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Understanding the interaction of small molecules with DNA has become an active research area at the interface between chemistry, molecular biology and medicine. Plant derived polyphenols possess diverse biological and pharmacological properties. Esculetin is a coumarin derivative polyphenolic compound having diverse pharmacological and therapeutic properties. However, its mode of interaction with DNA is still not well understood. In the present study, we have attempted to determine the mode of binding of esculetin with calf thymus DNA (Ct-DNA) through various biophysical techniques. Analysis of UV-visible absorbance spectra and fluorescence spectra indicates the formation of a complex between esculetin and Ct-DNA. The binding constant was found to be 1.87 × 10(4) M(-1). Thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures indicated that hydrophobic interactions and hydrogen bonding played major roles in the binding process. Several other experiments such as iodide induced quenching and competitive displacement studies with ethidium bromide, acridine orange and Hoechst 33258 suggested that esculetin possibly binds to the minor groove of the Ct-DNA. The strong dependence on ionic strength in controlling the binding of esculetin with Ct-DNA confirms the possibility of electrostatic interaction. These observations were further supported by DNA melting studies, viscosity measurements, CD spectral analysis and in silico molecular docking.

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DNA is one of the major intracellular targets for a wide range of anticancer and antibiotic drugs. Elucidating the binding between small molecules and DNA provides great help in understanding drug-DNA interactions and in designing of new and promising drugs for clinical use. The ability of small molecules to bind and interfere with DNA replication and transcription provides further insight into how the drugs control the expression of genes. Interaction of an antimetabolite anticancer drug 6mercaptopurine (6MP) with calf thymus DNA was studied using various approaches like UV-visible spectroscopy, fluorescence spectroscopy, CD, viscosity and molecular docking. UV-visible spectroscopy confirmed 6MP-DNA interaction. Steady state fluorescence experiments revealed a moderate binding constant of 7.48×103 M−1 which was consistent with an external binding mode. Competitive displacement assays further confirmed a non-intercalative binding mode of 6MP which was further confirmed by CD and viscosity experiments. Molecular docking further revealed the minimum energy conformation (−119.67 kJ/mole) of the complex formed between DNA and 6MP. Hence, the biophysical techniques and in-silico molecular docking approaches confirmed the groove binding/electrostatic mode of interaction between 6MP and DNA. Further, photo induced generation of ROS by 6MP was studied spectrophotometrically and DNA damage was assessed by plasmid nicking and comet assay. There was a significant increase in ROS generation and consequent DNA damage in the presence of light.

Enzyme engineering is a powerful tool to fine‐tune the enzymes. It is a technique by which the stability, activity, and specificity of the enzymes can be altered. The characteristic properties of an enzyme can be amended by immobilization and protein engineering. Among them, protein engineering is the most promising, as in addition to amending the stability and activity, it is the only way to modulate the specificity and stereoselectivity of enzymes. The current review sheds light on protein engineering and the approaches applied for it on the basis of the degree of knowledge of structure and function of enzymes. Enzymes, which have been engineered are also discussed in detail and categorized on the basis of their respective applications. This will give a better insight into the revolutionary changes brought by protein engineering of enzymes in various industrial and environmental processes.