Fingerprinting branches on supercoiled plasmid DNA using quartz nanocapillaries

We show detailed understanding of enzyme dependent structural changes in supercoiled DNA along with a quantitative analysis of its branches using nanopores.

Novel Benzimidazole Derived Imine-Based Ligand and its Co(III), Ni(II), Cu(II) and Pt(II) Complexes: Chemical Synthesis, Structure, Antimicrobial, DNA Interaction Studies and Nuclease Activity

Herein, we report the synthesis of a Schiff base ligand, 2-((E)-((1H-benzo[d]imidazol-2-yl)methylimino)methyl)-4,6-dichlorophenol (BMDC) and its Co(III), Ni(II), Cu(II) and Pt(II) coordinated compounds (henceforth named as M1, M2, M3, and M4, respectively). The synthesized compounds were subjected to various analytical and physical characterization techniques to confirm their molecular structures. Further, the DNA binding and cleavage propensity of the as-prepared metal complexes were evaluated using calf thymus DNA (CT-DNA) and supercoiled plasmid DNA (pBR322). The DNA interaction results revealed that the tested metal complexes bind with DNA through the covalent and non-covalent mode of interaction, which is investigated using absorption and fluorescence spectral studies followed by viscosity measurement. The scission of supercoiled plasmid DNA by the metal complexes suggested the potentiality of the molecules toward the cleavage of pBR322 DNA. Furthermore, the compounds were screened for in vitro antiproliferative activity, tested against the various cell lines such as A549, EAC, SIHA, and NIH3T3. Results revealed that compound M4 exhibited marked anti-proliferative activity against EAC cell line with a significant IC50 value of 10 µm compared to its parent ligand, BMDC, and other metal transition complexes under study. In addition, various hematological parameters (alkaline phosphate, creatinine, urea, RBC, and WBC) were studied, and significant results are obtained from the experiments.

Effect of Chromatographic Conditions on Supercoiled Plasmid DNA Stability and Bioactivity

The dysfunction of the tumor suppressor gene TP53 has been associated with the pathogenesis of the majority of the cases of cancer reported to date, leading the cell to acquire different features known as the cancer hallmarks. In normal situations, the protein p53 protects the cells against tumorigenesis. By detecting metabolic stress or DNA damage in response to stress, p53 can lead the cell to senescence, autophagy, cell cycle arrest, DNA repair, and apoptosis. Thus, in the case of p53 mutations, it is reasonable to assume that the reestablishment of its function, may restrain the proliferation of cancer cells. The concept of cancer gene therapy can be based on this assumption, and suitable biotechnological approaches must be explored to assure the preparation of gene-based biopharmaceuticals. Although numerous procedures have already been established to purify supercoiled plasmid DNA (sc pDNA), the therapeutic application is highly dependent on the biopharmaceutical’s activity, which can be affected by the chromatographic conditions used. Thus, the present work aims at comparing quality and in vitro activity of the supercoiled (sc) isoform of the p53 encoding plasmid purified by three different amino acids-based chromatographic strategies, involving histidine–agarose, arginine–macroporous, and histidine–monolith supports. The B-DNA topology was maintained in all purified pDNA samples, but their bioactivity, related to the induction of protein p53 expression and apoptosis in cancer cells, was higher with arginine–macroporous support, followed by histidine–monolith and histidine–agarose. Despite the purity degree of 92% and recovery yield of 43% obtained with arginine–macroporous, the sc pDNA sample led to a higher expression level of the therapeutic p53 protein (58%) and, consequently, induced a slightly higher apoptotic effect (27%) compared with sc pDNA samples obtained with histidine–monolithic support (26%) and histidine–agarose support (24%). This behavior can be related to the mild chromatographic conditions used with arginine–macroporous support, which includes the use of low salt concentrations, at neutral pH and lower temperatures, when compared to the high ionic strength of ammonium sulfate and acidic pH used with histidine-based supports. These results can contribute to field of biopharmaceutical preparation, emphasizing the need to control several experimental conditions while adapting and selecting the methodologies that enable the use of milder conditions as this can have a significant impact on pDNA stability and biological activity.

Polyoxometalates as artificial nucleases: hydrolytic cleavage of DNA promoted by a highly negatively charged ZrIV-substituted Keggin polyanion

A highly negatively charged ZrIV-substituted Keggin polyoxometalate [{α-PW11O39Zr(μ-OH)(H2O)}2]8− (ZrK 2 : 2) promoted the hydrolysis of phosphoester bonds in the supercoiled plasmid pUC19 DNA under physiological pH and temperature.