The base specificity and energetics of DNA binding of the phenazinium dyes phenosafranine and safranine-O have been studied using various biophysical tools. The guanine-cytosine base specificity of both compounds was established from binding affinity values and competition dialysis results and also from circular dichroism, thermal melting, and calorimetric studies. Both dyes bind to DNA with affinity of the order of 10(5) M(-1), but the values are significantly higher for the guanine-cytosine rich DNAs over adenine-thymine rich ones and for phenosafranine over safranine-O. Calorimetric studies revealed that the binding reactions were exothermic and favoured by negative enthalpy as well as predominantly large positive entropy contributions. The temperature dependence of enthalpy changes yielded negative heat capacity values, which were higher for phenosafranine, compared to safranine-O, suggesting substantial contribution from hydrophobic forces in the binding process. Enthalpy-entropy compensation behaviour was also observed for the binding of both dyes to DNAs, revealing the molecular aspects of the interaction. Taken together, the spectroscopic and calorimetric data reflect clearly the guanine-cytosine base specificity of these molecules and a stronger DNA binding of PSF over SO. The results also provide some insights into the role of a bulkier substituent in the phenazinium ring in the binding process.

The sequence selectivity of the DNA binding of the phenazinium dyes phenosafranin and safranin O have been investigated with four sequence-specific deoxyribopolynucleotides from spectroscopic and calorimetric studies. The alternating guanine-cytosine sequence selectivity of the dyes has been revealed from binding affinity values, circular dichroism, thermal melting, competition dialysis, and calorimetric results. The binding affinities of both the dyes to the polynucleotides were of the order of 10(5) M(-1), but the values were higher for the guanine-cytosine polynucleotides over adenine-thymine ones. Phenosafranin had a higher binding affinity compared to safranin O. Isothermal titration calorimetric studies revealed that the binding reactions were exothermic and favored by negative enthalpy and predominantly large positive entropy contributions in all cases except poly(dA)·poly(dT) where the profile was anomalous. Although charged, nonpolyelectrolytic contribution was revealed to be dominant to the free energy of binding. The negative heat capacity values obtained from the temperature dependence of enthalpy changes, which were higher for phenosafranin compared to safranin O, suggested significant hydrophobic contribution to the binding process. In aggregate, the data presents evidence for the alternating guanine-cytosine base pair selectivity of these phenazinium dyes and a stronger binding of phenosafranin over safranin O.

The interaction of bioactive protoberberine alkaloids berberine, palmatine, and coralyne with the DNA triplex poly(dT) · (poly(dA) · poly(dT)) was studied using biophysical and calorimetric techniques. All three alkaloids bound the triplex cooperatively. Berberine and palmatine predominantly stabilized the triplex structure, while coralyne stabilized both triplex and duplex structures as inferred from optical thermal melting profiles. Fluorescence quenching, polarization, and viscometric studies hinted at an intercalative mode of binding for the alkaloids to the triplex, coralyne being more strongly intercalated compared to partial intercalation of berberine and palmatine. The overall affinity of coralyne was two order higher (2.29 Â 10 7 m À 1 ) than that of berberine (3.43 Â 10 5 m À 1 ) and palmatine (2.34 Â 10 5 m À 1 ). Isothermal titration calorimetric studies revealed that the binding to the triplex was favored by negative enthalpy change (DH ¼ À3.34 kcal/mol) with favorable entropy contribution (TDS ¼ 4.07 kcal/mol) for berberine, favored by almost equal negative enthalpy (DH ¼ À3.88 kcal/mol) and entropy changes (TDS ¼ 3.37 kcal/mol) for palmatine, but driven by large enthalpy contributions (DH ¼ À25.62 kcal/mol and TDS ¼ À15.21 kcal/mol) for coralyne. These results provide new insights on the binding of isoquinoline alkaloids to the DNA triplex structure.

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