Self-structure induction in single stranded poly(A) by small molecules: Studies on DNA intercalators, partial intercalators and groove binding molecules

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

“…Each experiment was repeated twice with separate fillings. The DSC thermograms of excess heat capacity versus temperature were analyzed using the Origin 7.0 software to determine the calorimetric transition enthalpy (ΔH cal ) as described earlier [25,30]. This calorimetrically determined enthalpy is model-independent and unrelated to the nature of the transition.…”

“…A MicroCal VP-ITC unit (MicroCal, Inc., Northampton, MA, USA) using protocols developed in our laboratory and described in details previously [25,30,35,36] was used for all ITC experiments. A reverse titration protocol of injecting aliquots of DNA solution from a rotating syringe (290 rpm) into the calorimetric cell containing the thionine solution (1.4235 mL) under identical buffer conditions was adopted.…”

“…The excess heat capacities as a function of temperature were measured on a Microcal VP-differential scanning calorimeter (DSC) (MicroCal, Inc., Northampton, MA, USA) as described previously [25,30]. In a series of DSC scans, both the sample and reference cells were loaded with buffer solution, equilibrated at 20°C for 15 min and scanned from 40°to 120°C at a scan rate of 60°C/h.…”

“…Job plot [28,29] was constructed to determine the binding stoichiometry in each case from fluorescence spectroscopy results at 20 ± 1°C as described previously [25,30]. At constant temperature, the fluorescence signal was recorded for mixture of solutions where the concentrations of both DNA and thionine were varied while the sum of their concentrations was kept constant.…”

“…Melting curves of DNA and DNA-thionine complexes were measured on a Shimadzu Pharmaspec 1700 unit equipped with a peltier controlled TMSPC-8 model accessory (Shimadzu Corporation, Kyoto, Japan) as described earlier [25,30]. DNA sample (20 μM) was mixed with varying concentrations of the thionine in the degassed buffer in to the eight chamber micro optical cuvette of 1 cm path length and the temperature of the microcell accessory was raised at a heating rate of 0.5°C/min.…”