)TA(2))(4), in the pH range of 4.2 to 6. Calorimetric melting curves show biphasic transitions; both transitions are shifted to higher temperatures as the pH is decreased, indicative of cytosine protonation, which favors the formation of C*C(+) base pairs. Furthermore, the transition temperature, T(M), of the lower transition depends on strand concentration, while the T(M) of the higher transition is independent of strand concentration, indicating the following sequential melting: bimolecular complex(s)-->intramolecular complex-->random coil. The thermodynamic profiles for the formation of each complex, bimolecular and i-motif reveals small favorable free energy terms resulting from favorable enthalpy-unfavorable entropy compensations, uptake of protons, marginal uptake of counterions (i-motif) and marginal release of water molecules (i-motif). Furthermore, an enthalpy of 3.2 kcal/mol (bimolecular complex) and 5.0 kcal/mol (i-motif) is estimated for a single C*C(+)/C*C(+) base-pair stack.