Puja Paul

Biophysical studies on the base specificity and energetics of the DNA interaction of photoactive dye thionine: Spectroscopic and calorimetric approach

In this study absorbance, fluorescence, circular dichroic spectroscopy, viscosity, thermal melting and calorimetric techniques were employed to understand the binding of the phenothiazinium dye, thionine, with deoxyribonucleic acids of varying base composition. Strong hypochromic and bathochromic effects and quenching of fluorescence were observed that showed strong binding of thionine to the DNAs. The binding parameters evaluated from Scatchard analysis through McGhee-von Hippel analysis showed that the binding was non-cooperative and affinities of the order of 10(5)M(-)(1). The results of ferrocyanide fluorescence quenching studies and viscosity experiments, taken together suggested the intercalation of thionine while thermal melting, differential scanning calorimetry and circular dichroic studies provided evidence for the thermal stabilization and conformational perturbations associated with the binding. The thermodynamic parameters elucidated through sensitive isothermal titration calorimetric studies suggested that the binding was exothermic, characterized by negative enthalpy and large positive entropy changes and that the non-electrostatic contributions play a significant role for thionine association to DNA. The heat capacity changes obtained from the temperature dependence of enthalpy changes gave negative values suggesting substantial hydrophobic contribution in the DNA binding process of thionine. Further, an observation of enthalpy-entropy compensation in the DNA binding also suggested the involvement of multiplicity of non covalent interactions in the binding process. The base specificity of the complexation and energetics of the interaction of thionine to DNA are obtained for the first time from this study.

Spectroscopic studies on the binding interaction of phenothiazinium dyes toluidine blue O, azure A and azure B to DNA.

In this study a detailed characterization of the binding aspects of three phenothiazinium dyes, toluidine blue O (TBO), azure A and azure B with herring testes DNA is presented employing spectroscopic techniques. The absorbance and fluorescence properties of these dyes have been remarkably modified upon binding with DNA and the interaction is manifested through noncooperative binding as revealed form non-linear Scatchard plots with negative slopes at all binding ratios. The binding clearly revealed the high preference of TBO to DNA followed by the other two dyes azure A and azure B. The affinity of TBO was higher by about two times than that of the azures. From the series of studies using absorption, steady-state emission, the effect of ferrocyanide ion-induced steady-state fluorescence quenching, fluorescence polarization anisotropy, circular dichroism, the mode of binding of these dyes to the DNA double helix has been substantiated to be principally intercalative in nature. The stoichiometry of the association of these dyes to DNA was determined by the continuous variation analysis of Job from fluorescence data. The conformational aspects of the interaction was delineated from circular dichroism studies wherein higher perturbation was observed with TBO. Hydrodynamic study using viscosity measurements of linear rod like DNA confirmed that the binding was intercalative and strongest for TBO and weaker for azure A and azure B. The utility of the present work lies in exploring the potential binding applicability of these dyes to DNA for their development as effective therapeutic agents.

Self-structure formation in polyadenylic acid by small molecules: new insights from the binding of planar dyes thionine and toluidine blue O

Self-structure induction in single stranded poly(A) is a promising approach that can switch off protein production and pave a new route for the development of RNA based therapeutic agents. Utilising spectroscopic techniques and isothermal titration calorimetric methods, we examined the ability of two DNA binding phenothiazinium dyes, thionine (TH) and toluidine blue O (TB), to induce structural changes in ss poly(A). The cooperative binding of both the dyes to ss poly(A) was revealed from absorbance and fluorescence studies. The binding affinity was of the order of 106 M−1 at 50 mM [Na+] as determined from spectroscopic and calorimetric studies. Ferrocyanide quenching and viscosity studies showed intercalative binding of the dyes to poly(A). The binding perturbed the circular dichroism spectrum of poly(A) with concomitant formation of prominent induced CD bands in the 300–700 nm region for the dyes. Poly(A) forms self-structure in the presence of both TH and TB. The binding affinity and the ease of formation of self-structure increased with [Na+] in the presence of the dyes in the range 50–200 mM. The single stranded poly(A) binding affinity of TH is higher compared to TB. Poly(A) may be a potential bio-target of these dyes in their pharmacological application.

Targeting ribonucleic acids by toxic small molecules: Structural perturbation and energetics of interaction of phenothiazinium dyes thionine and toluidine blue O to tRNAphe

This study was designed to examine the toxic interaction of two phenothiazinium dyes thionine (TO) and toluidine blue O (TBO) with tRNA(phe) by spectroscopic and calorimetric techniques. While phenothiazinium dye complexation with DNA is known, their bindings to RNA are not fully investigated. The non cooperative binding of both the dyes to tRNA was revealed from absorbance and fluorescence studies. From absorption, steady-state emission, the effect of ferrocyanide ion-induced steady-state fluorescence quenching, circular dichroism, the mode of binding of these dyes into the tRNA helix has been substantiated to be principally by intercalative in nature. Both dyes enhanced the thermal stability of tRNA. Circular dichroism studies provided evidence for the structural perturbations associated with the tRNA structure with induction of optical activity in the CD inactive dye molecules. Results from isothermal titration calorimetry experiments suggested that the binding of both dyes was predominantly entropy driven with a smaller but favorable enthalpy term that increased with temperature. The binding was dependent on the Na(+) concentration, but had a larger non-electrostatic contribution to the Gibbs energy. A small heat capacity value and the enthalpy-entropy compensation in the energetics of the interaction characterized the binding of the dyes to tRNA. This study confirms that the tRNA(phe) binding affinity is greater for TO compared to TBO. The utility of the present work lies in understanding the potential binding and consequent damage to tRNA by these toxic dyes in their development as therapeutic agents.